HFP2019 Advanced Lecture Course

Molecular Mechanisms of Host-Pathogen Interactions and Virulence in Human Fungal Pathogens

May 18th-24th 2019

La Colle sur Loup, France

Contents

Important information 3

Organizing committee 5

Sponsors 6

General information 8

Program 12

Abstracts 24

Index 218

Please find the complete abstract booklet online:

https://hfp2019.febsevents.org

or

Important information

For questions or information regarding housing:

Belambra Clubs – Les Terrasses de Saint Paul de Vence

Chemin de Montmeuille

06480 La Colle sur Loup, France

Phone: +33 (0) 4 93 32 96 96

For questions or information regarding the conference:

Liesbeth Demuyser

liesbeth.demuyser@kuleuven.vib.be

Leni Vandoren

leni.vandoren@kuleuven.vib.be

Find us on Facebook and Twitter #HFP2019

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Organizing Committee

Organizers

Patrick Van Dijck (Belgium)

Jessica Quintin (France)

Alex Andrianopoulos (Australia)

Co-organizers

Liesbeth Demuyser (Belgium)

Nico Vangoethem (Belgium)

Leni Vandoren (Belgium)

International Scientific Advisory Board

Atilla Gaçser (Hungary) Xiarong Lin (USA)

Sarah Gaffen (USA) Mike Lorenz (USA)

Hubertus Haas (Austria) Donna MacCallum (United Kingdom)

Ilse Jacobsen (Germany) Robin May (United Kingdom)

Bruce Klein (USA) Joachim Morschhäuser (Germany)

Damian Krysan (USA) Julian Naglik (United Kingdom)

Oliver Kurzai (Germany) Clarissa Nobile (USA)

Salome Leibundgut-Landmann (Switzerland) Norman Pavelka (Singapore)

Anne Puel (France) Ana Traven (Australia)

Sponsors

The organizing committee would like to thank the sponsors for their contributions.

FEBS

FEMS

Burroughs Wellcome Fund

ISHAM

FWO – Research Foundation Flanders

SFMM

INRA-MICA

Opathy

Institut Carnot Pasteur MS

Départment de Mycologie – Institut Pasteur

Elsevier

VIB

Frontiers in Cellular and Infection

Microbiology

ECCM

Microbiology Society

Journal of Fungi

KU Leuven

Frontiers in Microbiology

Département de Mycologie

General information

Venue

Belambra Clubs – Les Terrasses de Saint Paul de Vence

Chemin de Montmeuille

06480 La Colle sur Loup, France

Phone: +33 (0) 4 93 32 96 96

Registration

The conference registration desk will be open on Saturday 18th of May, from 15:00 to 19:00 and the following mornings from 8:15 to 9:00 am the 19th – 21st of May.

Conference facilities

Meeting room and equipment

The meeting room (Riviera) is located near the main building of the complex. It is equipped with a digital projector, an overhead projector, and microphones. Two laptops will be available (Windows and Mac). Speakers should arrive in the meeting room 30 min. prior to their session to upload and check their presentation on these computers. It is strongly recommended that speakers bring their PowerPoint presentation on a memory stick so that all presentations can be loaded on one computer. If speakers use presentation software other than PowerPoint or Acrobat, they can bring their own laptop.

Posters

Poster boards are located in the three poster session rooms (Kandinsky, Miro and Monet). Posters should be posted on Sunday May 19th before the session starts and should be removed after the last poster session on Wednesday May 22nd.

Posters should have a maximum size of 100 cm x 120 cm (w x h). Participants should be at their posters on the following days:

Sunday May 19th: Posters with the letter A

Monday May 20th: Posters with the letter B

Wednesday May 22nd: Posters with the letter C

Material to attach the posters to the boards is provided.

Accommodation

General

Rooms have been booked at Club Belambra for the nights of May 18th through to May 23rd (6 nights), with departure on Friday May 24th. Accommodation will be either a studio (single) or small apartment with two bedrooms and a shared bathroom (two or three people).

Extra nights

If you require extra accommodation in addition to the nights included in the conference booking, please contact the venue directly. Extra nights are not covered in the registration package.

Meals

Breakfast will be served in buffet style from 7:30 am. Times for lunch and dinner are as shown in the conference program. Mineral water and coffee are served at each meal. Additional beverages can be purchased at the participants’ own expense.

Site services

Telephone calls

There are no telephones in the bedrooms. There are several public telephones at the conference site. These operate with calling cards that can be bought at the reception desk. A public telephone operating with coins is available in the main building.

Photocopies and faxes

Photocopies may be made, and faxes sent and paid for at the reception desk.

Computer facilities and internet connection

Internet access via Wifi is available in the lobby area and surrounds. There is an internet café in La Colle sur Loup.

Means of payment to the conference site

Credit cards are accepted by the Club Belambra reception desk (not at the bar) and all currency payments should be in Euro. It is not possible to change foreign currency at the venue. Al additional expenses, such as drinks (except those served during meals), telephone calls, tours, etc. are at the participants’ own expense and should be paid upon check-out from the Club Belambra.

Bank facilities

The nearest bank, the Caisse d’Epargne, is in La Coll sur Loup (2 km, 15 min walk). This bank has an ATM where withdrawals with an international credit card are possible. Exchange of foreign currencies or withdrawal of Euros with international credit cards is possible at Nice Airport.

Leisure activities and tourism

Weather

Weather in May is normally sunny with temperatures ranging from 15 to 20°C during the day and 8 to 12°C during the night. However, showers are also possible. Check the weather forecast to get the latest information.

On site

The venue is set within a 25 acres private pine-tree forest and provides numerous recreational and leisure facilities: bar with terrace, TV room, American pool table, outdoor swimming pool, volleyball and basketball courts, bowl pitch and 3 tennis courts.

The surrounding area

The countryside provides opportunities for hiking, rock-climbing and canyoning. La Colle sur Loup is close to the traditional village of Saint Paul de Vence with the Maeght Foundation displaying an impressive collection of modern art. It is also close to Nice with the Matisse museum and the Chagall museums, Vence with the Chapel of Rosary imagined and achieved by Henri Matisse, and Vallauris with the Picasso museum. It is also close to Biot famous for its glass workshop and Grasse famous for its perfume industry. Excursions will be organized during the free afternoon of May 21st.

Social program

Saturday May 18th – Welcome drinks

Tuesday May 21st – Excursions on free afternoon and evening

Thursday May 23rd – Conference dinner and aperitif followed by a party

For the excursions, three busses have been organized going to either Nice, Monaco or Saint Paul de Vence and Nice.

Participants will be asked to select their preferred excursion at registration as there is limited availability for each excursion (53 persons each). Busses will be organized to return to the site from the different locations. On the evening of May 21st, you will be free to make your own dinner arrangements.

Insurance

The meeting organizers do not provide insurance and cannot take responsibility for accidents or illnesses that might occur during the week, or in the course of travel to and from the venue. It is therefore the responsibility of each participant to check that they are adequately covered by health and travel insurance.

Shopping hours

The nearest shops (newspapers, toiletries, etc.) are in La Colle sur Loup, about 15 minutes walking distance from the Club Belambra. Major shopping can be done in Nice. Opening times of shops in France in general are Monday through Saturday: 09:00 to 12:00 and 14:00 to 19:00 but many close on Monday.

Program

Program at a glance

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Detailed program

Saturday 18th May 2019

15:00 - 19:00 Registration

16:30 - 18:00 Opathy – World café (discussion sessions)

19:00 - 20:30 Dinner

20:30 - 20:35 Welcome by Patrick Van Dijck

20:35 – 20:40 FEBS by Stefana Petrescu

20:40 – 20:45 FEMS by Carol Munro

20:45 – 21:30 Keynote lecture, sponsored and chaired by Opathy

Beyond resistance: insights into subpopulation responses to antifungals (T1)

Judith Berman, Tel Aviv University - Israel

21:30 - 23:30 Welcome drinks

Sunday 19th May 2019

07:30 - 08:30 Breakfast

Session 1. Pathogenic Fungi – Genomics, Evolution and Epidemiology

Sponsored by SFMM

08:30 - 08:50 Introduction by the chair – Geraldine Butler, University College Dublin - Ireland

08:50 - 09:20 Candida albicans genome diversity: mechanisms and consequences (T2)

Christophe d’Enfert, Institut Pasteur - France

09:20 - 09:50 Genome comparisons reveal the secret life of Candida glabrata: not so asexual, not so commensal (T3)

Toni Gabaldon, Center for Genomic Regulation Barcelona - Spain

09:50 - 10:20 Coffee break

10:20 - 10:50 Genome evolution of virulence traits and drug resistance in human fungal pathogens (T4)

Christina Cuomo, Broad Institute of MIT, Harvard - USA

10:50 - 11:20 Fungal genome and mating system transitions facilitated by chromosome translocation and fusion (T5)

Joe Heitman, Duke University - USA

11:20 - 11:50 Mechanisms driving C. albicans genome plasticity in the presence and absence of antifungal drugs (T6)

Anna Selmecki, Creighton University medical School - USA

12:15 - 13:30 Lunch

15:00 - 15:45 Opathy – Discussion results

Workshop talks 1 Chairs: Meleah Hickman (Emory University - USA) and Oliver Bader (University Medical center Göttingen - Germany)

15:45 - 16:00 Spore germination as a target for antifungal therapeutics (P31A)

Sébastien Ortiz, University of Wisconsin - USA

16:00 - 16:15 Comparative transcriptomics reveals generalized patterns of host-pathogen interactions between human and major Candida fungi (P34A)

Hrant Hovhannisyan, Centre For Genomic Regulation - Spain

16:15 - 16:30 Evidence for multiple in vivo selective pressures contributing to MRR1 variation in a C. lusitaniae population (P37A)

Elora Demers, Dartmouth College - USA

16:30 - 16:45 Studying the role of CgMIP1 in the evolution of Candida glabrata during adaption to the human host (P40A)

Sofia Siscar Lewin, Hans Knöll Institute - Germany

16:45 - 17:15 Coffee break

Elevator talks 1 Chairs: Meleah Hickman (Emory University - USA) and Oliver Bader (University Medical center Göttingen - Germany)

17:15 - 17:21 Multi-transcriptomic studies of Cryptococcus reveal the broad evolutionary utility of start codon context in the fungal kingdom (P1A)

Edward Wallace, University of Edinburgh - UK

17:21 - 17:27 Effect of temperature stress on transposon-mediated mutagenesis and drug resistance in Cryptococcus neoformans in a host model of infection (P4A)

Asiya Gusa, Duke University - USA

17:27 - 17:33 Comparison of stress resistance in Candida auris with other pathogenic Candida species (P7A)

Helen Heaney, University of Aberdeen - UK

17:33 - 17:39 Genotypic and phenotypic diversity in Candida tropicalis (P10A)

Caoimhe O’Brien, UCD Conway Institute - Ireland

17:39 - 17:45 Into the identification of genetic polymorphisms in C. albicans genome associated with the variability in the interaction of diverse clinical isolates with host keratinocytes by dual transcriptomics (P13A)

Abhilash Kannan, CHUV - Switzerland

17:45 - 17:51 How chromosomes are impacted by stress: Elucidating genome-wide LOH dynamics in Candida albicans (P16A)

Timea Marton, Institut Pasteur - France

17:51 - 17:57 Rampant transposition and loss of RNAi cause hypermutation and antifungal drug resistance in Cryptococcus neoformans (P19A)

Shelby Priest, Duke University - USA

17:57 - 18:03 Genomic patterns of Candida parapsilosis s.l. hybrids suggest a weak effect of Bateson-Dobzhansky-Muller incompatibilities (P22A)

Veronica de Pinho Mixao, Centro de Regulacion Genomica - Spain

17:03 - 18:09 Analysis of hotspot regions of the genes involved in primary resistance (erg11 and fks1) in C. inconspicua, C. rugosa and C. ciferrii (P25A)

Antonio Pérez Hansen, Medical University of Innsbruck - Austria

18:09 - 18:15 Functional implications of the genetic diversity in C. albicans (P28A)

Kontxi Martinez de San Vicente, University Zurich - Switzerland

19:00 - 20:30 Dinner

20:30 - 23:00 Poster session A

Monday 20th May 2019

07:30 - 08:30 Breakfast

Session 2. Molecular and Cellular Biology of Fungal Pathogens

08:30 - 08:50 Introduction by the chair – Joachim Morschhäuser, University of Würzburg - Germany

08:50 - 09:20 Cellular dynamics during Candida albicans morphogenesis (T7)

Martine Bassilana, Université Côte d’Azur Nice - France

09:20 - 09:50 From systems biology to damage limitation in Aspergillus-related disease (T8)

Elaine Bignell, University of Manchester - UK

09:50 - 10:20 Coffee break

10:20 - 10:50 Host sensing, nutrient acquisition and cryptococcal virulence (T9)

Jim Kronstad, University of British Columbia - Canada

10:50 - 11:20 Circuit diversification in a biofilm regulatory network (T10)

Aaron Mitchell, Carnegie Mellon University in Pennsylvania - USA

11:20 - 11:50 Chromatin dynamics - a double-edged sword in fungal virulence (T11)

Karl Kuchler, Medical University Vienna - Austria

12:15 - 13:30 Lunch

Workshop Talks 2 Chairs: Mary Ann Jabra-Rizk (University of Maryland Baltimore - USA) and Duncan Wilson (University of Aberdeen - UK)

15:45 - 16:00 Reducing animal use in antifungal drug development: near-infrared reporter strains and imaging (P29B)

Ambre Chapuis, University of Aberdeen - UK

16:00 - 16:15 Bacteria and fungi: how do they communicate? (P32B)

Filomena Nogueira, CCRI - Austria

16:15 - 16:30 The central repressor Tup1 is required for high-level expression of ALS3 and ECE1 in Candida albicans (P35B)

Sophia Ruben, Hans Knöll Institute - Germany

16:30 - 16:45 Protein mistranslation modulates hyphal initiation in Candida albicans (P38B)

Carla Oliveira, iBiMED - Portugal

16:45 - 17:15 Coffee break

Elevator talks 2 Chairs: Mary Ann Jabra-Rizk (University of Maryland Baltimore - USA) and Duncan Wilson (University of Aberdeen - UK)

17:15 - 17:21 At the cross-section of PKA activity and iron metabolism - riboflavin synthesis as a potential antifungal drug target (P2B)

Liesbeth Demuyser, KU Leuven/VIB - Belgium

17:21 - 17:27 Cell wall changes associated with echinocandin resistance across Candida species (P5B)

Giuseppe Buda De Cesare, University of Aberdeen - UK

17:27 - 17:33 A role for vesicular transport in fungal susceptibility to fluconazole (P8B)

Katrien Van Dyck, KU Leuven/VIB - Belgium

17:33 - 17:39 PolyP Mobilisation in the Pathobiology of Candida albicans (P11B)

Yasmin Ahmed, Biotechvana - Spain

17:39 - 17:45 Candida mixed species competitions in different pH, carbon, and media environments (P14B)

Brooke Esquivel, University of Missouri Kansas City - USA

17:45 - 17:51 Identifying novel antifungal agents against the drug-resistant pathogen Candida auris (P41B)

Kali Iyer, University of Toronto - Canada

17:51 - 17:57 Inhibition of spore germination by macrophages promotes A. fumigatus persistence and susceptibility to voriconazole (P17B)

Emily Rosowski, Clemson University - USA

17:57 - 18:03 Candida albicans quorum sensing molecule farnesol modulates staphyloxanthin production and activates the thiol-based oxidative stress response in Staphylococcus aureus (P20B)

Taissa Vila, University of Maryland Baltimore - USA

18:03 - 18:09 Characterisation of a novel zinc detoxification system and its role in the pathogenicity of Candida albicans (P23B)

Aaron Crawford, University of Aberdeen - UK

18:09 - 18:15 Antibody theranostics for fungal infections (P26B)

Louise Walker, University of Aberdeen - UK

19:00 - 20:30 Dinner

20:30 - 23:00 Poster session B

Tuesday 21st May 2019

07:30 - 08:30 Breakfast

Session 3. Advanced tools for human fungal pathogen research

08:30 - 08:50 Introduction by the chair – Hubertus Haas, Medical University Innsbruck - Austria

08:50 - 09:10 Spike in your RNA-Seq experiments. How to do it and what you might gain doing it (T12)

Guilhem Janbon, Institut Pasteur - France

09:10 - 09:30 Antibody-guided imaging of invasive fungal infections (T13)

Christopher Thornton, University of Exeter - UK

09:30 - 09:50 A non-invasive imaging toolbox to monitor fungal infection progression, dissemination and host response in small animal models (T14)

Greetje Vande Velde, KU Leuven MoSAIC facility - Belgium

09:50 - 10:10 How host cells respond to Candida hyphal growth (T15)

Sergio Grinstein, University of Toronto - Canada

10:20 - 10:50 Coffee break

Session 4. Mycobiome

10:50 - 11:10 Introduction by the chair – Ilse Jacobsen, Hans Knöll Institute - Germany

11:10 - 11:30 Gut colonization by Candida albicans (T16)

Christian Pérez, University of Würzburg - Germany

11:30 - 11:50 Fungi in the oral cavity; directors of the bacteriome? (T17)

Bastiaan Krom, ACTA - The Netherlands

12:30 - 13:30 Lunch

14:00 - 15:00 Free afternoon

Departure for sightseeing. Buses to Nice, Monaco and St Paul de Vence

Wednesday 22nd May 2019

07:30 - 08:30 Breakfast

Session 5. Host-pathogen interactions – the host perspective

08:30 - 08:50 Introduction by the chair – Julian Naglik, King’s College London - UK

08:50 - 09:20 Metabolic regulation of Candida-macrophage interactions (T18)

Ana Traven, Monash University - Australia

09:20 - 09:50 CLRs and anti-fungal immunity (T19)

Selinda Orr, Cardiff University - UK

09:50 - 10:20 Coffee break

10:20 - 10:50 Fungi in blood – how fungal pathogens interact with circulating immune cells (T20)

Oliver Kurzai, University of Würzburg - Germany

10:50 - 11:20 TBA (T21)

TBA

11:20 - 11:50 IL-17 in antifungal immunity: beyond Candida (T22)

Salomé Leibundgut-Landmann, University of Zürich - Switzerland

12:15 - 13:30 Lunch

13:30 - 15:00 Sports event organized by Opathy

Workshop talks 3

Chairs: Emily Rosowski (Clemson University - USA) and Bernard Hube (Hans Knöll Institute - Germany)

15:45 - 16:00 Exploring how a family of small-secreted effectors manipulates macrophages during Histoplasma capsulatum infection (P33C)

Rosa Rodriguez, UC San Francisco - USA

16:00 - 16:15 DHN-melanin of the human pathogenic fungus Aspergillus fumigatus intervene with cell-autonomous defenses of amoebae (P36C)

Iuliia Ferling, Hans Knöll Institute - Germany

16:15 - 16:30 Manipulation of the dendritic cell/T cell interface by the human fungal pathogen Cryptococcus neoformans inhibits antigen-mediated T cell proliferation (P39C)

Guillaume Desanti, University of Birmingham - UK

16:30 - 16:45 The genetic basis of Candida glabrata persistence within macrophages (P42C)

Sascha Brunke, Hans Knöll Institute - Germany

16:45 - 17:15 Coffee break

Elevator talks 3

Chairs: Emily Rosowski (Clemson University - USA) and Bernard Hube (Hans Knöll Institute - Germany)

17:15 - 17:21 Defining pH-sensing in fungal virulence (P3C)

Hannah Brown, Duke University - USA

17:21 - 17:27 The Pathogen Box: Using drug repurposing to identify inhibitors of Candida auris (P6C)

Gina Wall, University of Texas at San Antonio - USA

17:27 - 17:33 Exploring the effects of Interferon alpha on Cryptococcus neoformans infection (P9C)

Paula Seoane, University of Birmingham - UK

17:33 - 17:39 Exploring the host-pathogen transcriptomics of macrophages, Mucorales and their endosymbionts (P12C)

Poppy Sephton Clark, University of Birmingham - UK

17:39 - 17:45 Identification of novel Candida albicans transcription regulators with roles in host colonization (P15C)

Philipp Reuter-Weissenberger, University of Würzburg - Germany

17:45 - 17:51 Interaction of human neutrophil granulocytes and the melanin producing fungus, Curvularia lunata (P18C)

Eszter Judit Toth, University of Szeged - Hungary

17:51 - 17:57 Mechanical forces during filamentous growth of a human fungal pathogen (P21C)

Charles Puerner, University Côte d'Azur - France

17:57 - 18:03 Pseudomonas aeruginosa-derived volatile sulphur compounds promote distal Aspergillus fumigatus growth and a synergistic

pathogen-pathogen interaction that increases pathogenicity in co-infection (P24C)

Jennifer Scott, University of Manchester - UK

18:03 - 18:09 How lactobacilli antagonize pathogenicity of Candida albicans: Lessons from in vitro gut models and a dynamic organ-on-chip model (P27C)

Mark Gresnigt, Hans Knöll Institute - Germany

18:09 - 18:15 Pathogenic Candida species and the filamentous fungus Aspergillus fumigatus trigger distinct killing and feeding mechanisms in a fungivorous amoeba (P30C)

Falk Hillmann, Hans Knöll Institute - Germany

19:00 - 20:30 Dinner

20:30 - 23:00 Poster session C

Thursday 23rd May 2019

07:30 - 08:30 Breakfast

Session 6. Host-pathogen interactions – the pathogen perspective

08:30 - 08:50 Introduction by the chair – Mike Lorenz, University of Texas - USA

08:50 - 09:20 Sugars and surfaces – what’s important to Cryptococcus? (T23)

Tamara Doering, Washington University in St Louis - USA

09:20 - 09:50 Interactions between the emerging fungal pathogen Candida auris and the host (T24)

Guanghua Huang, Chinese Academy of Science Beijing - China

09:50 - 10:20 Coffee break

10:20 - 10:50 Surface molecules of Aspergillus fumigatus decisive for immune evasion (T25)

Axel Brakhage, Hans Knöll Institute - Germany

10:50 - 11:20 Fungal stress signalling pathways as mediators of virulence (and how we can exploit this) (T26)

Janet Quinn, University of Newcastle - UK

11:20 - 11:50 Exploring the host-pathogen arms race in cryptococcosis (T27)

Robin May, University of Birmingham - UK

12:15 - 13:30 Lunch

Session 7. Antifungal strategies

15:00 - 15:20 Introduction by the chair – Damian Krysan, University of Iowa - USA

15:20 - 15:40 Success and challenges of glucan synthase inhibitors (T28)

David Perlin, Hackensack Center for Discovery and Innovation - USA

15:40 - 16:00 Novel strategies to the treatment of fungal infections with emphasis on anti-virulence approaches (T29)

José Lopez-Ribot, University of Texas - USA

16:00 - 16:20 Cryptococcal meningitis: trials and tribulations (T30)

Tihana Bicanic, University of London - UK

16:20 - 16:50 Coffee break

16:50 - 17:50 Keynote lecture

Development of a Candida vaccine: next steps, scientific and development challenges, and implications for other fungi and bacteria (T31)

Jack Edwards, UCLA - USA

17:50 - 18:00 Award ceremony

19:00 - 20:30 Dinner

20:30 - 01:30 Farewell party

Friday 24th May 2019

07:00 - 09:00 Departure

Abstracts Talks

Beyond resistance: insights into subpopulation responses to antifungals

J. BermanI

ITel Aviv University, Ramat Aviv, Israel

Treatment of fungal infections in animals, or fungal infestations of crops, requires an understanding of how much of an antifungal agent needs to be applied, how often touse it and what types of responses to expect. In general, clinical studies have focused on the minimum inhibitory concentration: the concentration of drug at which 50%or more of the cells exposed to the drug do not grow. Above this concentration, cells are said to be susceptible. However, many patients infected with susceptibleisolates suffer persistent or recurrent infections despite being treated with drug concentrations above the MIC for the infecting organisms. Phenomena known astolerance, trailing growth, heteroresistance or persistence have been noted and are due to subpopulations of cells that grow slowly in the presence of the drug. Thesesubpopulations, together with the selective pressure of drug treatment acting upon them, have the potential to drive the evolution of new mechanisms for survival of thepathogen. We have been studying tolerance and heteroresistance in Candida albicans and Candida glabrata, respectively. This talk will highlight the growthdynamics, physiological responses and evolutionary responses of subpopulations that survive and grow, albeit slowly, in the presence of severe antifungal stress. One ofthe mechanisms of survival in drug involves changes in gene stoichiometry, often via aneuploidy or gene amplification, and its effects on the expression of specific genes.

Candida albicans genome diversity: mechanisms and consequences

C. d'EnfertI

IInstitut Pasteur, Paris, France

The fungal pathogen Candida albicans shows significant diversity at the genetic and phenotypic levels. Here, I will review our current knowledge of the C. albicansdiploid genome and its variability, the genetic structure of the C. albicans population and the mechanisms that are involved in C. albicans genome dynamics, with afocus on the parasexual cycle and loss­of­heterozygosity events. I will further explore the impact of genetic diversity and genome dynamics on C. albicans phenotypicdiversity. Finally, I will discuss how our current knowledge of C. albicans genetic diversity could be leveraged in the future in order to get insights in the mechanismsunderlying important biological attributes that are subject to variations across C. albicans isolates.

Genome comparisons reveal the secret life of Candida glabrata: not soasexual, not so commensal

T. GabaldonI

ICentre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain., Barcelona, Spain

Candida glabrata is an opportunistic yeast pathogen, whose incidence has increased over the last decades. Despite its genus name, this species is actually more closelyrelated to the budding yeast Saccaromyces cerevisiae than to other Candida pathogens, such as Candida albicans. Hence, C. glabrata and C. albicans must haveacquired the ability to infect humans independently, which is reflected in the use of different mechanism for virulence, and survival in the host. Yet, research on C.glabrata generally departs by adopting assumptions influenced by lessons learned from the more studied C. albicans. Regarding the adaptation of C. glabrata to thehuman host, the prejudice was that, just as C. albicans, C. glabrata is a natural human commensal that turns deadly when immune defenses weaken. It was alsoconsidered asexual, as no one has observed mating, diploids, or spores, despite great efforts. However, the recent analysis of whole genomes from globally distributedC. glabrata isolates have shaken these assumptions. C. glabrata seems to be only secondarily associated to humans, as indicated by a lack of co­evolution with itshost, and genomic footprints of recombination shows compelling evidence that this yeast is able to have sex. Here I will discuss the implications of this and other recentfindings and highlight the new questions opened by these results.

Genome evolution of virulence traits and drug resistance in humanfungal pathogens

C. CuomoI

IBroad Institute, Cambridge, MA, United States of America

Population genomic studies of human fungal pathogens reveal how natural variation contributes to important phenotypes including virulence and drug resistance. Wehave combined large scale genome sequencing and phenotyping to search for such associations. In Cryptococcus, we have used rare variant approaches to map genesassociated with human infection and identified new candidate genes involved in drug resistance. Population genomic analyses of these data identified regions underselective pressure and the extent of gene flow and recombination within this species. In parallel studies of recently emerging pathogens, genomic epidemiologicalapproaches identified major population subdivisions and mapped mutations that confer drug resistance. These studies have begun to characterize how evolution acrossrecent or more ancient timescales has impacted the genomes of fungal pathogen populations and how this natural variation contributes to phenotypes important forhuman infection.

Fungal genome and mating system transitions facilitated by chromosometranslocation and fusion

J. HeitmanI

IDuke Univeristy, Durham, United States of America

Cryptococcus neoformans is a global, environmental pathogenic basidiomycete fungus that infects immunocompromised humans causing significant morbidity/mortality.Cryptococcus gattii is a related pathogen, more geographically restricted and causes fewer infections but often in healthy individuals. Ten distinct species populate thismonophyletic pathogenic species complex. The pathogens are embedded in two broader species groups (sensu stricto, sensu lato), which are saprobic non­pathogens. The sensu stricto complex includes the pathogens and four additional species: Cryptococcus amylolentus, Cryptococcus depauperatus, Cryptococcuswingfieldii, and a new species Cryptococcus floricola. The sensu lato complex includes Kwoniella mangrovensis, Kwoniella botswanensis and others. Howmating type systems, genome organization, and pathogenesis evolved is being addressed by comparative genomics employing Illumina, PacBio, and Nanoporesequencing and chromoblot analysis. This enables complete genome assembly, revealing genes unique to pathogens or particular lineages, higher order genomicarchitecture, specific chromosomal loci (centromeres, telomeres), and chromosome number alterations. We discovered extant sexual cycles and defined mating­typeloci for multiple species. By comparing chromosome­wide complete genomes, the closest nonpathogenic sibling species (C. amylolentus, C. wingfieldii, C. floricola)all have 14 chromosomes like C. neoformans/C. gattii, but with translocations and intrachromosomal rearrangements. We defined extant tetrapolar sexual cycles forC. amylolentus and C. floricola and found the MAT loci are genetically linked to large regional repetitive centromeres of their host chromosomes. Inter­centromericrecombination has driven chromosomal translocations and may have facilitated transition from an ancestral tetrapolar outcrossing saprobic state to the derivedpathogenic bipolar configuration. Similar inter­centromeric chromosomal translocations are found in the C. neoformans/C. gattii genomes, and those of other speciesin the complex. These findings show recombination involving repeats within centromeres impacts evolutionary trajectories during mating­type locus evolution andspeciation. Remarkably, other species analyzed have fewer chromosomes, in some cases as few as 11, 8, or only 3, providing insights chromosomal translocation andfusion events that sculpted genomic organization in the evolution of pathogens from saprobes.

Mechanisms driving Candida albicans genome plasticity

R. ToddI, T. WikoffI, A. SelmeckiI

ICreighton University, Omaha, United States of America

Genome rearrangements resulting in copy number variation (CNV) and loss of heterozygosity (LOH) are frequently observed during the somatic evolution of cancer andpromote the rapid adaptation of fungi to novel environments. In the diploid, heterozygous fungal pathogenCandida albicans, CNV and LOH can confer increasedvirulence and antifungal drug resistance, yet the mechanisms driving these somatic genome rearrangements are not completely understood. Here, we examined the roleof genome architecture during the formation of genetic variation in the diploid, heterozygous fungal pathogen, C. albicans. Our genome­wide analysis identified longrepeat sequences that had prominent roles in generating genomic diversity across diverse strain backgrounds including clinical, environmental, and experimentallyevolved isolates. These long repeat sequences included previously uncharacterized repeat sequences, centromeric repeats, repeats found within intergenic sequences,and repeats that span multiple ORFs and intergenic sequences. Importantly, long repeat sequences were found at every CNV and sequence inversion breakpointobserved, and frequently occurred at LOH breakpoints as well. Furthermore, these results were independent of genetic background or source of isolation. Thus, longrepeat sequences found across the C. albicansgenome underlie the formation of significant genome variation that can increase fitness and drive adaptation.

Cellular dynamics during Candida albicans morphogenesis

A. WeinerI, R. WakadeI, D. StalderI, S. BoglioloI, P. SilvaI, C. PuernerI, M. Basante­BedoyaI, R. ArkowitzI, M. BassilanaI

IUniversity Côte d’Azur, Nice, France

Virulence of the human fungal pathogen Candida albicans depends in particular on the switch from budding to filamentous hyphal growth, which requires reorganizationof the cytoskeleton and Golgi, together with sustained membrane traffic, to maintain this highly polarized process. To understand the temporal and spatial regulation ofhyphal morphogenesis in C. albicans, we analyzed both the dynamics and organization of the secretory pathway, using live cell imaging and three­dimensional electronmicroscopy, focusing essentially on small GTPases, as these molecular switches play key roles in cytoskeleton organization and membrane trafficking in eukaryotic cells.Live cell imaging and genetic analyses show the interplay between different classes of small GTPases, as well as phospholipids, during initiation and maintenance ofhyphal growth. In addition, analyses of loss of function mutants highlight the importance of these small GTPases and their regulators in fungal growth and virulence.Furthermore, using three­dimensional electron microscopy, we demonstrate that the C. albicans secretory pathway is organized in different domains, including a sub­apical zone exhibiting distinct membrane structures. Our results also shed light on the organization and dynamics of the Spitzenkörper, a structure characteristic offilamentous fungi, and indicate that the organization of the secretory pathway in C. albicans facilitates short­range secretory vesicle delivery.

From systems biology to damage limitation in Aspergillus­related disease

E. BignellI

IManchester Fungal Infection Group (MFIG), Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicineand Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

Research in my lab seeks a mechanistic understanding of fungal disease with a view to developing novel diagnostics and antifungal therapies. Our approach to studyingthe host­pathogen interaction transcends multiple experimental scales to address disease outcomes at the molecular, cellular, tissue, organ and whole animal levels. Weare currently combining this suite of tools with a systems level approach to define pathogenicity in the major mould pathogen of humans, Aspergillus fumigatus. We arealso developing novel pH signalling inhibitors as novel antifungal drugs, and studying secreted fungal proteins as novel vaccine candidates and diagnostic tools. In thistalk I will present the data from a new genome­scale study addressing the A. fumigatus regulatory network driving stress adaptation and epithelial damage, andexamine how this knowledge might lead to novel therapeutic interventions.The human lung is continually exposed to spores of the airborne mould Aspergillus fumigatus. Inhaled spores are small enough to bypass mucociliary clearancemechanisms and reach the alveoli of the lung where interaction between host and pathogen cells can lead to fungal clearance, or to development of inflammatory orinvasive fungal diseases. A. fumigatus is an accidental pathogen whose encounters with the host, although frequent, are circumstantial in nature. The capacity of thisorganism to cause human disease is unique amongst a cohort of several hundred related Aspergillus species and relative to closest sequenced relatives there are no largescale genetic events which signify recent evolution of pathogenicity. Clinical relevance of aspergillus species does not correlate with fungal growth rate, but is positivelycorrelated with thermotolerance, and epithelial toxicity.Fuelled by the results of extensive fungal transcriptome research we have developed, in recent years, a programme of research which seeks the mechanistic basis of lungdamage during A. fumigatus infection. In order to find out why A. fumigatus is a pathogen and how infectionis cytotoxic to human epithelia we have observed theinteraction between host and pathogen in laboratory culture and in mouse disease, and measured epithelial decay, lytic death of host cells, host cell signalling andcytokine degradation in response to fungal challenge. This research has revealed multiple mechanisms involved in epithelial damage, at different stages of the host­pathogen interaction.

Host sensing, nutrient acquisition and cryptococcal virulence

J. Kronstad *I, G. Bairwa *I, M. Caza *I, L. Horianopoulos *I, G. Hu *I, E. Sánchez­León *I, F. Mayer *I

IMichael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

The pathogenic fungus Cryptococcus neoformans causes a meningoencephalitis disease that is estimated to result in 15% of deaths in people with HIV/AIDS. Themolecular mechanisms by which C. neoformanssenses and interprets nutrient signals to support proliferation in vertebrate hosts are poorly understood. We areapplying molecular genetic and transcriptomic approaches to identify functions required for iron sensing and uptake. Iron is particularly important for C.neoformansbecause the availability of this metal influences elaboration of the polysaccharide capsule that is the major virulence factor of the fungus. Part of our effortinvolves the identification of mutants with impaired ability to proliferate on inorganic iron or heme, or with increased susceptibility to non­iron metalloprotoporphyrins.This work revealed, for example, that mutants defective in clathrin­mediated endocytosis have altered susceptibility to non­iron metalloprotoporphyrins and aredefective in heme trafficking. Additionally, the Sec1/Munc18 (SM) protein Vps45, which has a regulatory role in vesicle trafficking through interactions with SNAREproteins, also contributes to endocytosis, iron acquisition, and virulence in a mouse model of cryptococcosis. In parallel, we are characterizing the regulatory factors thatlink iron sensing with virulence in C. neoformans. A related project in the lab focuses on characterizing the role of the cAMP signaling pathway in the control ofvirulence factor elaboration. This work revealed the proteasome as a target of PKA regulation, and the analysis of mutants with increased susceptibility to proteasomeinhibitors identified multiple functions that influence capsule formation. These include transcription factors, signaling components and, interestingly, the Cap60 protein thatis implicated in capsule formation. Overall, our approaches are providing novel insights into connections between nutrient sensing and cryptococcal virulence.

* The authors marked with an asterisk equally contributed to the work.

Circuit diversification in a biofilm regulatory network

M.Y. HuangI, C.A. WoolfordI, G. MayI, C.J. McManusI, A. MitchellI

ICarnegie Mellon University, Pittsburgh, United States of America

How diverse are genotype­phenotype relationships among members of a species? Knowledge of genotype­phenotype consistency is especially important for pathogens,because mutant phenotypes define both virulence traits and drug targets. We have addressed this question with the fungal pathogen Candida albicans, which formsbiofilms on implanted medical devices as a prelude to infection. Our understanding of biofilm formation comes primarily from studies of one clinical isolate, SC5314, andits marked derivatives. We used CRISPR­based methods to create mutations of four key biofilm regulatory genes – BCR1, UME6, BRG1, and EFG1 – in five diverseclinical isolates. Phenotypic analysis revealed that mutations in BCR1 or UME6 have variable impact across strains, while mutations in BRG1 or EFG1 had uniformlysevere impact. Gene expression, sampled with Nanostring probes and examined comprehensively with RNA­Seq, indicates that network relationships are highlyvariable among isolates. Our gene expression data lead to a simple hypothesis that explains variable impact of mutations of BCR1, and functional assays support thehypothesis. Our analysis provides a new approach to sample natural variation and illustrates the need to do so.

Chromatin Dynamics ­ A Double­Edged Sword in Fungal Virulence

K. KuchlerI

IMedical University Vienna, Max F. Perutz Laboratories, Center for Medical Biochemistry, Vienna Biocenter, Dr. Bohr­Gasse 9/2, 1030 Vienna, Austria

The increased incidence of invasive fungal infections and the constant raise of antifungal drug resistance adversely impacts both healthcare management and medicare.Candida albicans is the most prevalent opportunistic human fungal pathogen and the leading cause of fungal blood­stream infections. In addition to knownpathogenicity mechanisms, epigenetic adaptations constitute emerging mechanisms critically determining the outcome of invasive fungal infections. Candida albicans isan extraordinary example of how a commensal fungal pathogen can sense and integrate immune signals to allow for rapid adaptation and survival within distinct hostniches. Subtle transcriptional changes can arise from altered chromatin organization, which profoundly affects cell fate during the C. albicans yeast­to­hyphaemorphogenetic transition, a key virulence trait. Morphogenesis entails both transcriptional modulation and chromatin remodeling mediated by histone deacetylases(HDACs) and acetyl­tranferases (HATs), which regulate specific sets of histone­ as well as non­histone target proteins and their genetic regulatory networks.Importantly, HDACs/HATs act in concert with histone chaperones such as HIR to coordinate nucleosome remodeling through replication­dependent as well asreplication­independent mechanisms. Thus, chromatin dynamics enables adaptive survival strategies adopted by fungal pathogens to evade host immune surveillance orantifungal treatments. In my talk, I will discuss how certain HDACs and HATs, as well as the HIR chaperone, control pathogenicity of Candida albicans. In addition, Iwill also discuss some mechanisms by which the histone chaperone HIR enables immune evasion by C. albicans. Finally, I shall discuss the validity of an emergingparadigm change for the treatment of invasive fungal diseases, which is to modulate the host immune response rather than targeting the pathogen itself.

Spike in your RNA­Seq experiments. How to do it and what you mightgain doing it.

P.A. FerrarezeI, C. MaufraisII, C.C. StaatsI, G. JanbonII

IUniversidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, IIInstitut Pasteur, Paris, France

RNA­Seq analysis has become a classic of pathogenic fungi analysis in recent years. Because it became very cheap and because the first bioinformatics analyses arestraightforward, a large amount RNA­Seq data are produced every year. In most cases, a mutant strain is compared to the reference strain or a wild type strain isgrown under different conditions. Unusually, one looks for genes up and down regulated, tries to identify regulated pathways and raises some hypotheses concerning thefunction of the mutated gene or the action of a molecule added to the medium. The first step in the statistical analysis of the data consists in their normalization. To do so,the commonly used normalization pipelines assume that most genes are not affected by the mutation or the change in growth condition studied. They hypothesized thusthat just a small subset of genes have their level of expression altered. This hypothesis is true in number of cases and important discoveries have been made using thistype of data normalization. Yet, it can be completely wrong leading then to completely biased analyses. Spike in your RNA­Seq is then the best way to solve theproblem.

Antibody­guided imaging of invasive fungal infections

C. ThorntonI

IUniversity of Exeter, Exeter, United Kingdom

Invasive pulmonary aspergillosis (IPA) is a life­threatening lung disease of haematological malignancy and bone marrow transplant patients caused by the ubiquitousenvironmental fungus Aspergillus. Current diagnostic tests for the disease lack sensitivity or specificity, and culture of the fungus from invasive lung biopsy, consideredthe gold standard for IPA detection, is slow and often not possible in critically ill patients. Despite the limited specificity of standard Computed Tomography (CT) fordiagnosing IPA, or invasive fungal diseases in general, CT remains a trigger for commencing antifungal treatment in many haematology units. However, radiologicalabnormalities are not pathognomonic since other mould pathogens and bacterial pathogens capable of angio­invasion can produce similar appearances in a chest CT. Previously, we reported the development of a novel non­invasive procedure for IPA diagnosis based on antibody­guided positron emission tomography and magneticresonance imaging (immunoPET/MRI) using a [64Cu]­labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. The mAb binds to a diagnosticmannoprotein antigen which is secreted during active growth of the pathogen only, and so is able to discriminate between inactive spores present in inhaled air andinvasive hyphae that infect the lung of the neutropenic host. The mAb forms the basis of the Aspergillus Lateral­Flow Assay (LFA) which is CE­marked for IPAdiagnosis in humans using serum and bronchoalveolar lavage fluids.To enable translation of the tracer to the clinical setting, we have developed a humanised version of the antibody (hJF5), generated by grafting of the mJF5 CDRs into ahuman IgG1 framework. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to itsradiolabeled murine counterpart. Using reverse genetics of the pathogen, we have shown that the antibody binds to the antigenic determinant b1,5­galactofuranose(Galf) present in the diagnostic mannoprotein antigen. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities ofthe hJF5 antibody, means that the humanised tracer represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting, entering a first­in­humanclinical trial in Oct 2018.

A non­invasive imaging toolbox to monitor fungal infection progression,dissemination and host response in small animal models

G. Vande VeldeI

IKU Leuven – University of Leuven, Leuven, Belgium

To investigate fungal infection, their progression and dissemination, host response thereto and novel treatment options, investigators typically rely on end­stageprocedures on isolated tissues from animal models or survival curves that only provide snapshot data of processes that are essentially dynamic in time and space.Therefore, non­invasive imaging technology that allows to repeatedly evaluate the infection process is indispensable to grasp the dynamics of disease progression andtreatment. We develop ground­breaking integrated multimodal imaging approaches involving intravital fibered confocal fluorescence microscopy, magnetic resonanceimaging, computed tomography, whole­body in vivo and non­destructive ex vivo optical imaging. These novel technologies enable us to acquire novel observations andinsights in disease progression, dissemination, host response and therapy effects in live animal models of fungal infections. These techniques have immediateconsequences towards the reduction of animal numbers and suffering typically related to the research in the fungal infections field. Ultimately, through improving basicand translational fungal infections research, this technology will ultimately benefit patient care.

Regulation of phagosomal size and integrity during the control of fungalinfection

S. GrinsteinI

IThe Hospital for Sick Children, Toronto, Canada

Sergio Grinstein1,21Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada2Department of Biochemistry, University of Toronto, Toronto, Canada Professional phagocytes, such as macrophages, engulf invading fungal pathogens to prevent infection. However, in certain instances the engulfed microorganism survivesinside the phagosome and can even grow therein. In such cases, the phagosome needs to expand in order to contain the viable microbe inside the microbiostaticenvironment. Failure to expand as the microorganism grows and/or replicates would eventually result in rupture of the phagosomal membrane and leakage ofphagosomal contents, potentially leading to pathogen escape and macrophage cell death.However, it is unclear whether phagosomes can in fact sense and activelyregulate their size to accommodate enlarging or dividing pathogens. To address this issue, we used C. albicans, which is readily engulfed by macrophages and convertsfrom yeast to hyphal form within the phagosome, where it grows at a rate of several microns per hour. Strikingly, 4 hours after ingestion of a single C. albicans yeast,the expanded phagosome containing the growing hypha occupies ≈50% of the total macrophage volume. Remarkably, the macrophage senses the need to expand thephagosome, which grows pari passuwith the hypha. To this end, lysosomes migrate towards the phagosome, which is often located near the microtubule­organizingcenter (MTOC) and undergo fusion with the phagosomal membrane. After 4 hours, ≥80% of the cellular lysosomes have fused with the phagosomal membrane,contributing to its expansion. Translocation of lysosomes towards the phagosome is dependent on polymerized microtubules and requires dynein, a centripetal motorprotein. Depolymerization of microtubules or inhibition of dynein with ciliobrevin prevented lysosomal displacement and fusion with the phagosome. These manipulationsaccelerated phagosomal rupture, followed by leakage of phagosomal contents and eventual lysis of the macrophage.The activation of lysosomal biogenesis inmacrophages infected by C. albicans, and the possible role of calcium in these processes will be discussed.

Gut colonization by Candida albicans

C. PerezI

IUniversity Wuerzburg, Wuerzburg, Germany

The fungus Candida albicans resides in the gastrointestinal tract of most, if not all, human adults, seemingly living there as a harmless commensal. My laboratory isemploying gnotobiotic mouse models of gut colonization to investigate the interplay between C. albicans and other members of the microbiota as well as the mammalianhost. Through the use of FISH probes and immunohistochemistry, we have visualized the interface between the fungus and the intestinal mucosa in mono­ and co­colonized animals. While the fungus reaches comparable loads (in colony forming units per gram of stool) in both environments, our observations demonstrate that thelocation, distribution and morphology of the fungus are strongly influenced by the presence/absence of other microbes. Furthermore, we have identified several C.albicans transcription regulators that play roles in murine intestinal colonization. Through RNA­Seq and ChIP­Seq experiments, we are establishing the targets ofregulation of these proteins and the biological processes that they govern. In my presentation, I will describe in detail some of the gene circuits that we have discoveredand how they contribute to the interplay with the host.

Fungi in the oral cavity; directors of the bacteriome?

B. KromI

IDepartment of Preventive Dentistry ­ ACTA, Amsterdam, Netherlands

Fungi have long been known to colonize the oral cavity, with most research emphasis being placed on studying their role in development of oral diseases. Recently itbecame apparent that fungi play a potential role in maintaining oral health. Oral health is maintained by a balance between the total microbiota and the host. Dysbiosiscan be caused by presence of exogenous species or by increase colonisation of commensal species. Fungi can modulate the environment and interactions with the hostresulting in a balanced oral ecosystem and the mechanisms involved are slowly being unravelled.

Disruption of host metabolic homeostasis regulates Candida­macrophageinteractions

T. TuceyI, J. VermaI, T. LoI, A. TravenI

IDepartment of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia

Macrophages are an important component of innate immune responses to Candida albicans. C. albicans can evade macrophages by a number of mechanisms, and itcan trigger macrophage cell death which is, at least in part, related to inflammatory host responses. We have recently demonstrated that a major mechanism by which C.albicans triggers cell death of macrophages is by starving them of glucose (Tucey et al Cell Metabolism 2018). Here we asked how host glucose homeostasis regulatesmacrophage inflammatory responses to C. albicans. To answer this question, we used massively parallel, quantitative live cell imaging with high spatio­temporalresolution. We monitored inflammasome activation using macrophages in which the ASC inflammasome adaptor protein is fluorescently labelled, and correlated it tomacrophage cell death and glucose homeostasis. We combined this powerful imaging platform with a compendium of clinical isolates of C. albicans of varying hyphalmorphogenesis phenotypes, and genetic or chemical perturbations of pathogen or host pathways. With this approach, we show for the first time that perturbation of hostglucose homeostasis due to C. albicans infection is a major trigger of inflammasome activation. We delineate the requirements of this new mechanism, and show that thepreviously characterised hyphae­dependent inflammasome activation by C. albicans operates only under a certain set of experimental conditions. Our data suggeststhat, by linking inflammasome activation to host glucose homeostasis, macrophages can sense both the morpho­type of C. albicans (hyphae versus pseudohyphaeversus yeast) and fungal load, to control the extent and the timing of inflammasome activation. These mechanisms could help the host to carefully control inflammatoryresponses, and suggest that metabolic interventions could modulate inflammation in C. albicans infections.

C­type Lectin­like Receptors and Systemic Fungal Infections

S. OrrI

ICardiff University, Cardiff, United Kingdom

Fungal infections including invasive candidiasis and aspergillosis kill more people per year (~1.5M) than TB or malaria. Invasive fungal infections are particularly aproblem for immunosuppressed patients and associated mortality rates are unacceptably high (30­95%) in these patients. Fungal pathogens engage various pathogenrecognition receptors (PRRs) including C­type lectin­like receptors (CLRs) such as Dectin­1, Dectin­2 and Mincle. Using novel multi­CLR KO mice generated in ourlab, we have uncovered a vital role for CLR collaboration to mediate anti­fungal immune responses including recognition, phagocytosis, respiratory burst and cytokineproduction. Collaboration between these CLRs varies between different innate immune cells however, it is required to mediate the protective effect of inflammatorymonocytes during systemic infection with C. albicans.

Fungi in blood ­ how fungal pathogens interact with circulating immunecells

O. KurzaiI, A. HäderII, V. SchüllerIII, K. HünnigerII, J. GehlenIV, B. BürfentIV, S. SchäubleV, T. WolfII, J. SchumacherIV

IUniversity of Würzburg, Institute for Hygiene and Microbiology, Würzburg, Germany, IIHans­Knöll­Institute, Jena, Germany, IIIInstitut für Medizinische Biometrie,Informatik und Epidemiologie, Bonn, Germany, IVZentrum für Humangenetik, Marburg, Germany, VHans­Knöll­Institut, Jena, Germany

Human blood is a central niche for some pathogens that disseminate via hematogenous spread during systemic infection. This group of pathogens includes fungi likeCandida spp. and Cryptococcus spp. Other pathogens are rarely found in blood but will be confronted with circulating immune cells when those extravasate andmigrate to foci of infection. This group includes fungal pathogens like Aspergillus spp. or Mucorales. Using a human whole blood model of infection we analyze host­pathogen interactions in a situation close to the in vivo relevant host niche "blood". Our assay provides time­resolved data on cell activation, localization andphysiological state of the pathogen and immune activation using flow­cytometric, transcriptomic or proteomic analysis tools and require minimal pre­analytical handlingof the cells. In addition, biomathematical modeling allows quantification of parameters that remain inaccessible to direct experimental quantification. Based on theseanalyses, we show that (i) immune activation in human blood differs considerably from activation patterns found in isolated immune cells and (ii) the host response inblood differs between fungal and bacterial pathogens. As early transcriptional responses in human blood are largely determined by monocytes we have additionallystudied individual variation in monocyte activation in mor detail. By parallel assessement of transcriptional patterns and genetic polymorphisms expression quantitativetrait loci (eQTLs) that govern transcriptional responses to infection in human monocytes could be identified.

TBA

IL­17 in antifungal immunity: beyond Candida

S. LeibundGut­LandmannI

IUniversity of Zurich, Zurich, Switzerland

The skin is home to millions of microbes that play an essential role in immune homeostasis, but in certain conditions can also mediate inflammation and disease. Whetherfungal communities that constitute an integral part of the skin microbiome exert similar functions as their bacterial counterparts is poorly understood. By far the mostcommon fungi on the mammalian skin are those of the genus Malassezia. Aside from their commensal lifestyle, Malassezia spp. are associated with commoninflammatory skin disorders such as atopic dermatitis. However, the causative relationship between fungal commensalism and disease manifestation remains poorlyunderstood. Using a novel epicutaneous infection model in mice, we found Malassezia spp. to selectively induce IL­17 and related cytokines and that this response waskey for preventing fungal overgrowth on the skin. Under conditions of impaired skin integrity mimicking the situation in the atopic skin, Malassezia spp. dramaticallyaggravated cutaneous inflammation in an IL­23­ and IL­17­dependent manner emphasizing an active contribution of the fungus to skin pathology. Consistent with themurine data, we found Malassezia­responsive memory T cells in healthy individuals in the CCR6+ Th17 subset and their frequency was increased in atopic dermatitispatients. Thus, the Malassezia­induced IL­23/IL­17 immune axis is pivotal in orchestrating antifungal immunity and in actively promoting skin inflammation.

Sugars and surfaces – what’s important to Cryptococcus?

T. DoeringI

IWashington University School of Medicine, SAINT LOUIS, United States of America

As this group of researchers knows, Cryptococcus neoformans is an opportunistic pathogen that causes serious infections in hundreds of thousands of people eachyear, primarily in developing areas of the world. Cryptococcosis is contracted by inhalation of the infectious particle, which grows in the lungs; depending on the hostimmune status the infection may be cleared, remain latent in the lungs, or disseminate. Dissemination to the central nervous system results in a potentially lethalmeningoencephalitis, with mortality rates in some areas close to 70%.

Like other yeast species, C. neoformans produces a predominantly carbohydrate cell wall and also glycosylates proteins and lipids, although its repertoire of sugarmoieties is notably broader than that of the model yeast Saccharomyces cerevisiae. In addition, C. neoformans bears a large polysaccharide capsule surrounding itscell wall; capsule polysaccharides are also shed copiously into the environment. The capsule, a structure that is unique among fungal pathogens, is required for virulence.Capsule thickness (and probably capsule structure as well) is extremely sensitive to environmental conditions.

This presentation will discuss aspects of capsule synthesis and capsule regulation. For example, we have recently described several nucleotide sugar transporters thatmove activated precursor molecules into the secretory pathway, where they serve as the donors for capsule synthetic reactions. The absence of these transporterssignificantly perturbs cryptococcal pathogenesis and also alters the host response to this pathogen. We are also working to define capsule regulation at the transcriptionallevel and to relate the transcriptional network to the underlying biology of capsule synthesis, particularly the production and transport of nucleotide sugars.

C. neoformans replicates both extracellularly and within host phagocytes. The interactions between yeast and host cells, such as those leading to fungal internalization,largely determine the course of infection. These processes are highly influenced by the surface characteristics of the yeast. This presentation will also describe image­based approaches that we are using to identify key fungal processes that influence cryptococcal surfaces and therefore fungal:host cell interactions.

Interactions between the emerging fungal pathogen Candida auris andthe host

H. YueI, G. HuangII

IState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, Beijing, China, IIInstitute of Microbiology,Chinese Academy of Sciences, Beijing, China

The emerging human fungal pathogen Candida auris has been recognized as a multidrug resistant species and is associated with high mortality. Recently, we report thefirst isolate of C. auris from the bronchoalveolar lavage fluid (BALF) of a hospitalized woman in China. Under different culture conditions, this strain exhibits multiplemorphological phenotypes including round­to­ovoid, elongated, and pseudohyphal­like forms, but is much less virulent than Candida albicans in both mouse systemicand invertebrate Galleria mellonella models. Morphological plasticity has historically been an indicator of increased virulence among fungal pathogens allowing rapidadaptation to changing environments. We further found that C. auris is able to undergo filamentation and has three distinct cell types: typical yeast, filamentation­competent (FC) yeast, and filamentous cells. These cell types form a novel phenotypic switching system that contains a heritable (typical yeast­filament) and a non­heritable (FC­filament) switch. Intriguingly, the heritable switch between the typical yeast and the FC/filamentous phenotype is triggered by passage through amammalian body, whereas the switch between the FC and filamentous phenotype is non­heritable and temperature­dependent. Low temperatures favor the filamentousphenotype, whereas high temperatures promote the FC yeast form. Systemic in vivo and in vitro investigations were used to characterize phenotype specific variationsin global gene expression, secreted aspartyl proteinase (SAP), and changes to virulence; indicating potential for niche specific adaptations. Taken together, our study notonly sheds light on the pathogenesis and biology of C. auris but also provides a novel example of morphological and epigenetic switch.

Surface molecules of Aspergillus fumigatus decisive for immune evasion

A. BrakhageI,II

ILeibniz Institute for Natural Product Research and Infection Biology ­ Hans Knoell Institute, Jena, Germany, IIDepartment of Microbiology and Molecular Biology,Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany

The filamentous fungus Aspergillus fumigatus is the most important air­borne fungal pathogen causing 90 % of all systemic Aspergillus infections in humans. The smallconidia of A. fumigatus with a diameter between 2­3 µm easily reach the lung alveoli where they form the first contact with the host. The contact zone between conidiaand host consists of many surface molecules of different types. By transcriptome analyses, proteome analyses, HPLC and mass spectrometry we discovered severalproteins like CcpA and natural products, in particular the conidial pigment consisting of dihydroxynaphthalene (DHN) melanin, as important virulence factors becausethese molecules are able to modulate the host response and allow for immune evasion of the fungus. A. fumigatus is able to interfere with important defense processeslike phagocytosis, formation of mature phagolysosomes and LC3­associated phagocytosis. In my talk, I will focus on recent findings of my laboratory concerning theinteraction of surface molecules with lipid­raft microdomains of phagolysosomes whose formation is essential for immunity against A. fumigatus. In addition, I willdiscuss the discovery of antifungal extracellular vesicles produced by neutrophils for defense against A. fumigatus.

Fungal stress signalling pathways as mediators of virulence ­ and how wecan exploit this.

J. QuinnI

IInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom

The ability to adapt to diverse and often hostile environments within the host is essential for the virulence of a broad range of pathogenic fungi, including the human fungalpathogen Candida albicans. We are interested in the stress­sensing and signalling mechanisms that C. albicans evokes to counter a range of host­imposed stresses.Central to stress adaptation and virulence in multiple pathogenic fungi is the Hog1 stress activated protein kinase (SAPK). However, such SAPKs are evolutionarilyconserved and there is a high level of structural and functional conservation between the fungal and analogous human (p38/JNK) SAPKs. This makes the developmentof antifungal drugs that will specifically target fungal, and not human, SAPKs challenging. Because of this, there is much interest in identifying fungal­specific regulators ofSAPKs as potential drug targets. Such targets include two­component related phosphorelay systems which are used by fungi, but not mammals, to sense and relaystress signals to SAPK modules. Here I will present recent findings from my group which have dissected the role of key phosphorelay proteins in regulating Hog1, andhow we are exploiting this knowledge to screen for compounds that inhibit activation of this key kinase. In addition I shall present our recent work revealingunanticipated roles for phosphate acquisition in promoting stress­resistance and virulence in C. albicans, and the work currently ongoing to identify compounds thatprevent acquisition of this essential macronutrient. Our goal is to exploit our mechanistic dissection of fungal stress­adaptation pathways to identify potential novelantifungals that prevent activation of key processes essential for virulence.

Exploring the host­pathogen arms race in cryptococcosis

R. MayI

IUniversity of Birmingham, birmingham, United Kingdom

My group are interested in host­pathogen interactions and, in particular, in understanding how some pathogens are able to subvert the innate immune system. Most ofour work focuses on phagocytic cells, which some microorganisms are able to use as a ‘safe house’ within which to replicate. We try and understand how suchpathogens can survive inside this hostile environment and the effect this intracellular reservoir has on disease progression.In this talk, I will discuss our recent work reveal how inflammatory signals and coinfections change the way in which host phagocytes deal with Cryptococci, and howwe might ultimately be able to manipulate this process to help treat this disease. From the “pathogen side” I will talk about our recent work on ‘long­range virulencecontrol’ in Cryptococci and how secreted products from the fungus may have profound effects on systemic immunity in infected people.

Success and challenges of glucan synthase inhibitors

D. PerlinI,II

IRutgers University, Newark, United States of America, IICenter for Discovery and Innovation, Nutley, New Jersey, United States of America

Drugs that target the cell wall as a primary cell vulnerability have been coveted since the origin of antifungal therapy. In 2001, the echinocandin caspofungin, whichtargets glucan synthase, was approved as a first in­class inhibitor of cell wall biosynthesis to treat invasive fungal infections. Subsequently, micafungin and anidulafunginwere also approved. The echinocandin class antifungals are highly active against Candida yeasts and show differential activity with many moulds. Due to theireffectiveness and safety, they are recommended first­line agents to treat invasive candidiasis. Newer agents in clinical development include Rezafungin, an echinocandinwith extended PKs and Ibrexafungerp (formerly SCY­078), which is the first representative of a novel class of structurally­distinct glucan synthase inhibitors,triterpenoids. These agents impose significant stress on the cell inducing well­defined molecular cellular responses, including genetic, epigenetic and post­translationalevents, that alter cell wall integrity pathways. Cellular­induced modulation of glucan synthase is known to influence the development of drug tolerance and drugresistance with clinical impact.

Novel Strategies to the Treatment of Fungal Infections with Emphasis onAnti­virulence Approaches

J. Lopez­RibotI

IUTSA, San Antonio, United States of America

Fungal infections are an increasing threat to a growing number of immune­ and medically­compromised patients. Candidiasis remains the most common fungal infection.The current arsenal of antifungal drugs is very limited and emergency of resistance is a problem. Clearly, new strategies and therapeutics are urgently needed. Our grouphas taken a multipronged approach to tackle this problem. First, we are developing anti­virulence strategies by performing large­scale screening of over 50,000 smallmolecule compounds to identify inhibitors of C. albicans filamentation and biofilm formation, the two most important virulence factors during candidiasis. Second, unlikethe tortuous and costly path of de novo drug discovery, drug repurposing is gaining traction as an alternative path to accelerated drug development. Thus, we have alsoembarked in a “repurposing” campaign, in order to identify already existing drugs with activity against fungal biofilms. Third, we have determined the activity of differentnanoparticle preparations against C. albicans biofilms, as well as against mixed fungal/bacterial biofilms that are particularly difficult to treat with conventionalantimicrobial therapy. Lastly, our group has developed a novel technique consisting of nano­scale culture of microbial biofilms on a microarray platform. Using thistechnique, hundreds to thousands of microbial biofilms, each with a volume of approximately 30­50 nanolitres, can be simultaneously formed on a standard microscopeslide, which allows for the implementation of true throughput screening techniques that can speed up discovery of novel antifungals. Development of these novelantifungal approaches should have a profound impact on the management of patients suffering from these devastating infections.

Cryptococcal meningitis­ trials and tribulations

T. BicanicI

ISt George's University of London, London, United Kingdom

This talk will cover recent developments in the antifungal treatment of HIV­associated cryptococcal meningitis. Topics covered will include the background and rationalefor combination antifungal therapy for cryptococcal meningitis, recently completed and ongoing phase II and III clinical trials, novel and repurposed anticryptococcalagents in the drug development pipeline, and what makes for an attractive anti­cryptococcal drug.

Development of a Candida Vaccine: Next Steps, Scientific andDevelopment Challenges, and Implications for Other Fungi and BacteriaJ. Edwards *

Presently, we are not aware of any known human fungal vaccines approved by the Federal Drug Administration (FDA), or other known regulatory agencies. Onlytwo double­ blind, placebo­controlled trials of fungal vaccines have been conducted in humans, to date. Of these two trials, only one has shown a signal of efficacy. The trial was an exploratory 1b/2a trial demonstrating efficacy in a subset analysis of women with recurrent Candida vulvovaginitis under the age of 40 years. Theantigen used in this trial was the N­terminus of the cell surface protein encoded by the gene Als3 of Candida albicans. Alum was formulated as the adjuvant. The N­terminus of Als3p has extremely high 3­D (3­Dimensional) homology with cell surface proteins on Staphylococcus aureus, which are known adhesins. Because of thishomology, a trial in military recruits has been conducted to determine if the vaccine will reduce colonization of recruits by S. aureus (including MRSA). Enrollment ofthis trial has recently been completed. Of additional interest is another cell surface protein on C. albicans, Hyr1p, which shares 3­D structural homology to MRSA SraP, an adhesin to platelets. Hyr1palso shares striking structural homology with a hemagglutinin/hemolysin of MDR Acinetobacter baumannii (AB) and carbapenemase­producing Klebsiellapneumoniae (CPKP). Active and passive vaccination with Hyr1p protects mice from a lethal challenge of S. aureus, MDR Acinetobacter baumannii, andKlebsiella pneumoniae. Of still further interest is that three orthologs of Als3p­N exist on C. auris. Sera from mice that have been vaccinated with the Als3p­N vaccine recognize theseorthologs on C. auris and vaccination of mice with the Als3p­N vaccine significantly improves survival from a lethal challenge of C. auris. When the vaccine is used inconjunction with the antifungal mycafungin, significant improved survival is seen, suggesting an increase in the efficacy of the antifungal drug. The findings of 3­D homology in the N­terminus of surface proteins being common across several species including resistant Staphylococcus, Acinetobacter, andKlebsiella species, and the finding of significantly improved survival in mice challenged with lethal inocula of these drug resistant fungi and bacteria, opens the door forfurther exploration of fungal vaccines with 1. multiple antigens, 2. newer adjuvants, 3. and now passive immunization with polyclonal/monoclonal antibodies.

* The authors marked with an asterisk equally contributed to the work.

Posters

Multi­transcriptomic studies of Cryptococcus reveal the broadevolutionary utility of start codon context in the fungal kingdom

E. WallaceI, C. MaufraisII, F. MoyrandII, J. Sales­LeeIII, P. NagarajanIV, H. MadhaniV, G. JanbonII

IUniversity of Edinburgh, Edinburgh, United Kingdom, IIInstitut Pasteur, Paris, France, IIIUniversity of California, San Francisco, San Francisco, United States ofAmerica, IVUniversity of California San Francisco, San Francisco, United Kingdom, VUniversity of California San Francisco, San Francisco, United States of America

Fungi have evolved gene expression programs that dynamically respond to their environment and that are crucial for fungal pathogenesis. Genomic sequence determinesboth protein sequence and its regulation, and the 5’ region of messenger RNA encodes choice and efficiency of start codon usage. Here, we combine new genome­wide datasets in the major fungal pathogen Cryptococcus with re­analysis of data from diverse fungi, to show that start codon sequence context has a strong effect onprotein production and localization across the fungal kingdom.In both C. neoformans H99 and C. deneoformans JEC21, we measured mRNA 5’ ends (transcription start sites), 3’ ends (polyadenylation sites), isoform­levelabundance, and translation (ribosome profiling). These data show that upstream open reading frames repress translation, particularly when they start with a goodcontext (Kozak sequence) AUG. Start codon context is highly constrained in highly translated genes including ribosomal proteins. Genes with a poor­context annotatedAUG and a good­context downstream AUG are enriched in predicted N­terminal mitochondrial targeting signals: a small proportion of initiating ribosomes produce thelonger mitochondrial form, while most ribosomes wait to initiate at a downstream start codon and produce the cytoplasmic form. In Cryptococcus, 11 of the 20 tRNAsynthetase activities are each encoded by a single genomic locus with this dual­targeting structure. We predict that hundreds of such dual­targeted proteins areconserved across fungi and are essential for mitochondrial function, which is in turn essential for pathogenesis.In S. cerevisiae, by contrast, start codon context has a weak effect genome­wide, 5' UTRs are relatively short and AUG­depleted, and dual­targeted proteins tend toinitiate at a non­AUG codon instead of a poor­context AUG. These features vary in the few other fungal species with mapped 5’ UTRs and/or ribosome profiling dataavilable. Eukaryotic translation initiation factor 1, which enforces the accuracy of start codon selection, has sequence divergences in fungi at locations known to affectstart codon selection in Saccharomyces, and the Cryptococcus homolog additionally has two unstructured insertions. Our results highlight start codon context as amajor architectural feature of fungal genomes, a locus of biologically relevant regulation, and a factor to be considered in genome annotation.

At the cross­section of PKA activity and iron metabolism ­ riboflavinsynthesis as a potential antifungal drug target.

L. DemuyserI,II, I. PalmansI,II, P. VandecruysI,II, P. Van DijckI,II

IVIB­KU Leuven Center for Microbiology, Leuven, Belgium, IILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven,Belgium

Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare, are substantial.Riboflavin is a yellow pigment, also termed vitamin B2. Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate thattargeting riboflavin production is a promising novel strategy against fungal infections.

In this project, we first characterized the riboflavin produced by the laboratory C. albicans strain, SC5314, based on its unique absorbance and fluorescence spectra.We confirm that the iron level in the growth medium inversely correlates to the production of riboflavin by this fungus. Protein kinase A (PKA) activity regulates thisprocess as well, although the exact mechanism remains undetermined. Based on gene expression analysis, we consider CaRib1 as the central regulatory hub forriboflavin synthesis. Deletion of this gene causes C. albicans to become completely auxotrophic for the vitamin. By heterologous expression of the, until thenuncharacterized, CaRIB1 gene in a S. cerevisiae rib1∆ strain, we confirm the role of this enzyme in riboflavin synthesis in the pathogenic fungus. As overexpression ofthe CaRIB1 gene, in contrast to overexpression of any other CaRIB gene, increases production of the pigment, we hypothesize that any upstream regulation wouldinvolve CaRib1. Based on literature, the transcription factor Sef1 is involved in regulation ofRIB gene expression. We confirm that a Casef1∆/∆ strain is hampered inPKA­dependent regulation of riboflavin synthesis and completely fails to overproduce this pigment under iron­limiting conditions. The involvement of PKA in thisprocess is further supported by data showing that a mutant strain altered in the putative PKA­phosphorylation sites of the CaSEF1 gene shows similar phenotypes inthese assays.

In conclusion, we show that CaRib1 is essential for growth of C. albicans in low­riboflavin containing medium, as well as it is the likely hub of upstream regulationthrough CaSef1. Apart from iron metabolism, we show that PKA activity plays a role in this process.

Fungal Plasma Membrane Homeostasis is Required for the Response toAlkaline pH

H. BrownI, J.A. AlspaughI

IDuke University, Durham, United States of America

A key virulence attribute of any microbial pathogen is the ability to adapt to the conditions of the infected host. One of these host­associated conditions is neutral/alkalineextracellular pH. Fungal pathogens use the Rim/Pal signal transduction pathway to sense and respond to host pH. Mutants in this pathway are attenuated for survival atalkaline pH and in the infected host. The human fungal pathogen, Cryptococcus neoformans is an important cause of opportunistic infections in immunocompromisedpatients. This pathogen uses the Rim pathway to regulate cellular changes necessary for growth at alkaline pH. This pathway was first described in the ascomycetephylum. Interestingly, C. neoformans, a basidiomycete, lacks genes encoding many of the components of the Rim pathway, including much of the membrane pH­sensing complex. Additionally, the mechanism by which C. neoformans senses an increase in extracellular pH and how this mechanism might involve the plasmamembrane is unknown. This work aims to identify proteins in the C. neoformans Rim pathway that sense changes in pH and investigate how C. neoformans andrelated basidiomycetes use the organization and composition of the plasma membrane to sense and respond to an elevated extracellular pH. I have completed aninsertional mutagenesis screen to identify novel genes required for growth at host pH. These mutants were prioritized based on a sensitivity to alkaline pH that is rescuedby active Rim101, suggesting that they are upstream components of the Rim pathway. From this screen I identified a lipid flippase regulatory subunit, Cdc50, as having arole in the initial propagation of the alkaline pH signal through the Rim pathway. I will continue to test if the C. neoformans Cdc50 flippase, and maintenance of plasmamembrane asymmetry, are required for Rim pathway activation. I have also completed deep RNA­sequencing analysis to define the transcriptome of the rim101∆ strainto identify targets of the Rim101 transcription factor, the most downstream component of the Rim pathway. The results from this screen reveal a connection between thecomposition of the plasma membrane and the ability for C. neoformans to respond to external pH changes. Together, these experiments will elucidate the molecularinteractions that drive environment­sensing in a large and biologically diverse group of fungi.

Effect of temperature stress on transposon­mediated mutagenesis anddrug resistance in Cryptococcus neoformans in a host model of infection

A. GusaI, A. AveretteI, S. SunI, J. HeitmanI, S. Jinks­RobertsonI

IDuke University, Durham, United States of America

Cryptococcus neoformans is a human fungal pathogen causing infections primarily in immunocompromised individuals, such as those with HIV/AIDS or cancerpatients undergoing chemotherapy. Dissemination of Cryptococcus to the brain causes meningoencephalitis, a condition with a high mortality rate and limited treatmentsuccess due to drug resistance. Previous studies have shown temperature­induced mutagenesis and increased survival of C. neoformans serotype D (XL280α) in thepresence of antifungal drugs in culture. Compared to growth at 30˚C, there was a 25­fold increase in mutagenesis and drug resistance when Cryptococcus was grownat 37˚C (human host temperature), largely due to insertions by transposable elements identified as the DNA transposon T1 and retrotransposon Tcn12. Using a murinemodel of infection, this study provides the first evidence that, in vivo, transposon­mediated mutagenesis gives rise to XL280α isolates that are resistant to the drug 5­fluoroorotic acid (5­FOA). Cryptococcus mutants resistant to 5­FOA (insertions in the URA3 or URA5 genes) were recovered from the organs of mice infectedintravenously at 3 and 7 days post­infection. The number of resistant isolates was compared to the total number of colonies recovered from the lung, brain, spleen andkidney tissue to determine the mutation frequency. Significantly, nearly half of the loss of function mutations in the URA3 and URA5 genes were due to transposoninsertions. Both the T1 and Tcn12 transposable elements were present at insertion sites, supporting previous results that showed these elements to be highly mobile at37˚C in vitro. These results suggest that transposon­mediated mutagenesis at elevated temperatures may provide a selective advantage for the survival ofCryptococcus in the presence of antifungal drugs or other hostile host conditions. To explore the effect of temperature on host survival in a cryptococcal infection,Galleria mellonella larvae were infected with XL280α then incubated at 30˚C or 37˚C. Killing of the larvae was significantly more rapid at 37˚C, with the majority oflarvae dying between Day 6 and 10 post­infection. Future experiments are planned to determine if there is a difference in the frequency of transposition in vivo at 30˚Cand 37˚C and whether transposition is important for the survival and pathogenesis of Cryptococcus in the host.

Cell wall changes associated with echinocandin resistance across Candidaspecies

G. Buda De CesareI, D. SteadI, C. MunroI

IUniversity of Aberdeen, Aberdeen, United Kingdom

One of the problems related to the treatment of fungal infections is the development of drug resistance. Generally, these infections are associated with high mortality sothe choice of antifungal drug, based on clinical trial’s evidence and the identification of the fungal species, is crucial. Variation in the response to antifungals occurs acrossthe Candida genus. At the species level, intrinsic resistance confers ability to tolerate the stress caused by drug exposure; at population level, acquired mutations enableto reduce (or eliminate) the effects of the drug. Echinocandins, in particular caspofungin, are the first­line treatment for candidaemia in clinical settings and targetspecifically fungal cell wall β (1,3)­glucan. Tolerance and resistance mechanisms to this class of antifungals has been described in C. albicans, C. glabrata and C.tropicalis. However, the cross­species similarities of such mechanisms do not reflect the differences in the minimum inhibitory concentration and therapy outcomesbetween species and isolates.In this work, the cell walls of C. albicans, C. glabrata and C. tropicalis clinical isolates were characterised and compared to the respective reference strains, in orderto identify the different responses to caspofungin. Fungal cultures were grown in the presence or absence of drugs and MIC values measured to quantify the drug effectson the growth of the selected strains. Mass spectrometry analyses were performed to detect changes in protein expression between drug­resistant and ­susceptibleisolates. The strains were characterised by high pressure liquid chromatography, flow cytometry and electron microscopy to look at differences in cell wall componentsand architecture. Differences were detected in the cell walls from resistant and sensitive strains, such as the expression of proteins related to cell wall biosynthesis andmaintenance. Common trends in the exposure of different cell wall components were observed amongst echinocandin­resistant and ­susceptible isolates, as well asdifferent adaptations of the wall in response to caspofungin treatment.This study shows the complexity of the antifungal response, the understanding of which is required to identify new drug targets and biomarkers for antifungal resistance.

The Pathogen Box: Using Drug Repurposing to Identify Inhibitors ofCandida auris

G. WallI, N. HerreraI, J.L. Lopez­RibotI

IUniversity of Texas at San Antonio, San Antonio, United States of America

Candida auris, an opportunistic fungal pathogen, made its first identified appearance in humans in 2009 in an ear infection in Japan, and since then it has spreadthroughout the world. In contrast to antibacterial antibiotics, there are currently only three main classes of FDA­approved antifungals. C. auris is known to be resistantto fluconazole (a member of the azole class) in most cases, and some isolates display multidrug resistance against all classes of currently available antifungals. Whenconsidering the resistance seen in not only C. auris but also other fungal species, it is clear that new treatment options are urgently needed. Besides its drug resistanceproperties, C. auris is also extremely difficult to eliminate, as it can live on surfaces for up to 4 weeks and has caused outbreaks in several ICUs around the world.Given these problems, there is great concern that this yeast could become a common source of nosocomial infections. In this project, we chose to use a drugrepurposing approach for the identification of novel antifungals against C. auris, as this process can be much shorter and less costly than de novo drug development. Tothat end, we decided to screen the Pathogen Box repurposing library from Medicines for Malaria Ventures to identify compounds with activity against C. auris, forwhich we used the 96­well plate method of biofilm formation designed in our lab and CLSI methodologies for planktonic growth. These screenings identified severaldrugs with >50% inhibitory activity with no previous clinical use as antifungals. We further evaluated the inhibitory activity of the identified hits in dose response assays,and we chose to focus on two of them: Miltefosine and Iodoquinol. Miltefosine was found to possess potent activity against C. auris biofilms, both biofilm formationand pre­formed biofilms, as well as against planktonic cells. Iodoquinol was only effective against C. auris under planktonic growing conditions. We are currentlyexpanding our studies on Miltefosine and Iodoquinol and testing their activity against multiple C. auris isolates and other Candida species. Our results for thesecompounds so far indicate that they represent potential repositionable candidates for the treatment of C. auris infections.

Comparison of stress resistance in Candida auris with other pathogenicCandida species

H. HeaneyI, L. PatersonII, J. LaingII, D.M. MacCallumI, A.W. WalkerI, E. JohnsonIII, A.J.P. BrownI

IUniversity of Aberdeen, Aberdeen, United Kingdom, IINHS Grampian, Aberdeen, United Kingdom, IIIMycology Reference Laboratory, Bristol, United Kingdom

Candida auris has rapidly emerged in the last decade as a clinically significant human pathogen. C. auris infections can be associated with high mortality rates (~66%),multi­drug resistance and a propensity for transmission in healthcare environments. We tested whether enhanced resistance to environmental stresses contributes to theability of C. auris to spread in healthcare environments. We compared the C. auris resistance to single stresses and combinations of stresses with other pathogenicCandida species. Stress resistance was examined using in vitro assays on solid media and also on hospital linen. Results suggest that some C. auris strains arerelatively thermotolerant, some displaying growth at 42 ºC. All of the C. auris strains examined in this study were salt tolerant, and resistant to pHs between 4 and 13,but sensitive to pH 2 and to certain types of oxidative stress. When contaminating hospital linen, C. auris displayed resistance to desiccation, but sensitivity tocombinations of low pH, elevated temperature and salt. The data offer potential strategies to limit the spread of C. auris via contamination of hospital linen.

A role for vesicular transport in fungal susceptibility to fluconazole

L. Demuyser *I,II, K. Van Dyck *I,II, B. TimmermansI,II, P. Van DijckI,II

IVIB­KU Leuven Center for Microbiology, Leuven, Belgium, IILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven,Belgium

There are over 600 different fungal pathogens able to cause either minor or invasive infections in humans. The majority of human fungal infections are caused by speciesof the Candida genus including Candida albicans and Candida glabrata. In most healthy individuals, they are both harmless commensals of the oral cavity,gastrointestinal tract and/or urogenital tract. However, in conditions involving a reduced immune competence or an imbalance of the microflora, they are able to causeinfections. The most commonly used antifungal drugs are azoles. These fungistatic drugs, with the most studied member being fluconazole, target the ergosterolbiosynthesis pathway. However, the limited amount of available therapies together with the development of resistance against these drugs, strongly emphasize the needfor novel therapeutic strategies. As it is quite time­consuming to introduce completely new drugs to the market, potentiating the efficacy of existing drugs would be abetter strategy. Therefore, it is important to identify cellular pathways involved in the development of drug resistance or tolerance.We found that vesicular transport is involved in fungal susceptibility to the most widely used antifungal drug, fluconazole. We identified specific complexes in the vesiculartransport pathway which contribute to fluconazole resistance or tolerance in the model organism Saccharomyces cerevisiae. Furthermore, we confirm our findings inthe clinically relevant fungi Candida albicans and Candida glabrata. Finally, we show that the combination of fluconazole with a specific inhibitor of the vesiculartransport pathway (Sortin2) increases the susceptibility of Candida species, indicating the potential of using vesicular transport as a target in combination therapy.

* The authors marked with an asterisk equally contributed to the work.

Exploring the effects of Interferon alpha on Cryptococcus neoformansinfection

P.I. SeoaneI, L.M. Taylor­SmithI, D. SterlingII, L.C.K. BellII, M. NoursadeghiII, D. BaileyIII, R.C. MayI

IInstitute of Microbiology and Infection, University of Birmingham, birmingham, United Kingdom, IIDivision of Infection and Immunity, University College London,London, United Kingdom, IIIThe Pirbright Institute, Surrey, United Kingdom

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularlyrelevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungi to survive andreplicate within the phagosome of macrophages, as well as its ability to escape via a novel non­lytic mechanism known as vomocytosis. The impact of vomocytosis onthe outcome of infection is not fully understood, although it has been seen to affect dissemination of the fungal pathogen.

Given its relevance within HIV+ patients, we have been exploring whether viral infection affects the interaction between C. neoformans and macrophages. Here weshow that viral infection enhances cryptococcal vomocytosis without altering phagocytosis or intracellular proliferation of the fungus. The effect was observed usingdifferent viral infections, and is recapitulated when macrophages are stimulated with the anti­viral cytokine interferon alpha (IFNα). The doses of IFNα used correspondto those produced during active HIV infection. Importantly, the effect is abrogated when type­I interferon signalling is blocked, thus underscoring the importance oftype­I interferons in this phenomenon.

Our results highlight the importance of incorporating specific context cues while studying host­pathogen interactions. By doing so, we found that acute viral infection maytrigger the release of latent cryptococci from intracellular compartments, with significant consequences for disease progression.

Genotypic and phenotypic diversity in Candida tropicalis

C. O'BrienI, J. Oliveira­PachecoI, G. ButlerI

IUCD Conway Institute, Dublin, Ireland

BackgroundCandida tropicalis is an emerging human pathogen with considerable mortality rates. Although it is well known as a human pathogen, it is also capable of survival in avariety of different environmental niches. In this study, we sequenced 75 C. tropicalis isolates with the aim of studying the intra­species diversity of this pathogen at agenetic and phenotypic level. These isolates were collected from clinical and environmental sources and sequenced using Illumina technology.

ResultsPhylogenetic analysis of the isolates using SNP trees and PCA analysis reveals several multi­isolate clusters, which are unrelated to geographical origin. Overall, thegenomes of the isolates were stable, with only 4 out of 75 isolates demonstrating any aneuploidy. However, the genomes are highly heterozygous, with one variant, onaverage, every 177 bases. Variant analysis revealed three highly divergent isolates, with one variant, on average, every 7 bases. Further examination of these isolatesrevealed that they are the products of hybridisation between two different, but related, parents.

Hybridisation has been observed in several other fungal species, but has not previously been observed in C. tropicalis. Haplotype phasing revealed two potentialparents; one which is almost identical to the reference strain (>99.9%), and a second, unidentified parent, which is approximately 4.5% different in its sequence to thereference strain. All three putative hybrids are heterozygous at the mating type­like locus (MTL), i.e. they possess both MTLa and MTLalpha idiomorphs. The MTLaidiomorph appears to have originated from the parent that is similar to the reference strain, and the MTLalpha idiomorph appears to be derived from the alternativeparent. Loss of heterozygosity (LOH) is relatively rare in these isolates, which implies recent or ongoing hybridisation events. Furthermore, differences in sequence andLOH patterns indicate that these hybrids may have arisen from separate hybridisation events.

ConclusionsIn this study, we have successfully resequenced 75 isolates of C. tropicalis from a variety of sources. Our study reveals the existence of hybrid isolates in this species,likely to have arisen from mating between two different but related parents.

PolyP Mobilisation in the Pathobiology of Candida albicans

Y. AhmedI, M. IkehII, K. WaldronI, J. QuinnI

IInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom, IIUC Merced, Merced, United States of America

Pivotal to the pathogenic success of C. albicans is its adaptability to survive host imposed stresses which allows for this fungus to proliferate in virtually every anatomicalniche within the immunocompromised host. A facet of this adaptability is the ability of C. albicans to assimilate essential nutrients, such as phosphate (Pi) from hostmicroenvironments. In C. albicans Pi acquisition is mediated via the transcription factor Pho4. Recently, we made the unexpected finding that Pho4­mediated Piaccumulation is also vital for resistance to a pleiotropy of stress­inducing agents. Pho4 also regulates the synthesis of the Pi storage molecule polyphosphate (polyP), andunder Pi limiting conditions Pho4 activation is delayed until the internal Pi stores are liberated from polyP. Here we wanted to explore the importance of Pi mobilisationfrom polyP in the pathobiology of C. albicans. A search of the C. albicans genome data base revealed two putative polyphosphatases, Ppn1 and Ppx1, which arepredicted to regulate the mobilisation of polyP stores. Interestingly, we discovered that a level of functional redundancy exists as both PPN1 and PPX1 needed to beinactivated before polyP mobilisation was impaired. Cells lacking both polyphosphatases displayed similar stress phenotypes as pho4Δ cells, including impaired stressresistance to metal cations, alkaline pH and superoxide stress which was exacerbated in Pi limiting conditions. Unlike pho4Δ cells, the ppx1Δppn1Δ double mutant wasalso highly sensitive to replication stress indicative of a role of polyP mobilisation in the Pi­demanding process of DNA replication. Phenotypic characterisation of thepolyphosphatase deletion strains also uncovered morphological defects and impaired yeast to hypha switching which were exacerbated in cells lacking both PPN1 andPPX1. Collectively our data supports key roles of polyP mobilisation in many aspects of C. albicans pathobiology including, stress resistance, DNA replication andmorphogenesis.

Exploring the host­pathogen transcriptomics of macrophages, Mucoralesand their endosymbionts

P. Sephton­ClarkI, J. MunozII, K. VoelzI, C. CuomoII, E. BallouI

IUniversity of Birmingham, birmingham, United Kingdom, IIBroad Institute of MIT and Harvard, Cambridge, MA, United States of America

Mucormycosis is a disease which can be caused by many members of the Mucorales order, to varying degrees of severity and progression. Understanding the processof infection is key to understanding mucormycosis and developing new treatments. It is unclear how Mucorales spores, the causative agents of mucormycosis, interactwith the innate immune system to cause acute, chronic, or resolving infection. Here we present the differences between two species in the Mucorales order bycharacterising their differential interactions with the innate immune system. We present the transcriptional responses of Rhizopus delemar and Rhizopus microsporus,two of the most commonly diagnosed species, to macrophages, captured through RNA­Seq. We also present the response of macrophages, captured with single cellRNA­Seq. We reveal the variation in these responses, given the presence or lack, of bacterial endosymbionts hosted by the fungi. We show that the fungal response isdriven by the presence or lack of interaction with macrophages, however the fungal response is not affected by the presence of bacterial endosymbionts. In contrast, weshow that the macrophage response varies, depending on the species they interact with, but also depending on endosymbiont status. The most ‘successful’ macrophageselicit a pro­inflammatory response, and germination of the fungal spores can be inhibited through the inhibition of chitin synthase, which enhances macrophage survival.

Into the identification of genetic polymorphisms in C. albicans genomeassociated with the variability in the interaction of diverse clinical isolateswith host keratinocytes by dual transcriptomics

A. KannanI, J. GrandaubertII, G. LavalII,III, C. D’EnfertII, M. Elisabeth BougnouxII, D. SanglardI,IV

ICHUV, Lausanne, Switzerland, IIInstitut Pasteur, Paris, France, IIIInstitut Pasteur, CNRS, C3BI, Bioinformatics and Biostatistics Hub, Paris, France, IVUniversity ofLausanne, Lausanne, Switzerland

Candida albicans clinical isolates intrinsically display high diversity in their population genetic structure. Regulation of several host factors and development of C.albicans diseases can be influenced by diverse genetic backgrounds of the fungus. Using a collection of genome­sequenced isolates, we aim here to understand by so­called Expression Quantitative Trait Loci (eQTLs), the influence of C. albicans genome diversity on fungal transcriptomic profiles in contact with human keratinocytes(TR146 cells). In this setting, the impact of the fungal genomes on the host transcriptome can be probed. Given that a collection of 96 C. albicans isolates will be used,attempts to reduce the number of replicate samples were undertaken. We first addressed with 5 distinct isolates intra­variability of triplicate C. albicans cultures incontact with TR146 cells for 6h. Compared to the reference isolate SC5314, a large number of genes were differentially regulated between the different isolatesemphasizing the large variability between isolates when in contact with host cells. Additionally, low variations between triplicates were obtained, suggesting that the useof one biological replicate from each of the remaining isolates may be sufficient for further eQTL mapping analysis.Compared to SC5314, 153 C. albicans genes were found to be differentially regulated (2­fold) in all four isolates. Based on the overall summary of top 200 variableC. albicans genes among all the five isolates groups, we observed that the isolate CEC3665 most varied from SC5314 upon infection. We observed that 642 geneswere differentially regulated in CEC3665­infected TR146 cells compared to SC5314­infected cells. C.albicans genes involved in filamentous growth weredownregulated in CEC3665. Concerning the host response, genes enriched for innate immune response sets were activated upon infection with the aboveisolates. Based on our findings, the transcriptomic response of CEC3665 at several time points (0.3­, 1­,2­ and 6h) were analyzed and compared to SC5314.Although, the biological replicates from each time point clustered together, the variation between the replicates were not as low as observed at 6h. The number ofdifferentially expressed genes in both these isolates increased with longer co­incubation times with TR146 cells. These findings suggested that the best time of co­incubation for the eQTL analysis to be 6h.

Candida Mixed Species Competitions in Different pH, Carbon, andMedia Environments

B. EsquivelI, E. WillisI, S. BrownI, M. ColbertI, E. McMichaelI, T.C. WhiteI

IUniversity of Missouri Kansas City, Kansas City, United States of America

C. albicans has been the predominant cause of human oral and vaginal candidiasis for decades, however there has been a recent increase in infections caused by non­albicans Candida species. The recognition that many oral, vaginal, bloodstream and gut fungal infections are caused by non­albicans Candida species has resulted in agreater appreciation for the diversity and dynamic nature of the human microbiome. Perturbations to the mucosal environment such as changes in pH, antimicrobial drugtreatment, altered immune function, and even diet can have dramatic effects on the microbial landscape. Fungal flora composition and diversity may directly affecttreatment outcome in clinical cases of infection. We developed an assay to measure interspecies dominance, non­dominance, or cohabitation in a mixed­species population between the seven yeast species C.albicans, C. glabrata, C. krusei, C. tropicalis, C. dublinienses, C. neoformans and S. cerevisiae. We characterized competition within the mixed­speciespopulations under a variety of variables: media buffered to pH 4, 6 or 7; carbon sources coming from commercial artificial sweeteners, sucrose, or glucose; rich andminimal media; and a range of growth temperatures. We found that we can modulate the composition of mixed­species cultures by changing the growth environments. All yeast species grew in single species cultures, butcertain species can repeatedly and predictably outcompete other species and dominate the growth of a mixed­species culture. Other species were able to coexisttogether in a mixed population.This preliminary survey of mixed species interactions and environmental adaptations can potentially be used to detect trends and make predictions about conditions thataffect species fitness and proliferation. Ultimately we would like to determine the mechanisms of species dominance such as cell­cell interactions, secretion of smallmolecules or peptides, flexibility in carbon usage, and nutrient acquisition. These analyses may provide useful insights for oral, gut, bloodstream and vaginal microbiomecomposition.

Identification of novel Candida albicans transcription regulators withroles in host colonization

P. Reuter–WeissenbergerI, L. BöhmI, J.C. PérezI

IInstitute for Molecular Infection Biology, Julius Maximilians University, Würzburg, Germany

Candida albicans is a normal resident of the oral cavity and intestinal tract in most humans. While a few C. albicans genes have been reported to influence theproliferation of the fungus on these mucosae, the general biology of the organism on these surfaces remains underexplored. Our laboratory has screened a collection ofC. albicans transcription regulator mutants in mouse models of oral and gut colonization. These studies have identified previously undescribed regulators with roles inpromoting or reducing the fitness of the fungus in one or both sites of colonization. We are characterizing the function of one of the identified factors, the zinc clustertranscription regulator ZCF8, a determinant of colonization in both niches. ChIP­Seq and RNA­Seq analyses indicate that Zcf8p controls the acquisition of specializedsources of nitrogen which may be relevant during proliferation in the mammalian host. Furthermore, Zcf8p appears to work in concert with other two proteins toregulate subcellular mRNA localization and the response to serum, a filamenting cue. We posit that the cellular processes governed by Zcf8p may be significant duringproliferation of the fungus in the oral mucosa and in the intestinal tract.

How chromosomes are impacted by stress: Elucidating genome­wideLOH dynamics in Candida albicans

T. MartonI,II, C. MaufraisIII, C. d’EnfertI, M. LegrandI

IInstitut Pasteur, INRA, Labex IBEID, Unité Biologie et Pathogénicité Fongiques, Paris, France, IIUniv. Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, rue duDocteur Roux, Paris, France, IIIInstitut Pasteur, CNRS, C3BI, Bioinformatics and Biostatistics Hub, Paris, France

The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss­of­heterozygosity (LOH) events, which can beseen on all 8 chromosomes and affect both laboratory and clinical strains. Genomic studies in C. albicans have highlighted the occurrence of homozygosis biases forcertain chromosomes, explained by the presence of lethal and/or deleterious recessive alleles on these chromosomes. LOH, which are often the consequence of DNAdamage repair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecularmechanisms at their origins. Although LOH are frequent and ubiquitous, little is known regarding their importance in the biology of C. albicans. In diploidSaccharomyces cerevisiae, LOH facilitate the passage of recessive beneficial mutations through Haldane’s sieve allowing rapid evolutionary adaptation. This appearsalso true in the predominantly clonal organism C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as recently illustrated inthe case of antifungal resistance and niche adaptation.To understand the genome­wide dynamic of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on adifferent chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneousLOH events to be detected by monitoring the loss of one of the fluorescent marker using flow cytometry. This collection was characterized through in vitro growthphenotyping and whole­genome sequencing (of note, we confirmed the mutagenic properties of transformation, as long­tract LOH and/or aneuploidies were detected in60% of the transformants tested, often occurring on chromosomes other than the one targeted by the transformation). We are assessing spontaneous LOH frequency ineach reporter strain as well as LOH frequency upon physiologically relevant stresses (pH, heat, oxidative and drug treatments). Our project will allow addressing thefollowing fundamental questions: Do all chromosomes possess the same potential of undergoing LOH events? Does LOH frequency correlate with chromosome size?Are there stress­specific hotspots for LOH? These answers will provide a significant insight in the potential coordination of LOH events, occurring at random or ascontrolled events.

Inhibition of spore germination by macrophages promotes A. fumigatuspersistence and susceptibility to voriconazole

E. RosowskiI,II, N. KellerI, A. HuttenlocherI

IUniversity of Wisconsin­Madison, Madison, WI, United States of America, IIClemson University, Clemson, SC, United States of America

The role of macrophages in Aspergillus pathogenesis remains obscure, with reports of macrophages as both required and dispensable for host resistance. We employeda larval zebrafish macrophage­deficient model to investigate this question, taking advantage of the vast imaging capabilities of this host. We find that depending on thespecific context, macrophages can possess either pro­ or anti­fungal functions, both primarily due to the ability of macrophages to inhibit spore germination.Macrophages act as a protective niche for spores against neutrophil­mediated killing, and faster germinating strains of A. fumigatus are killed more efficiently in amacrophage­deficient host. On the other hand, when larvae are treated with voriconazole, macrophages are required for the drug’s efficacy. By inhibiting sporegermination, macrophages give the drug more time to act in vivo. These results position macrophages as a key regulator of A. fumigatus­induced pathology anddisease.

Interaction of human neutrophil granulocytes and the melanin producingfungus, Curvularia lunata

E.J. TóthI,II, M. TakóII, C. VágvölgyiII, T. PappI,II

IHAS­USZ Fungal Pathogenicity Mehanisms Research Group, Hungarian Academy of Science, University of Szeged, Szeged, Hungary, IIUniversity of Szeged,Department of Microbiology, Szeged, Hungary

Curvularia lunata is an opportunistic human and plant pathogenic, melanin producing filamentous fungus. Members of the genus Curvularia typically causephaeohyphomycoses in humans, which can manifest as local infections (e.g. keratitis, sinusitis) in immunocompetent or invasive mycoses in immunocompromisedpatients. Neutropenia is a critical condition in term of phaeohyphomycoses. Although the plant­fungal interactions of these fungi have been intensively studied, there isonly little information available about the host response to them in human infections. The aim of this study was to investigate the neutrophil granulocytes’ response to thehyphae of C. lunata in comparison with that to the well­studied Aspergillus fumigatus.In the present study, C. lunata SZMC 23759 and A. fumigatus SZMC 23245, both isolated from human eye infection, were examined. ROS production by neutrophilgranulocytes were measured in the presence and the absence of the supernatant of germinating conidia and after serum treatment. Activation of neutrophils and killingefficiency towards C. lunata were checked after the interaction. Expression of haloperoxidase coding genes of C. lunata was measured after H2O2 induction of thefungus.Hydrogen peroxide production of neutrophils in interaction with the fungi were compared. Activation of neutrophils was observed after serum opsonisation and in thepresence of the supernatant of germinating conidia as well. C. lunata was able to induce hydrogen peroxide release only after serum opsonisation, just like A.fumigatus. However, viability of C. lunata did not decrease when the hydrogen peroxide production was detected. The survival of the fungus can be explained by theability of haloperoxidase production; indeed, an elevated level of haloperoxidase gene expression was detected after H2O2 induction.This research was supported by the grants the UNKP­18­3 New National Excellence Program of the Ministry of Human Capacities” „Lendület” LP2016­8/2016 andGINOP­2.3.2­15­2016­00035.

Rampant transposition and loss of RNAi cause hypermutation andantifungal drug resistance in Cryptococcus neoformans

S. PriestI, C. RothI, J. GranekI, P. MagweneI, J. HeitmanI

IDuke University, Durham, United States of America

To successfully survive and compete within their environmental niches, microorganisms must stochastically acquire mutations or face evolutionary stagnation. Althoughincreased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of a strong selective pressure. Toexplore how hypermutation arises in nature and elucidate its consequences, we employed a recently assembled collection of 387 sequenced clinical and environmentalisolates of Cryptococcus neoformans, a fungal pathogen responsible for approximately 15% of AIDS­related deaths annually. HIV­positive individuals diagnosed withcryptococcal meningitis face unacceptably high mortality rates: up to 70% in low income nations and 30% in North America. This high mortality is attributable to adearth of antifungal treatment options, so limited because of the conserved homology between many essential fungal and human proteins, and to high rates of drugresistance. Preliminary screening for the ability of each isolate to acquire resistance to otherwise lethal concentrations of diverse antifungal drugs has identified 30hypermutator strains, including two robust hypermutators. Characterization of the resistant colonies these two isolates produced revealed that insertion of a singletransposable element (TE) was largely responsible for de novo drug resistance. Long­read whole genome sequencing (WGS) revealed that both hypermutator genomesencode >600 copies of this TE and harbor a nonsense mutation in the first exon of an RNAi component known to be involved in TE silencing, ZNF3. Quantitative traitloci mapping of F1 segregants from a genetic cross between one of the hypermutators and the laboratory reference strain identified a single significant peak associatedwith hypermutation that includes the mutant znf3 allele. Therefore, our central hypothesis is that hypermutability due to frequent transposition in these isolates isattributable to the presence of a novel TE in the C. neoformans lineage as well as an RNAi defect. We will apply classical genetic techniques, perform additional WGSanalysis, and conduct in vivo survival and drug resistance assays in the Galleria mellonella and mouse models to determine the genetic and molecular basis as well asthe evolutionary trajectory of this elevated transposition and define its impact on the fitness of these strains and their ability to acquire drug resistance in host­relevantconditions.

Candida albicans Quorum Sensing Molecule Farnesol ModulatesStaphyloxanthin Production and Activates the Thiol­Based OxidativeStress Response in Staphylococcus aureus

T. VilaI, M.A. Rizk *I

IUniversity of Maryland Baltimore, Baltimore, Maryland, United States of America

Microbial species utilize secreted signaling molecules to coordinate their behavior. Our previous studies characterized a dynamic fungal­bacterial interaction betweenCandida albicans and Staphylococcus aureus in biofilm. Recently, we demonstrated that the quorum­sensing molecule farnesol secreted by C. albicans modulates S.aureus response to antimicrobials. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureus behavior within the context of inter­species interactions. To mimic mixed biofilm dynamics, a farnesol­sensitized S. aureus phenotype was generated through sequential exposure to farnesol. The sensitizedcells exhibited a dramatic loss of the typical S. aureus yellow pigment, which was identified as staphyloxanthin, an important virulence factor synthesized by the Crtoperon. Additionally, farnesol exposure resulted in accumulation of intracellular ROS. Farnesol­induced oxidative stress was also indicated by transcriptional analysisdemonstrating major transcriptomic alterations in redox sensors and stress response machinery and importantly, in major regulators of virulence. However, expression ofenzymes in staphyloxanthin biosynthesis pathway was not altered. Based on theoretical binding modeling, we demonstrated that farnesol may block staphyloxanthinbiosynthesis via competitive binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity between farnesol and the CrtM substrate.Finally, we showed that S. aureus growth with C. albicans in the absence of farnesol supplementation similarly induces bacterial cell depigmentation. In contrast, growthwith a farnesol­deficient C. albicans strain did not impact S. aureus cell pigmentation. Collectively, the findings demonstrate that a fungal secreted molecule acts as aredox cycler eliciting a stress response by a bacterial cell via activation of the thiol­based redox system under control of global regulators of virulence. Therefore,farnesol­induced transcriptional modulations of key regulatory networks in S. aureus may modulate the pathogenesis of C. albicans­S. aureus co­infections.

* The authors marked with an asterisk equally contributed to the work.

Mechanical forces during filamentous growth of a human fungalpathogen

C. PUERNER *I, N. KUKHALEISHVILI *I, D. THOMSON *I, S. SCHAUBI, X. NOBLINI, A. SEMINARAI, M. BASSILANAI, R. ARKOWITZI

IUniversity Côte d’Azur, Nice, France

Physical forces appear to be critical for host tissue penetration by the human fungal pathogen Candida albicans, as well as its escape from host immune cells (1). C.albicans filamentous growth and its ability to invade or colonize solid surfaces, such as medical implants and human tissues are critical for its pathogenicity. With the goalof determining the biophysical properties of C. albicans hyphae, we have generated PDMS microchambers (2, 3), in which C. albicans are entrapped andsubsequently monitored filamentous growth. Furthermore, we have altered the stiffness of these microchambers to mimic that of human cells and tissues and havecharacterized surface (non­invasive) and invasive growth, using time­lapse microscopy. We observed a decrease in the percentage of invasive filamentous growth withan increase in PDMS stiffness. Strikingly, there appears to be a stiffness threshold where hyphae no longer are able to penetrate. This threshold is likely to be at thegrowth­stalling force, a value, which is balanced by the cell turgor pressure (2). When unable to penetrate the PDMS, hyphae bend upon extension, eventually filling themicrochamber. We have followed filament invasion within the substrate and show that they extend with a reduced rate compared to surface growing cells. There is afurther reduction in extension rate with an increase in PDMS stiffness. Additionally, we have measured a significant increase in cell diameter concomitant with a decreasein cell compartment length during invasive growth, yet, compartment volumes are indistinguishable to that of surface growing cells. These results suggest that invasivegrowth results in a disruption of polarity, as has been recently observed upon perturbation of growth in Schizosaccharomyces pombe (4). To further investigate cellmorphology, as well as the distribution and dynamics of intracellular compartments, we are currently following a range of reporters, in particular during filament exit fromthe PDMS into a neighboring microchamber. These studies provide a framework for analyzing the interplay between mechanical constraints and polarity during invasivefilamentous growth.

1. Sheppard and Filler (2015) CSH Perspect Med. 5: a019687.2. Minc et al. (2009) Curr Biol. 19: 1096­101.3. Minc (2014) Meth Cell Biol. 120: 215­26.4. Haupt et al. (2018) Curr Biol. 28: 1­10.

* The authors marked with an asterisk equally contributed to the work.

Genomic patterns of Candida parapsilosis s.l. hybrids suggest a weakeffect of Bateson­Dobzhansky­Muller incompatibilities

V. de Pinho MixaoI,II, T. GabaldónI,II,III

ICentre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain., Barcelona, Spain,IIUniversitat Pompeu Fabra (UPF), Barcelona, Spain, IIIICREA, Barcelona, Spain

Inter­species hybrids are chimeric organisms with highly heterozygous genomes. The co­existence of the two parental genomes provides a genomic plasticity that may

favor adaptation to new environments. At the same time, evolutionary models (i.e. Bateson­Dobzhansky­Muller) predict that the high levels of heterozygosity in hybrids

are likely to result in numerous deleterious epistatic interactions that would compromise their fitness. Under this scenario, loss of heterozygosity events affecting genomic

regions comprising such negative interactions are expected to be favored by selection. To test this hypothesis, we compared the genomic sequences of fourty­five strains

belonging to the Candida parapsilosis clade and resulting from at least five distinct natural hybridization events. Comparison of the different events of loss of

heterozygosity among the different clades suggests that Bateson­Dobzhansky­Muller incompatibilities are not sufficiently strong to drive genomic evolution in these

pathogenic hybrids. This high capacity to buffer the effect of negative epistatic interactions may explain the relative abundance and success of fungal hybrids.

Characterisation of a novel zinc detoxification system and its role in thepathogenicity of Candida albicans

A. CrawfordI, D. WilsonI

IUniversity of Aberdeen, Aberdeen, United Kingdom

During both pathogenic and commensal growth, Candida albicans encounters a large range of microenvironments and nutritional landscapes. These nutritionalbottlenecks are imposed actively or passively by the host in a process known as Nutritional Immunity. The host possesses the ability to weaponize essential nutrientssuch as zinc, starving pathogens of them or pooling nutrients to toxic levels; inhibiting growth or killing the pathogen. The ability to overcome these hurdles is thereforefundamental to pathogens’ ability to survive and cause disease.Our group have identified and characterised a novel zinc detoxification system in C. albicans. When challenged with a relative increase in environmental zinc,C. albicans cells exhibit a rapid accumulation of zinc­containing vesicular compartments known as zincosomes. These compartments accumulate zinc at an increasedrate compared to the vacuole. Here we demonstrate the first evidence of zincosomal zinc detoxification and through mutant analysis we have identified that the zinctransporter Zrc1 is directly associated with zincosomal zinc accumulation. Furthermore, utilising a Venus YFP fluorescent tag, we have successfully visualised Zrc1’ssubcellular localisation. C. albicans strains lacking ZRC1 exhibit reduced proliferation in a murine model of systemic infection, providing the first evidence that zinctolerance contributes to C. albicans virulence in the murine liver. We hypothesise that the impaired growth of the zrc1Δ mutant is due to host­mediated zinc toxicity.Using these tools and data, we hope to further characterise and demonstrate the importance of mammalian nutritional immunity and the mechanisms that C. albicans hasevolved to overcome it.

Pseudomonas aeruginosa­derived volatile sulphur compounds promotedistal Aspergillus fumigatus growth and a synergistic pathogen­pathogeninteraction that increases pathogenicity in co­infection

J. ScottI, M. Sueiro­OlivaresI, W. AhmedII,III, C. HeddergottIV, C. ZhaoI, R. ThomasI, M. BromleyI, J. LatgéIV, S. KrappmannV, S. FowlerII,VI, E. BignellI, J.AmichI

IManchester Fungal Infection Group (MFIG), Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicineand Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom, IIRespiratory and Allergy Research Group,Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester,Manchester, United Kingdom, IIIManchester Institute of Biotechnology, University of Manchester, Manchester, United Kingdom, IVUnité des Aspergillus, InstitutPasteur, Paris, France, VMikrobiologisches Institut ­ Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich­Alexander­Universität Erlangen­Nürnberg, Erlangen, Germany, VINIHR Manchester Biomedical Research Centre and Manchester Academic Health Science Centre, ManchesterUniversity Hospitals NHS Foundation Trust, Wythenshawe, Manchester, United Kingdom

Pathogen­pathogen interactions in polymicrobial infections directly impact disease outcomes. Co­infection with Pseudomonas aeruginosa and Aspergillus fumigatus,respectively the most common bacterial and fungal pathogens isolated from cystic fibrosis patient airways, leads to a worse prognosis. Recent in vitro studies havedemonstrated that P. aeruginosa­derived volatile sulphur compounds (VSCs) can promote A. fumigatus growth. In this study we investigate the mechanistic basis ofsuch cross­talk and its relevance during co­infection. We show that A. fumigatus assimilates VSCs as sulphur sources via cysteine­(CysB) or homocysteine­(CysD)synthase. Consequently, P. aeruginosa­derived VSCs trigger growth of wild­type A. fumigatus, but not of a ΔcysBΔcysD mutant, on sulphur­limiting media. GC­MS­mediated volatile analysis confirmed the production of relevant VSCs in P. aeruginosa infected Galleria mellonella. In the wax­worm model, P. aeruginosa and A.fumigatus wild­type co­infection triggered a ~5 fold increase in mortality compared to single infections. Interestingly, co­infection with ΔcysBΔcysD and Pseudomonasresulted in no more than a 2 fold increase in mortality, implying that the ability of A. fumigatus to utilise VSCs is critical to the synergistic effect of co­infection.Furthermore, co­infection with A. fumigatus wild­type, but not ΔcysBΔcysD triggered increased fungal and bacterial burdens, suggesting a VSCs dependentinterspecies cross­talk during infection. We found that a P. aeruginosa strain truncated in a methionine aminotransferase encoding gene (ΔybdL) produced less VSCsand had reduced capacity to enhance distal fungal growth in vitro. A P. aeruginosa ΔybdL­A. fumigatus wild­type co­infection resulted in the same moderate increasein mortality (~1.5 fold) observed in the P. aeruginosa wild­type­ΔcysBΔcysD co­infection. Therefore, both the ability of P. aeruginosa to produce VSCs and of A.fumigatus to assimilate them are crucial for the synergistic effect of co­infection on mortality. We conclude that P. aeruginosa produces VSCs during infection whichpromote fungal growth and trigger an inter­species cross­talk that increases the pathobiology of co­infection.

Analysis of hotspot regions of the genes involved in primary resistance(erg11 and fks1) in C. inconspicua, C. rugosa and C. ciferrii

A. Pérez HansenI, V. MixaoII, T. GabaldónII, C. Lass­FlörlI, M. LacknerI

IDivision of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria, Innsbruck, Austria, IICentre for Genomic Regulation (CRG), TheBarcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain., Barcelona, Spain

Introduction: Our prior comprehensive susceptibility study and the literature indicate that C. inconpicua, C. rugosa and C. ciferrii have low levels of susceptibility tothe commonly used antifungal drugs (azoles and echinocandins). However, since C. inconpicua, C. rugosa, and C. ciferri only represent a minor portion of allCandida infections, the molecular mechanism of their primary resistance is still unknown.Objective: The objective of this study is to characterize the amino­acid (AA) sequence of the hotspot regions of the genes involved in primary resistance against azoles(erg11) and echinocandins (fks1) in C. inconspicua, C. rugosa and C. ciferrii. We aim to identify amino acid changes that are linked with the limited activity of azoleand echinocandin drugs.Method: One representative strain per species was whole genome sequenced. Homologue genes of erg11 (lanosterol 14 alpha­demethylase) and fks1 (1 3­beta­glucansynthase) were identified by BLAST comparison with C. albicans genome. Primers were designed to amplify and sequence homologous “hotspot” regions in erg11 andfks1. Comprehensive amino acid alignments were generated for our collection of clinical isolates (n=202). Amino acid changes in the hotspot regions were correlatedwith resistance phenotypes and compared with literature.Results and conclusion: All tested species had amino acid changes in at least one of the “hotspot” regions of erg11 and fks1 in comparison with C. albicans. Erg11mutation at position D153E (erg11 HS1) present in C. ciferrii were previously associated with low azole susceptibility. The mutation H283N also in C. ciferrii has notbeen described yet, but other amino acid substitution in the same position (e.g., H283D, H283R– ERG11 HS2) was previously associated with fluconazole resistance.Mutations at position P649 (HS1) in the FKS1 gene were previously linked with a moderate increase in echinocandin resistance though the particular AA changepresent in our species haven’t been described before. The mutations occurring in FKS1 HS2 identified in this study have not yet been linked to echinocandin resistance.The high species­specific AA substitutions points towards an evolutionary conserved intrinsic resistance, structural and functional studies are needed to evaluate theimpact of these AAs on the enzyme.

Antibody theranostics for fungal infections

L. WalkerI, I. RaziunaiteI, D. MacCallumI, A. JensenII, L. ErwigI, N. GowI, F. RudkinI

IUniversity of Aberdeen, Aberdeen, United Kingdom, IIHiFiBio SAS, Paris, France

Invasive fungal infections kill 1.5 million people every year. Clinicians urgently require more accurate, discriminative and efficient diagnostics as well as improvedtherapeutic strategies to reduce the high mortality rates. Monoclonal antibodies (mAbs) have revolutionised the way many diseases are diagnosed and treated but haveyet to be fully exploited in the setting of fungal infections.

We have developed a unique set of fully human mAbs against Candida with diagnostic and therapeutic potential. A novel single B cell technology was employed tofacilitate the isolation of human anti­Candida mAb genes directly from single B cells derived from donors with a history of Candida infection. Characterization of thesemAbs revealed morphology specific, high avidity binding to the cell wall of Candida species. The host’s first line of defense against systemic candidiasis includes immunecells such as macrophages which recognise, ingest and digest fungal cells. We have shown that our pan‑Candida mAbs generated strong opsono­phagocytic activity ofmacrophages against C. albicans in vitro and demonstrated protection in vivo in a murine model of disseminated candidiasis. Recently, there has been a rise in drugresistance to the main classes of clinically used antifungals. In particular Candida glabrata and the emerging pathogen, Candida auris, are prone to developing multi­drug resistance. We have demonstrated that our mAbs bind with equal affinity to both drug­resistant clinical isolates of C. albicans, C. glabrata and C. auris. As aresult a significant increase in opsono­phagocytic activity with macrophages was also observed when drug­resistant isolates of each species were exposed to our pan­Candida mAbs. In addition, treatment with these mAbs demonstrated protection against infection with drug­resistant clinical isolates of C. albicans, C. glabrata andC. auris in the Galleria mellonella model.We have also shown that these anti­Candida mAbs were effective in recognizing Candida antigens in a number of diagnostic formats. Our work highlights the value ofthis technology in generating theranostic mAbs to address the urgent requirements for new fungal diagnostic and therapeutic strategies.

How lactobacilli antagonize pathogenicity of Candida albicans: Lessonsfrom in vitro gut models and a dynamic organ­on­chip model

M. Gresnigt *I, A. Last *I, K. Graf *I, J. DroegeI, M. MaurerII, S. LindeIII, M. WestermannIII, M. GrögerII, A. MosigII,IV, B. HubeI,V

IHans Knöll Institut, Microbial Pathogenicity Mechanisms, Jena, Germany, IICenter for Sepsis Control and Care (CSCC), University Hospital Jena, Jena, Germany,IIICenter for electron microscopy Jena University Hospital, Jena, Germany, IVInstitute of Biochemistry II, Jena University Hospital, Jena, Germany, VFriedrich SchillerUniversity, Jena, Germany

The intestine is the main origin of Candida albicans cells that can cause systemic infections in immunocompromised patients. In this reservoir, the fungus normally existsas a harmless commensal. However, removal or disturbance of the bacterial microbiota by antibiotic treatment can initiate fungal overgrowth and favour pathogenicity inimmunocompromised patients. Current in vitro gut models used to study pathogenesis investigate the state where C. albicans behaves as a pathogen rather than acommensal.

We established an in vitro gut model where we reduced the fungal pathogenicity to a minimum by increasing biological complexity of the model. In this model,enterocytes represent the epithelial barrier and inclusion of mucus­secreting goblet cells limit C. albicans adhesion and invasion. Protection against C. albicans­induceddamage was achieved by inclusion of a microbiota of antagonistic lactobacilli. This required bacterial growth and was time­, dose­, and species­dependent, butindependent on competition for adhesion sites. Lactobacilli reduced hyphal elongation and we identified shedding of hyphae from the epithelial surface as a main andnovel mechanism of damage protection. An increase of the model complexity by introducing macrophages resulted in a complete absence of viable translocated fungalCFU’s. Using a microfluidic 3D gut­on­chip model that includes an epithelium, vascular endothelium, and resident macrophages we validated the protective effects of L.rhamnosus on reducing C. albicans mediated damage and invasion of the bloodstream.

Collectively, we established an in vitro gut model, which can be used to experimentally dissect commensal­like interactions of C. albicans with a bacterial microbiotaand the host epithelial barrier. We discovered fungal shedding as a novel mechanism by which bacteria contribute to the protection of epithelial surfaces. Further, thedynamic gut­on­chip model can be used for studying C. albicans pathogenicity and to investigate interactions with various host cell types and intestinal microbiota.

* The authors marked with an asterisk equally contributed to the work.

Functional implications of the genetic diversity in C. albicans

K. Martinez de San VicenteI, C. d’EnfertII, M. BougnouxII,III, S. LeibundGut­LandmannIV

I1. Section of Immunology, Vetsuisse Faculty, University of Zürich,, Zurich, Switzerland, II2. Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris,France, III3. Unité de Parasitologie­Mycologie, Service de Microbiologie clinique, Hôpital Necker­Enfants­Malades, Assistance Publique des Hôpitaux de Paris(APHP), Paris, France, IV1. Section of Immunology, Vetsuisse Faculty, University of Zürich, Zurich, Switzerland

The opportunistic fungal pathogen Candida albicans has a highly diverse population structure. It has become clear that the genetic background of C. albicans greatlyinfluences the interaction between the fungus with the mammalian host and it may thereby act as a critical determinant of disease outcome and severity in humans. Across a collection of 200 genetically diverse C. albicans isolates, we observed widely different capacities to induce cellular damage and to release of pro­inflammatory cytokines such as IL­1α in contact with keratinocytes. Using a small set of these isolates, we previously showed that the capacity to induce host damagewas linked to rapid neutrophil recruitment and rapid clearance of the fungus from the oral mucosa of experimentally infected mice in vivo. Here we extend this analysisto a larger number of C. albicans isolates and confirm the correlation between the epithelial response in vitro and the extent of inflammation in vivo, which is inverselycorrelated with their ability to persist as commensals in the oral epithelium. Using pairs of phylogenetically closely related strains that display divergent host­pathogenphenotypes, we aim at identifying the relevant polymorphisms causing the observed functional differences. For this, we combine phenotypic, transcriptomic and genomicanalyses together with computational approaches. This shall reveal novel pathogenic determinants of C. albicans, some of which may serve as novel biomarkers ortherapeutic targets.

Reducing animal use in antifungal drug development: near­infraredreporter strains and imaging.

A. ChapuisI, E. BallouII, D. MacCallumI

IUniversity of Aberdeen, Aberdeen, United Kingdom, IIInstitute of Microbiology and Infection, University of Birmingham, birmingham, United Kingdom

Fungal infections are a major burden on global health. Fungal diseases range from minor infections to life­threatening systemic infections. Difficulties in diagnosis andlimited therapeutic options increase the risk of mortality and there is a clinical need for new therapies. Traditional in vivo investigation of infection and new therapies inmice is carried out using post mortem methodologies. However, fluorescent proteins have become valuable tools for biomedical research. In particular, reporterproteins in the near­infrared spectrum (650­900 nm) allow good results for in vivo imaging. Developing in vivo imaging using near­infrared reporter strains addressethical concerns, as they allow a significant reduction in the number of animals required to evaluate a new drug, if the same animals are re­imaged during treatment.We are developing novel near­infrared reporters for imaging fungal infection in live mice. Two proteins in the near­infrared spectrum have been selected: near­infraredfluorescent protein (iRFP) and Katushka. Both reporters have been codon­optimised for optimal expression in Candida albicans and constructs have been created toencourage integration into the genome, creating near­infrared expressing fungi which have been tested in vitro. These strain will be used in infection models in liveanimals. Imaging will be carried out using the Bruker MS FX Pro in vivo imager. Future work will focus on refining the protocol for evaluating antifungal therapysuccess in living mice.

Pathogenic Candida species and the filamentous fungus Aspergillusfumigatus trigger distinct killing and feeding mechanisms in afungivorous amoeba

S. RadosaI, I. FerlingI, J.S. SpragueI, R. TóthII, T. WolfIII, J. LindeIII, A. GácserII, F. HillmannI

ILeibniz Institute for Natural Product Research and Infection Biology, Junior Research Group Evolution of Microbial Interactions, Jena, Germany, IIUniversity ofSzeged, Department of Microbiology, Szeged, Hungary, IIILeibniz Institute for Natural Product Research and Infection Biology, Systems Biology and Bioinformatics,Jena, Germany

Resistance to phagocytic attacks by innate immune cells can be considered as a common virulence attribute of most human fungal pathogens. The strategies how toescape from innate immune cells can be highly diverse, but size, diverse morphologies, and variable surface recognition patterns are among the primary challenges forphagocytes when encountering fungal prey. While filamentous fungi escape phagocytic killing by pure physical constraints, unicellular conidia and yeasts can maskmolecular surface patterns or arrest phagocytic processing.Here we show that such attributes may also serve as a selective advantage in natural habitats to escape predation. We have recently isolated the amoeba Protosteliumaurantium and characterized its fungivorous life­style. The amoeba readily internalized yeast­like fungi from the major fungal groups of basidiomycetes andascomycetes including Cryptococcus neoformans and several pathogenic Candida sp., but not Candida albicans whose mannoprotein coat was essential to escaperecognition. C. parapsilosis served as the preferred food source and killing occured via rapid intra­phagolysosomal cell­lysis within minutes after uptake. Functionalgenomics on C. parapsilosis revealed fungal copper­ and redox homeostasis as main targets during phagocytic killing.Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized by P. aurantium, but swelling and the onset of germination inducedinternalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip. Structures resembling the frustratedphagosomes of innate immune cells were resolved by an actin­mediated invasion of fungal filaments and followed by absorption of the entire cytoplasmic content of thehypha. Our results demonstrate that even a single amoeba species can impose predatory pressure on yeasts and filamentous fungi and suggests that covering molecularsurface patterns may provide protection during predator­prey as well as host­pathogen interactions.

Spore Germination as a Target for Antifungal Therapeutics

S. OrtizI, M. HuangI, C.M. HullI

IUniversity of Wisconsin, Madison, Madison, WI, United States of America

Spores are essential cell types required for long­term survival of most fungi. For fatal human fungal pathogens like Cryptococcus, germination is the key differentionprocess required for spores to initiate vegetative growth and ultimately cause disease. Because germination is required for pathogenesis, identifying novel compoundsthat inhibit germination could provide opportunities for new therapeutics. Such compounds could be developed into low­toxicity drugs that could be used in theprevention and/or treatment of cryptococcosis (and other fungal spore­mediated diseases).Using a novel luciferase­based high throughput assay, we screened a library of ~1200 FDA­approved drugs and identified several germination and yeast growthinhibitors. These drugs have been approved for treatment of a variety of conditions not related to invasive fungal disease but could be repurposed for use asantigermination and/or antifungal drugs. Importantly, we have demonstrated the ability of one of the drugs (CNX­16) to lower fungal burdens in mice infectedintranasally with Cryptococcus when used in either prophylaxis or treatment. Due to its ability to lower fungal burdens and its low intranasal toxicity, CNX­16 is a leadcandidate for repurposing for use as an antifungal drug, specifically as an agent to prevent cryptococcal meningoencephalitis in immunocompromised patients. Not onlyis CNX­16 a lead candidate for repurposing, but it is also a prime example of the potential of using germination as a target for prophylactic antifungal drug development.

Bacteria and fungi: how do they communicate?

F. NogueiraI, M. TschernerII, S. JenullII, N. PopitschI, L. PereiraI, K. KuchlerII, T. LionI

ICCRI, Vienna, Austria, IIMFPL, Vienna, Austria

The interactions between pathogens occurring during polymicrobial infections remain poorly understood. Only recently, with the expansion of microbiome research,scientists became aware of the complex relationships between microorganisms. Importantly, opportunistic bacteria and fungi that have been living as commensals mayturn into pathogens. Such alterations cause an imbalance of the microbiota that must be taken into account for diagnostics and treatment of infections. Often, bacteriaand fungi occupy the same niches which results in either a synergistic or antagonistic interaction. Bacteria and fungi communicate via secretion of quorum sensingmolecules and production of virulence factors which may also impact the response of the immune system and induce alterations in the host. Therefore, we aim tocharacterize the interaction of relevant human pathogens that occupy the same niches such as Aspergillus and Klebsiella. The capacity of biofilm formation andstructural integrity of bacteria and fungi alone and in co­culture were assessed by crystal violet staining, confocal microscopy and qPCR. Our data have shown asuppressive effect on fungal growth and biofilm formation by the bacteria in an in vitro co­culture. However, fungal growth could be restored upon antibiotic treatment.Analysis of the cell viability has also shown that the bacteria do not kill the fungus but rather exert a suppressive effect. Furthermore, omics analysis revealed key factorsmediating Aspergillus and Klebsiella interaction. Molecules identified as regulators of this interaction will provide novel insights potentially exploitable for diagnosticsand treatment of polymicrobial infections.

Exploring how a family of small­secreted effectors manipulatesmacrophages during Histoplasma capsulatum infection

R. RodriguezI, D. AzimovaI, A. SilI

IUC San Francisco, San Francisco, United States of America

Histoplasma capsulatum (Hc) is a thermally dimorphic intracellular human pathogen. Endemic to the Ohio and Mississippi River Valleys, Hc causes approximately500,000 new infections annually in the US. In the environment, Hc grows as a saprophytic mold. When the soil is disturbed, fragments of the mold are aerosolized andcan be readily inhaled by a mammalian host. The subsequent increase in temperature to 37°C is sufficient to induce a morphological and molecular transition to the yeastform. Once in the host, Hc is phagocytosed by macrophages where it is able to counteract and evade macrophage innate immune defenses and replicate to high levelswithin the host phagosome. Preliminary evidence from mass spectrometry revealed a handful of Hc secreted proteins that access the host cytosol during macrophageinfection, making them high priority candidates for Hc virulence factors. The genes that encode these proteins are expressed by the yeast form of the organism, but notthe mold form. One of these genes encodes a protein we have named She1, for Secreted Histoplasma effector. She1 is a small protein of unknown function with apredicted secretion signal and a 6­cysteine pattern in the C­terminus. SHE1 shows differential expression in yeast­form cells. Further bioinformatic analysis identifiedtwo paralogs of She1, paralog1 and paralog2, both of which also contain a C­terminal 6­cysteine pattern. We have determined that paralog2 is found in all sequencedHc strains while She1 and paralog1 are only found in the North American 2 and the Panama Hc clades. Furthermore, She1 family proteins are only found inHistoplasma species and not in any other closely related pathogenic fungi. Using CRISPR­Cas9, we have generated disruption mutants for each individual gene anddetermined that the mutant lacking paralog2 has an intermediate lysis defect in bone marrow derived macrophages. An analysis of the virulence of double and triplemutants will be presented. Our preliminary evidence suggests that She1 proteins may play an important role in the virulence of Hc. Studying this family of proteins mayelucidate the mechanisms through which Hc manipulates the macrophage.

Comparative transcriptomics reveals generalized patterns of host­pathogen interactions between human and major Candida fungi

H. HovhannisyanI,II, M. PekmezovicIII, E. IracaneIV, J. PachecoIV, S. BrunkeIII, G. ButlerIV, B. HubeIII, T. GabaldónI,II,V

ICenter for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain, IIUniversitat Pompeu Fabra, Barcelona, Spain, IIIDepartment ofMicrobial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, Jena, Germany, IVSchool ofBiomedical and Biomolecular Science and UCD Conway Institute of Biomolecular and Biomedical Research, Conway Institute, University College Dublin, Dublin,Ireland, VICREA, Barcelona, Spain

Fungal pathogens from the Candida clade are among the most common cause of opportunistic fungal infections worldwide. Despite numerous studies in the last decadeto elucidate mechanisms underlying the pathogenesis of Candida infections, we still lack understanding about the interactions of the diverse Candida species with thehost. To address this issue we have performed a time­series dual RNA­Seq analysis of the four most common Candida species, namely C. albicans, C. glabrata, C.parapsilosis and C. tropicalis, interacting with human vaginal epithelial cells and studied the course of transcription in both counterparts. This experimental setting allowedfungal cells to deploy their whole pathogenic arsenal without being restricted by the host immune system. On the human side, the transcriptome shows a quick andextensive up­regulation in response to all species – on average 235 up­regulated genes (log2 fold change > 1.5, FDR < 0.01) versus 12 down­regulated genes (log2fold change < ­1.5, FDR < 0.01) 3 h after infection. At this stage of infection the host transcriptional response was largely uniform against all four Candida species,which implies similar molecular mechanisms of initial sensing and interaction with phylogenetically distinct species. However, at later stages (12 h and 24 h) the hostresponse diversified, and the extent of damage caused by different fungal species acted as a significant factor driving the differences in the host transcriptional response.In contrast, the transcriptomic response of the different species upon exposure to human epithelial cells appears to be specific for each of the Candida species. Evenbased on reciprocal orthologous genes between the four species, the fungal transcriptomes demonstrate distinct patterns of differential expression involving variousmolecular pathways. Moreover, the set of infection­specific genes (genes which are differentially expressed during the infection, but not in control samples), is almostexclusive for each species. This finding supports the hypothesis that pathogenicity Candida species towards humans emerged multiple times independently throughouttheir evolutionary history.

The central repressor Tup1 is required for high­level expression of ALS3and ECE1 in Candida albicans

S. RubenI, J. MorschhäuserII, B. HubeIII, R. MartinIV, O. KurzaiI,IV

ISeptomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology –Hans Knoell Institute, Jena, Germany, IIInstitute for MolecularInfection Biology, University of Würzburg, Würzburg, Germany, IIIMicrobial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and InfectionBiology – Hans Knoell Institute (HKI), Jena, Germany, IVInstitute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany

The ability of Candida albicans to reversibly switch its morphology between yeast and filamentous stages is critical for its virulence. Formation of hyphae isaccompanied by upregulation of the core filamentation response genes ALS3 and ECE1, which play roles in pathogenesis, including adhesion, invasion, host celldamage, and iron acquisition. The global repressor Tup1 and its cofactor Nrg1 are considered the main antagonists of hyphal development and associated geneexpression. Employing a split­GFP assay, we observed an in vivo interaction of these two regulators in yeast cells. However, this interaction diminished after theinitiation of hyphal growth. Further experiments revealed that Tup1 is not only involved in the repression of ALS3 and ECE1 in yeast cells, but also required for fullexpression levels of these genes during in hyphal growth. Additionally, we identified the transcription factor Ahr1 as essential for high transcription levels of both genes.A hyperactive AHR1 allele was able to induce the transcription of both genes in the absence of the normal cues. This induction required the presence of Tup1 and wasindependent of the key hyphal regulators Efg1 and Cph1. In the absence of Ahr1, ALS3 and ECE1 were only expressed at intermediate levels. However, this couldpartially be restored by the overexpression of Ahr1’s known interaction partner Mcm1, but MCM1 overexpression alone did not bypass the absence of Tup1.Based on these observations, we conclude that Tup1 is not only important for the repression of ALS3 and ECE1 in yeast cells, but also for their activation in hyphalcells. While interacting with Nrg1 under yeast growth conditions, it is possible that Tup1 and Ahr1 act together under hyphal growth conditions to induce transcription ofALS3 and ECE1. Thus Tup1 represents an important central regulator of these virulence­associated genes, having both, repressor and activator functions.

DHN­melanin of the human pathogenic fungus Aspergillus fumigatusintervene with cell­autonomous defenses of amoebae

I. FerlingI,II, J.D. DunnIII, A. FerlingIV, A.A. BrakhageV, T. SoldatiIII, F. HillmannI

IEvolution of Microbial Interaction, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), Jena, Germany, IIFriedrichSchiller University, Jena, Germany, IIIDepartment of Biochemistry, University of Geneva, Geneva, Switzerland, IVTechnische Schule Heid­Tech, Heidenheim, Germany,VMolecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI), Jena, Germany

Inhalation of conidia from the filamentous fungus Aspergillus fumigatus can cause fatal lung infections in immunocompromised individuals. Highly specific virulencefactors are absent in this fungus, but its ability to withstand innate immune killing is regarded as one of its essential survival strategies. The fungal pigmentdihydroxynaphthalene­melanin covering the conidial surface was found to be a key mediator of survival by targeting LC3­associated phagocytosis and acidification ofphagolysosomes in alveolar macrophages. Considering its natural habitat, it is plausible that such virulence attributes have emerged long before the appearance of innate immune systems of vertebrates to avoidenvironmental predators. To test this hypothesis, we confronted conidia of A. fumigatus with the fungivorous amoeba Protostelium aurantium and found that conidialacking DHN­melanin were killed at higher efficiencies while in the non­fungivorous model amoeba Dictyostelium discoideum, DHN­melanin supported intracellularretention of conidia. Conidia, covered with the green pigment DHN­melanin, were internalized at far lower rates when compared to those lacking the pigment, despitehigh rates of initial attachment. Immediately after uptake of the fungal conidia, nascent phagosomes were formed through sequential membrane fusion and fission events.Using single­cell assays and fluorescent reporter cell lines for the V­ATPase, we could show that acidification of the phagolysosome was transient and correlated to theproduction of reactive oxygen species following the assembly of the NADPH oxidase complex on the surface of the phagosome. Acidification was followed byneutralization, and finally the expulsion of the conidium. The total outcome of the fungal infection for the whole population of the amoebae was further supported by acomputational model which integrated the results of the different dynamics in internalization and phagosome maturation and predicted DHN­melanin to be a crucialintracellular retention factor. We identified autophagy and ESCRT­III as two highly conserved pathways within the cell­autonomous defenses of amoebae that makepotential targets of DHN­melanin indicating that A. fumigatus cannot only escape killing but can also establish latent infections in environmental phagocytes.

Evidence for multiple in vivo selective pressures contributing to MRR1variation in a C. lusitaniae population

E. DemersI, J. StajichII, D. HoganI

IDepartment of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States of America, IIDepartment of Microbiology andPlant Pathology and Institute for integrative Genome Biology, University of California­Riverside, Riverside, CA, United States of America

Management of the limited number of antimicrobials currently available requires the identification of infections that contain drug resistant isolates and the discovery offactors that promote the evolution of drug resistance. Using a unique collection of antifungal naïve Clavispora (Candida) lusitaniae isolates obtained from a singlechronic fungal infection in a subject with cystic fibrosis, we have shown that numerous subpopulations differed in their alleles of a single gene that impacts drugresistance. Analysis of genomes from hundreds of these closely­related C. lusitaniae isolates, through individual and pooled isolate sequencing methods revealed atleast twenty­five nonsynonymous mutations in MRR1, which encodes a transcription factor capable of inducing fluconazole resistance in Candida species. Some ofthese mutations result in hyperactive Mrr1 variants while others result in a decrease or loss of Mrr1 activity compared Mrr1 encoded by the ancestral allele containingnone of the above mutations, suggesting that different conditions within an infection may favor different levels of Mrr1 activity. Retrospective characterization of sputumand lung populations by pooled sequencing found that mutations shown to confer fluconazole resistance, via increased expression of the MDR1­encoded efflux pump,were a minority in each pool, highlighting the potential importance for low Mrr1 activity variants within this infection. While exploring biologically relevant stressorsincluding hydrogen peroxide and diamide we found that while hyperactive Mrr1 variants confer resistance to diamide, they are more sensitive to hydrogen peroxide. Incontrast, we found that low activity Mrr1 variants are sensitive to diamide but have increased resistance to hydrogen peroxide, in some cases exhibiting even higherresistance than isogenic strains complemented with an unmutated MRR1 allele. Further analysis of one MRR1 allele which confers low Mrr1 activity revealed that of thetwo mutations present, the initial mutation alone confers high Mrr1 activity, but that this high activity was subsequently dampened by the secondary mutation whichresulted in Mrr1 truncation. Together these data suggest that multiple, perhaps varying, pressures present in vivo may select for changes in Mrr1 activity and that thiscan consequently affect antifungal resistance within fungal populations which have never been treated with antifungals.

Protein mistranslation modulates hyphal initiation in Candida albicans

C. Oliveira *I, E. LopesI, M. SantosII, S. RochaII, M. SantosI, A.R. Bezerra *I

IiBiMED – Institute of Biomedicine, University of Aveiro, AVEIRO, Portugal, IIMass Spectrometry Centre, Department of Chemistry & QOPNA, University ofAveiro, AVEIRO, Portugal

Translation of mRNA by the ribosome is a high­fidelity biological process whose error rate is globally low, ranging from 10­3 to 10­4 in eukaryotic cells. However, recentstudies show that mistranslation rates in vivo are variable and can increase up to tenfold in response to stress. For instance, production of reactive oxygen species(ROS) by the host in response to pathogens targets the protein quality control machinery by promoting charging of tRNAs with oxidized amino acids. C. albicans takesthese ambiguities to the extreme by translating the CUG codon as both leucine (3%) and serine (97%), using a tRNA that is charged by both seryl and leucyl­tRNAsynthetases. In this study, we show that changes in the environment, including exposure to antimicrobials and macrophages, increases Leu misincorporation levels from3% to approximately 50% and these hypermistranslators display remarkable morphological plasticity. Germ­tube (GT) assays in non­inducing conditions show thathypermistranslator strains have enhanced hyphal initiation compared to wild­type strains. During hyphal initiation, two independent pathways are involved indownregulation of the major hyphal repressor (Nrg1). Transcriptional downregulation requires the activation of the PKA pathway, whereas Nrg1 protein degradationrequires release from farnesol inhibition. Hypermistranslating strains show no alterations in the cAMP­PKA transcription pathway but the pathway involving degradationof the hyphal repressor Nrg1 is altered as hypermistranslating strains produce less farnesol than control strains. Studies are currently underway to understand howmistranslation alters the quorum sensing mechanism of this pathogen in order to decrease the production of farnesol.

This work is supported by the Portuguese Foundation for Science and Technology, POCI­ COMPETE2020 and FEDER through grants SFRH/BD/131637/2017,PTDC/IMI­MIC/5350/2014 and UID/BIM/04501/2013.

* The authors marked with an asterisk equally contributed to the work.

Manipulation of the dendritic cell/T cell interface by the human fungalpathogen Cryptococcus neoformans inhibits antigen­mediated T cellproliferation.

G. DesantiI, P. Seoane *I, E. BallouI, R. MayI

ISchool of Biosciences, University of Birmingham, birmingham, United Kingdom

Cryptococcus neoformans is a globally distributed fungus that mainly infects human beings via the airways. In healthy individuals, C. neoformansis a potent immunogenthat generates a protective and long­lasting adaptive immune response during early childhood. However, several lines of evidence suggest that antigen presentation isdisrupted during an active cryptococcal infection; a finding that may have major implications for coinfections during asymptomatic colonisation. We hypothesize that thisdisruption originates from a C. neoformans­mediated inhibition of dendritic cell (DC) and T cell interactions taking place during an immune response. We loaded GM­CSF mouse bone marrow derived DCs (mBMDCs) with chicken ovalbumin peptide or full protein in the presence or absence of a hypo­proliferative ras1Δ C.neoformans strain (H99 background). CFSE­labelled Ovalbumin­specific CD4 T cells were added to the culture and their proliferation monitored by flow cytometryafter 4 days of co­culture. The presence of IL­13 and IFNγ in these co­culture supernatants were measured by ELISA. We demonstrated that C. neoformans activelyinhibits T cell proliferation following antigen stimulation. This inhibition does not rely on physical competition between C. neoformans and leucocytes and is, at leastpartially, driven by capsule production and shedding. Intriguingly, antigen­driven production of IL­13 and IFNγ is conserved, indicating a specific defect in T cellproliferative responses. We are now investigating the impact of C. neoformanson mBMDCs surface expression of MHC and co­stimulatory molecules and areextending our cytokine expression profile analysis to six other soluble factors in order to map out the molecular impact of Cryptococci on adaptive immunity.

* The authors marked with an asterisk equally contributed to the work.

Studying the role of CgMIP1 in the evolution of Candida glabrata duringadaption to the human host.

S. Siscar LewinI, S. BrunkeII, T. GabaldónIII, B. HubeII

IHans Knöll Institut, Microbial Pathogenicity Mechanisms, Jena, Germany, IIDepartment of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural ProductResearch and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany, IIIDepartment of Bioinformatics and Genomics, Centre for Genomic Regulation (CRG),The Barcelona Institute of Science and Technology, Barcelona, Spain

Candida glabrata is the second most prevalent cause of candidemia worldwide, which is largely due to its high intrinsic and rapidly acquired antifungal resistances.Phylogenetically, C. glabrata is more closely related to Saccharomyces cerevisiae than to the most common pathogenic Candida species, C. albicans. Recentevolutionary studies have shown that the gene CgMIP1 may have been under positive selection during C. glabrata's evolution as a human pathogen. The S. cerevisiaeortholog MIP1 encodes a mitochondrial polymerase. Defective mitochondrial functions lead to petite phenotype, characterized by high fluconazole resistance due to anupregulation of the transcription factor Pdr1 and the efflux pumps Cdr1 and Cdr2.The aim of this study is to elucidate whether the selection pressure on MIP1 is due to the adaptation of C. glabrata to the human host. We created a Cgmip1∆ mutantwhich showed a petite phenotype. We found it to be resistant to endoplasmic reticulum (ER) stresses and to exhibit an upregulation of plasma membrane stressresponse genes, an altered exposure of cell wall components and an increased survival upon phagocytosis by human macrophages. A possible explanation for theresistance phenotype is the upregulation of the genes PDR1, CDR1, and CDR2. However, a Cgmip1∆+pdr1∆ double mutant still shows an intermediate resistantphenotype. These results demonstrate that the loss of mitochondrial function can trigger a response beyond PDR1 upregulation: we surmise that ER and membranestress promotes cell wall remodeling and, in turn, results in the observed different phagocytosis and survival rate of petite mutants. Therefore loss of mitochondrialfunction can confer an at least transient adaptive advantage to C. glabrata during infection. Overall, loss of MIP1 thus seems to be beneficial for C. glabrata undercertain stress conditions.We hypothesize that the fast­evolving CgMIP1 gene is undergoing mutations or epigenetic silencing under stress conditions that trigger transient partial loss ofmitochondria and “switch on” of the resistant petite phenotype, contributing to survival under high stress conditions, like in the human host.

Identifying Novel Antifungal Agents Against the Drug­ResistantPathogen Candida auris

K.R. IyerI, K. CamaraII, M. Daniel­IvadI, R. TrillesII, J.A. PorcoII, L.E. BrownII, L.E. CowenI

IUniversity of Toronto, Toronto, Canada, IIBoston University, Boston, United States of America

Fungal infections are a major contributor to infectious disease related deaths across the globe. Currently, only three classes of antifungal drugs are available for treatmentof systemic infections: azoles, echinocandins, and polyenes. Recently, there has been a global emergence of the fungal pathogen Candida auris. This is cause forconsiderable concern as the majority of C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some are resistant to drugs fromall three antifungal classes. Combination therapy is a promising approach to combat this problem as two compounds can synergize and increase potency, lower hosttoxicity, broaden the therapeutic range, and mitigate the emergence of resistance. By screening chemical libraries in combination with sub­inhibitory concentrations offluconazole, we can identify compounds that are bioactive alone and those that potentiate azole activity against resistant clinical isolates. In particular, when 2,456compounds from a diversity­oriented synthesis collection (BU­CMD collection) were screened, two distinct bioactive chemotypes were identified. The first chemotypedisplays cidal single agent activity against C. auris, and functions by inhibiting translation in C. auris but not in its close pathogenic relative Candida albicans. Thisspecies­specific activity is contingent on a single amino acid change in the drug target, Tif1. Interestingly, these compounds impede translation through a mechanism thatseems to result in apoptosis­like cell death in C. auris but not C. albicans, suggesting divergence in programed cell death pathways between these two organisms. Thesecond chemotype shows low mammalian cytotoxicity and potently abrogates fluconazole resistance against C. auris clinical isolates but not other Candida speciessuch as C. albicans and Candida glabrata. However, this chemotype does not inhibit drug efflux or synergize with other antifungal compounds, suggesting it targets amechanism specific to azole resistance in this pathogen. In order to elucidate mechanism of action, affinity purification coupled with mass spectrometry is being leveragedto identify the cellular target of the chemotype in C. auris. Overall, this work will identify and characterize compounds with novel bioactivity against the inherentlyresistant pathogen C. auris, which will aid in a better understanding of this pathogen and pave the way for new effective therapeutic strategies.

The genetic basis of Candida glabrata persistence within macrophages

S. Brunke *I, D. Fischer *I, B. HubeI,II

IHans Knoell Institute, Jena, Germany, IIFriedrich Schiller University, Jena, Germany

A striking feature of Candida glabrata is its ability to survive inside macrophages and even replicate inside the phagosome. It has been proposed that this allowsC. glabrata to persist in the host for extended periods of time, hidden from other immune effector cells and triggering only a low cytokine response. In fact, C.glabrata, often associated with mononuclear cells, can be isolated even after four weeks from systemically infected mice which are otherwise symptom­free. If the sameis true for human infections, this ability to persist in the presence of phagocytes may be an important contributor to the pathogenicity of C. glabrata, and a worthwhiletarget for closer investigations.We therefore developed a long­term interaction model of C. glabrata with human monocyte­derived macrophages. In this model, we can follow fungal survival insidethe phagocytes for a week and determine the genetic basis of this persistence phenotype. We investigated both the initial transcriptome in the phase leading toward thepersistence and used amplicon sequencing to analyse the survival of a large collection of barcoded deletion mutants.Our initial pool­based screen indicated the involvement of chromatin modifications in C. glabrata's persistence, as well as factors involved in cell wall composition andmRNA stability. We selected individual mutants for more in­depth tests and confirmed that genes with homologs involved in modifications specific for spore cell walls ofSaccharomyces cerevisiae are important for long­term survival of C. glabrata in macrophages. Furthermore, mutants lacking the RNA regulator protein Ssd1 (whichin S. cerevisiae represses translation and determines mRNA localization) are similarly getting depleted over time in the phagosome.These data show that intraphagosomal survival of C. glabrata not only requires the immediate stress response to survive, but also specific genes and adaptations whichallow the fungi to make phagocytes their long­term home.

* The authors marked with an asterisk equally contributed to the work.

Defining arrestin­regulated adaptive cellular responses in fungalvirulence

C. TelzrowI, J.A. AlspaughI

IDuke University, Durham, United States of America

In order to effectively cause disease, pathogens must sense and adapt to dynamic environments. Arrestin proteins are potent regulators of these adaptive cellularresponses. Arrestins are a structurally specialized and functionally diverse group of proteins that modulate intracellular responses to extracellular signals. Previous studieson fungal arrestins have demonstrated their roles in classical arrestin functions, such as G protein­coupled receptor desensitization and membrane protein endocytosis,but the mechanisms by which arrestin­regulated processes are involved in fungal virulence remained unexplored. Based on protein structure homology to humanarrestins, we identified a family of four putative arrestin proteins – Ali1, Ali2, Ali3, and Ali4 – in the human fungal pathogen Cryptococcus neoformans. The arrestinloss­of­function mutants exhibited varying degrees of sensitivity to cell surface stressors and high temperature. Additionally, macrophage co­culture assays indicated thatthe arrestin family supported virulence. To probe these shared phenotypes, we have focused initial studies on Ali1. We observed that Ali1 localized to the septum, andthat the Ali1 loss­of­function mutant displayed cytokinesis defects. These data implicate Ali1 in the regulation of cytokinesis. Based on preliminary co­immunoprecipitation experiments, Ali1 potentially interacted with multiple proteins involved in cell surface synthesis. Collectively, we hypothesize that Ali1 acts as anadaptor protein for enzymes that synthesize and rebuild the cell membrane and cell wall during cytokinesis. Future studies will focus on mechanisms, such asubiquitination, by which Ali1 regulates the localization and function of these enzymes throughout the cell cycle. By functionally characterizing each of the C. neoformansfour arrestin proteins, this work will enhance the basic biological understanding of arrestin functions, as well as establish the active role of fungal arrestins in virulence.

Development of a CRISPR gene editing system for Candida tropicalis

J. Olivera­PachecoI, L. Lombardi *I, G. ButlerI

IUniversity college dublin, Dublin, Ireland

Candida tropicalis has emerged as one of the most clinically relevant Candida species in terms of virulence and epidemiology, particularly in South America. C.tropicalis is a diploid organism, whose genome was originally sequenced in 2009 (strain MYA­3404). C. tropicalis belongs to the CUG­Ser1 clade, which encodesthe CUG codon as serine rather than leucine. This fact, together with the lack of a sexual cycle and the diploid genome, makes generating deletion strains adifficult process, because each allele needs to be targeted independently. With the advent of CRISPR technology, many gene editing tools were developed in many yeastspecies, including some Candidaspecies. We explored the possibility of using replicating plasmids to devise a CRISPR­based gene editing strategy for C. tropicalis.We designed a plasmid that expresses CAS9 and the guide RNA (gRNA), and contains a selectable marker (nuorseothricin resistance). The gRNA is introduced in asingle step by designing two 20 base oligonucleotides, and is released by self­cleaving. Gene editing is carried out in a single transformation step, by introducing theplasmid together with a repair template. The CAS9 gene is expressed only when the plasmid is present, and it can be cured easily from transformed strains. Noselectable marker remains in the genome following the editing step. The system can therefore be used to edit multiple genes in the same isolate, and to edit the samegene in multiple isolates.

* The authors marked with an asterisk equally contributed to the work.

Host immune function impacts genome and ploidy dynamics of Candidaalbicans

M. HickmanI, A. ShurzinskeI

IEmory University, Atlanta, United States of America

Candida albicans is the predominate opportunistic fungal pathogen of humans and causes a broad range of infection. In healthy individuals, superficial C. albicansinfections, such as oropharyngeal and vaginal candidiasis occur, whereas individuals with compromised immune function, are susceptible to severe bloodstreaminfections that result in death 30­50% of the time. A remarkable feature of this diploid fungal pathogen is its highly labile genome, in which large­scale genomeperturbations, including recombination, chromosomal aneuploidy, and whole­shifts in ploidy, are frequent and potentially facilitate adaptation to the host environment.Indeed, many clinical isolates obtained from both healthy and immunocompromised patients are aneuploid or tetraploid. To investigate how immune function impacts C.albicans genome ploidy dynamics, we infected healthy and immunocompromised C. elegans hosts with diploid and tetraploid C. albicans strains of laboratory orclinical origin and subsequently extracted C. albicans for genome analysis. In diploid C. albicans associated with healthy hosts, we detect ~100­fold increase in loss ofa heterozygous (LOH) marker, as well as rampant aneuploidy and even tetraploidy. Furthermore, we detect very rapid ploidy reduction in tetraploid C. albicansextracted from healthy hosts. We tested whether host innate immune function drives C. albicans genome instabitliy by extracting diploid and tetraploid isolates fromimmunocompromised hosts and found that both diploidy and tetraploidy were maintained, and the frequency of LOH was similar to that observed in vitro. Takentogether, these results suggest that host immune function stresses C. albicans and increases genome and ploidy instability which has meaningful clinical and evolutionaryimplications for this important human fungal pathogen.

Using forward genetics to uncover principles underlying macrophagesusceptibility to Histoplasma capsulatum infection

A. CohenI, E. JengII, M. BassikII, A. SilIII

IUniversity of California at San Francisco, San Francisco, United States of America, IIStanford University, Stanford, CA, United States of America, IIIUniversity ofCalifornia at San Francisco, San Francisco, CA, United States of America

Intracellular pathogens employ complex strategies to manipulate their hosts and cause disease. The pathogenic fungus, Histoplasma capsulatum (Hc), successfullyparasitizes macrophages, key effector cells of the mammalian innate immune system. Following internalization via phagocytosis, Hc evades microbicidal effectorfunctions of the macrophage, replicates inside of a modified phagosome, and actively triggers macrophage programmed cell death by apoptosis. We are interested inidentifying host genes that are manipulated by Hc to allow fungal replication and/or host­cell death. To this end, we have conducted a pooled CRISPR­Cas9 basedforward genetics screen in a murine macrophage­like cell line to identify genes required for macrophage susceptibility to Hc infection. Our results suggest involvement ofseveral host­cell processes, including protein maturation in the ER, nutrient stress sensing, and phagosome maturation in macrophage sensitivity to Hc infection. Theseresults point to host pathways targeted by the fungus to successfully parasitize macrophages, and are of high significance to understand manipulation of mammalian cellsby fungal pathogens.

Investigation of the Candida­host interaction using dual RNA­seq

E. IracaneI, H. HovhannisyanII, J.M. Oliveira PachecoI, M. PekmezovicIII, B. HubeIII, T. GabaldónII, G. ButlerI

IUniversity college dublin, Dublin, Ireland, IICentre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003Barcelona, Spain., Barcelona, Spain, IIIHans Knöll Institut, Microbial Pathogenicity Mechanisms, Jena, Germany

Candida species are commensal yeasts but are also responsible of life­threatening infection in at­risk populations, like new­born or immunocompromised patients.Candida albicans is the most common causative species, and the most studied. Moreover, non albicans Candida species as Candida glabrata, Candidaparapsilosis and Candida tropicalis cause a large proportion of infections.In our study we investigated the interaction between four Candida species and human vaginal epithelial cells A431 by using a dual RNA­seq method. Our aim is toidentify the different transcriptomic response of each yeast, and of the host, during the infection of human cells.Gene Ontology analysis of up­regulated genes in the yeasts during infection implicated the ergosterol (ERG) pathway in C. parapsilosis only. We therefore investigatedthe role of the ERG pathway in the three Candida species in which it is currently possible to generate gene knockouts. The transcriptional factor Upc2 is the mainregulator of ERG gene expression in C. albicans and C. parapsilosis. C. glabrata has two UPC2 orthologs, called CgUPC2A and CgUPC2B.We found that deleting CgUPC2A or CgUPC2B or both together does not reduce the damage inflicted by C. glabrata on host cells. However, deleting UPC2 in C.albicans greatly reduces damage. Deleting UPC2 in C. parapsilosis appears to reduce damage of host cells; however further investigation is required.

Our results show the that the role of ergosterol pathway in the host pathogen interaction differs between Candida species.

Building a reporter gene toolkit for the fungal pathogen Cryptococcusneoformans

L. TuckI, G. JanbonII, E. WallaceI

IUniversity of Edinburgh, Edinburgh, United Kingdom, IIInstitut Pasteur, Paris, France

The fungal pathogen Cryptococcus neoformans is the most common cause of fungal meningitis. Although C. neoformans is a unicellular haploid yeast, tools to permitthe investigation of molecular and cellular processes lag far behind those available in more commonly employed yeast model organisms, such as Saccharomycescerevisiae. Having a fluorescent reporter gene system that is easy to design and use is necessary to understand more about how Cryptococcus neoformans grows,and ultimately how it causes disease.Our approach to building a flexible reporter system in Cryptococcus neoformans is to re­engineer a Golden Gate­based modular cloning system (Andreou andNakayama, 2018) for expression in C. neoformans.We efficiently integrate our modular transcriptional units into the C. neoformans genome using a modifiedCRISPR­Cas9 system (Fan and Lin, 2018). We have designed codon­adjusted fluorescent reporters, to maximise the intensity of fluorescence for improved ease ofuse in downstream applications. This system will allow easier study of the cell biology of C. neoformans, interrogation of regulatory sequences such as promoters andterminators, and controlled expression of exogenous genes.

Localization and Function of Histoplasma capsulatum Secreted ProteinEffectors During Macrophage Infection

D. AzimovaI, B. EnglishI, M. VoorhiesI, A. SilI

IUCSF, San Francisco, United States of America

Intracellular pathogens strategically secrete protein effectors to evade detection by the immune system, scavenge nutrients, and spread effectively through the host.Histoplasma capsulatum (Hc) is a dimorphic fungal pathogen of humans that is able to infect and kill macrophages. Within the macrophage, it resides and replicates ina modified phagosomal compartment, and the ultimate outcome of infection is host­cell death. How Hc manipulates the host cell to evade detection and promote killingfrom within the phagosome is not understood. Our work suggests that Hc utilizes secreted protein effectors to achieve a successful infection. We are particularlyinterested in identifying the localization and function of putative secreted effectors during Hc infection.

Hc has a few known secreted effectors but their localization during infection is largely uncharacterized. The most abundant yeast phase­specific protein secretedby Hc is Calcium Binding Protein 1 (Cbp1), a critical virulence factor required for lysing macrophages in vitro and required for virulence in the mouse model of Hcinfection. We used subcellular fractionation of infected macrophages to show that Cbp1 produced by phagosomal Hc yeast cells is able to access the host cytosol.Using mass spectrometric analysis of the cytosol of infected cells, we have also found several additional Hc secreted factors that access the cytosol. To our knowledge,this is the first instance of effectors of an intracellular fungal pathogen of humans being found in the cytosol of the host cell. We are also pursuing the function of novel secreted effectors from Hc. We are particularly interested in a new family of Hc putative small cysteine­rich secretedeffectors that is expanded in Hc and related species. These effectors have a cysteine spacing that closely resembles “knottin” or cysteine­knot proteins. We are mutatingthe Hc knottin proteins and assessing their role in pathogenesis. Taken together, our data implicated small secreted proteins as being key virulence effectors in Hcpathogenesis.

Phenotypic characterization of Candida albicans protein kinases withunknown functions

P. BrandtI,II, B. Ramírez­ZavalaIII, J. MorschhäuserIII, T. WolfIV, M.M. Esfahani IV, G. PanagiotouIV, S. VylkovaI,II

INWG Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany, IISeptomics Research Centre, Leibniz Institute for Natural Product Research and InfectionBiology – Hans Knoell Institute, Jena, Germany, IIIInstitute for Molecular Infection Biology, Julius Maximilians University, Würzburg, Germany, IVSystems Biology andBioinformatics, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Jena, Germany

Adaptation of Candida albicans to different host­relevant environmental conditions, including nutrient availability, pH and stresses is tightly controlled via regulation ofgene expression and posttranslational modifications. Activation and inactivation of genes is often accompanied by phosphorylation or dephosphorylation of proteins byprotein kinases and phosphatases, respectively. C. albicans genome encodes for 108 predicted protein kinases, some of which are crucial for fungal viability orvirulence. Since the exact role of 59 of the kinases is currently unknown, this study aimed to perform a detailed functional analysis of the uncharacterized C. albicansprotein kinases. Of these, some have orthologs to Saccharomyces cerevisiae protein kinases, whereas others are specific to Candida spp.. The set was used in broadphenotypic screen, which included utilization of various nutrients, exposure to host­relevant stress conditions and to chemicals by using the Biolog PhenotypeMicroArrayTM technology. We were able to examine the metabolic activity of the protein kinase mutants under 850 different nutrient conditions and compare them tothe SC5314 wild­type phenotypes. Furthermore, the mitochondrial function and the sensitivity to 42 different cell wall stressors and chemicals were examined. Selectedphenotypes were further validated using standard spot dilution assays. By using the phenotypic microarrays we were able to identify novel phenotypes for theuncharacterized protein kinases, e.g. for strain lacking the gene encoding for the Candida spp.­specific orf19.35, which showed prominent defect in metabolism of keyintermediates of the TCA cycle and reduced mitochondrial activity. The best hit of orf19.35 in S. cerevisiae is Sky1, a protein kinase involved in the regulation ofmRNA metabolism and cation homeostasis. C. albicans has an orthologous gene SKY1, encoding for a predicted protein kinase with unknown function. Thephenotypic screen of C. albicans sky1∆ revealed a broader growth defect compared to orf19.35, suggesting that the two predicted protein kinases are very distinct inorigin and functionality. Further characterization will include epigenetic analysis and phosphoproteomics to identify effector proteins, in parallel to testing their interactionwith host cells and virulence using animal models of candidiasis.

Architecture of in vitro Fungal Morphotypes Dictates Lesion Morphologyand Disease Progression Through a Novel Collagen­Like Protein

C.H. KowalskiI, J.D. KerkaertI, C. NadellII, J.E. StajichIII, R.A. CramerIV

IDepartment of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States of America, IIDepartment of Biological Sciences,Dartmouth College, Hanover, NH, United States of America, IIIDepartment of Microbiology and Plant Pathology and Institute for integrative Genome Biology,University of California­Riverside, Riverside, CA, United States of America, IVDepartment of Microbiology and Immunology, Geisel School of Medicine at Dartmouth,,Hanover, NH, United States of America

While intra­species heterogeneity has become increasingly appreciated in the field of fungal pathogenesis, for many pathogenic fungi, the most obvious and easilyobservable heterogeneous phenotype, that of colony morphology, has yet to be directly linked to aspects of disease progression and clinical outcomes. For the humanpathogen Aspergillus fumigatus, recent work has highlighted intra­species morphotype heterogeneity specifically during low oxygen growth. For A. fumigatus, theability to grow in low oxygen is required for virulence, suggesting a relationship between fungal colony morphology, adaptation to hypoxic stress, and fungal diseaseprogression. To identify the genetic factors that connect these aspects of A. fumigatus biology we utilized an experimental evolution approach in host­like low oxygenconditions. A strain, herein called EVOL20, arose that is more fit in low oxygen, more virulent in mouse models of pathogenesis, and has an intriguing altered colonymorphology; a morphology reminiscent of some clinical isolates of A. fumigatus. Through genome sequencing, we have characterized a hypothetical protein, hrmA(hypoxia­responsive­morphology factor A), that is necessary for the morphology, but is also required for increased virulence, and hypoxia fitness of the EVOL20 strain.We have characterized hrmA and discovered that it is a nuclear­localized regulator of a gene cluster located within a sub­telomeric region. This gene cluster encodes amember of an unstudied collagen­like protein (CLP) family found in bacteria. In A. fumigatus, HrmA drives expression of this CLP, cgnA, resulting in an altered hyphalsurface that prevents extracellular matrix attachment and reduces hyphal interactions within in vitro biofilms and in vivo lesions. Our data indicate that the impact ofhrmA on colony morphology, hypoxia fitness, and virulence are all dependent on cgnA, making this the first CLP studied in human pathogenic fungi known to impactdisease progression and virulence.

The transcription factor Stp2 interconnects amino acid metabolism tobiofilm formation in the fungal pathogen Candida albicans

B. BöttcherI,II, E. GarbeI,II, D. DrieschIII, S. DietrichIV, B. HoffmannIV, M.T. FiggeV, N. Engert­EllenbergerVI, I.D. JacobsenVII, S. VylkovaI,II

IHost­Fungal Interfaces, Friedrich Schiller Universität, Jena, Germany, IIZIK Septomics, Leibniz­Institut für Naturstoff­Forschung und Infektionsbiologie – Hans­Knöll­Institut (HKI), Jena, Germany, IIIBioControl Jena, Jena, Germany, IVApplied Systems Biology, Leibniz­Institut für Naturstoff­Forschung und Infektionsbiologie – Hans­Knöll­Institut (HKI), Jena, Germany, V Applied Systems Biology, Leibniz­Institut für Naturstoff­Forschung und Infektionsbiologie – Hans­Knöll­Institut (HKI), Jena,Germany, VIMicrobial Immunology, Leibniz­Institut für Naturstoff­Forschung und Infektionsbiologie – Hans­Knöll­Institut (HKI), Jena, Germany, VII MicrobialImmunology, Leibniz­Institut für Naturstoff­Forschung und Infektionsbiologie – Hans­Knöll­Institut (HKI), Jena, Germany

The human pathogen Candida albicans colonizes different host niches, where rapid adaptation to various environmental factors is crucial to virulence. Hyphaeformation together with adherence is critical for colonization, infection and biofilm formation. The transcriptional factor Stp2, a regulator amino acid sensing and uptake,is essential for escape from macrophage phagosomes and for full virulence. Environmental signals, including neutral pH and metabolism of alternative carbon sources,can trigger hyphal morphogenesis. Stp2 appears crucial for environmental neutralization and filamentation upon C. albicans growth on amino acids as the sole carbonsource. Therefore, Stp2 could provide an important link between C. albicans amino acid metabolism to virulence­related processes, such as biofilm and hyphaeformation. Indeed, the stp2Δ mutant was hyperfilamentous under nitrogen­limiting conditions, suggesting the activation of starvation responses. In contrast, hyphal lengthwas reduced in RPMI and other host­relevant nutrients, as well as within the macrophage phagolysosome, a glucose­limited environment. Further investigations revealedthat loss of STP2 reduces C. albicans ability to adhere to abiotic polystyrene surfaces under steady and shear flow conditions, but not to human oral epithelia. In thelatter assay, filamentation of the stp2Δ strain was similar to the wild­type pointing towards a niche­specific role of Stp2. Maturation of stp2Δ biofilms on abiotic surfaceswas delayed, but reached wild­type levels by approximation. Since adhesion to abiotic surfaces and hyphal formation are essential for C. albicans biofilm formation, weperformed transcriptomics of stp2∆ mature biofilms and compared them to the SC5314 wild type. Interestingly, beside the Stp2 control of genes involved in amino acidutilization and metabolism, we noted lower expression of hypha­specific genes and adhesins in the stp2Δ biofilms. Currently, we are investigating the impact of thetranscriptional changes on biofilm architecture in vitro and ex vivo. Altogether, our data describe a novel role for the transcription factor Stp2 in initiation andmaturation of C. albicans biofilms, thereby linking carbon utilization and hyphal morphogenesis, processes essential for C. albicans virulence.

Isocyanide biology in Aspergillus fumigatus

N. RaffaI, F.Y. LimI, T.H. WonII, F.C. SchroederII, A. HuttenlocherI, N.P. KellerI

IUniversity of Wisconsin, Madison, WI, United States of America, IICornell University, Ithaca, NY, United States of America

Toxic secondary metabolites produced by Aspergillus fumigatus play a major role in the development of the deadly disease, invasive aspergillosis. Fouruncharacterized isocyanide synthases contained within three biosynthetic gene clusters (BGC) have been recently discovered in Aspergillus fumigatus with two of theclusters being differentially regulated by copper availability. Overexpression of the putative transcription factor present within one of the clusters, xanC, results inincreased expression of the genes present in the xan BGC. Subsequent chemical analysis of culture extracts indicates an accumulation of derivatives of xanthocillin, apreviously isolated molecule that contains a characteristic isocyanide moiety. The overexpression mutant also displays a pigmentation defect consistent with a copperdeficiency that is remediated with copper supplementation. Further data indicates that product(s) of the cluster are secreted and have the ability to bind copper. Giventhese data, we hypothesize that products of the xan BGC are involved in copper homeostasis and may provide protection during toxic encounters with the host immunemachinery.

The Mitotic Cycle of Cryptococcus neoformans Titan Cells

S. AltamiranoI, K. NielsenI

IUniversity of Minnesota, Minneapolis, United States of America

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life­threatening meningitis primarily in immunocompromised individuals. It is one of theleading mycotic killers of individuals with HIV­1. Interestingly, C. neoformans cells form polyploid cells, known as titan cells, during infection. Titan cells arecharacterized by their increase in ploidy level and their enlarged cell size. The enlarged size, along with cell surface alterations, protects titan cells from the host’s immuneresponse. Perhaps most intriguing, titan cells are able to undergo budding and cytokinesis, producing typical­sized daughter cells that are haploid or aneuploid. Titancells and titan daughter cells exhibit increased resistance to fluconazole, nitrosative, and oxidative stress, suggesting that these cells with altered ploidy are critical for hostadaptation. Despite evidence that titan cells are critical during infection, little is known about the mechanisms underlying how titan cells are formed, and how they areable to undergo division to produce daughter cells with reduced ploidy levels. Using time­lapse microscopy with cells expressing fluorescently tagged mitotic markerswe have monitored titan cell mitosis.

Deciphering the role of the Candida albicans transcription factor Stp2using a combinatory Omics approach

E. GarbeI,II, D. DrieschIII, S. VylkovaI,II

INWG Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany, IISeptomics Research Centre, Leibniz Institute for Natural Product Research and InfectionBiology ­ Hans Knoell Institute, Jena, Germany, IIIBioControl Jena, Jena, Germany

The dimorphic human fungal pathogen Candida albicans colonizes several distinct host niches, which confront the fungus with various environmental conditions, likeniche­specific pH or nutrient availability. Therefore, in order to persist in the host, C. albicans has developed a remarkable adaptive potential and metabolic flexibility.One abundant host nutrient source are amino acids, which can be utilized both as a nitrogen and a carbon source by the fungus. The transcription factor Stp2 is crucialfor amino acid utilization by controlling the expression of amino acid permeases. In addition, Stp2 regulates the transcription of ammonium transporters, involved in therelease of ammonia generated as a by­product of amino acid metabolism. This leads to rapid and notable increase of environmental pH and to hyphal morphogenesis,an important virulence factor. Interestingly, a transcriptional profiling of stp2Δ vs. SC5314 wild­type cells grown on amino acids as the sole carbon source, suggests amore global role for this factor, since aside from the prominent shift in amino acid metabolism we observed differential regulation of genes involved in various stressresponses, utilization of other alternative carbon sources and filamentous growth. For example, arginine biosynthesis was activated in the stp2Δ strain compared to thewild­type, while arginine catabolic genes were downregulated. Since it is unclear if and to which extent theses transcriptional changes are reflected on the metabolic leveland to further characterize the metabolic rearrangements in the cell upon growth on different nutrient sources, we currently perform phenotypic microarrays andmetabolomic analysis of wild­type and stp2Δ cells and their extracellular milieu to link the transcriptome data to the actual metabolic state of the cell. Additionally, wecurrently utilize ChIP­Sequencing to understand if the Stp2­dependent regulation of hyphae­induced and stress response genes is direct or indirect. With this method wewill be able to identify the exact binding motif(s) of Stp2 and to monitor potential dynamics in its genomic occupancy under different conditions. Taken together, thiswork will contribute to our understanding about metabolic adaptation in C. albicans and its implications on virulence.

Characterization of Aspergillus­induced Immune Cell­derivedExtracellular Vesicles

A. ZimmermannI,II, F. RivieccioI,II, T. KrügerI, M.G. BlangoI, A.A. BrakhageI,II

ILeibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute (HKI), Jena, Germany, IIFriedrich­Schiller­Universität Jena, Jena, Germany

Exposure to the mold Aspergillus fumigatus is continuous and can cause various diseases, ranging from allergic reactions to severe infections in immunocompromisedpatients. The small conidia of A. fumigatus, with a diameter between 2­3 µm, easily reach the lung alveoli, where they come in contact with epithelial cells, alveolarmacrophages, and neutrophils. The interaction between immune cells and pathogens can lead to intracellular killing of A. fumigatus by macrophages and neutrophils.Importantly, in some instances the fungus is also capable of surviving inside the host by using its many virulence factors. To elucidate and further prevent Aspergillus­associated infections, further knowledge of the pathogenicity is required. Intercellular communication between the pathogen and the host is an essential element indetermining the outcome of the infection as well as signaling between host cells. Besides direct cell­to­cell contact and transfer of secreted molecules like chemokinesand cytokines, a third way for communication centers on biomolecule transport by extracellular vesicles (EVs). This mode of information transfer remainsuncharacterized in Aspergillus infections. In this study we explore the role of EVs during an infection of RAW 264.7 macrophages with A. fumigatus. We established astreamlined system for isolation of spontaneous and infected cell­derived EVs from cell culture supernatant, resulting in pure and consistent EV samples. We nextcharacterized EV size and concentration and performed SEM imaging, where we ascertained that Aspergillus­induced EVs are more abundant after infection.Furthermore, we identified selected cell surface markers present on EVs, and therefore we could show that the isolated EVs are cell­derived. Additionally,transcriptomic and proteomic analyses of spontaneous and infected cell­derived vesicles revealed new insights into the host­pathogen interaction. A more detailedunderstanding of how EVs interact with the host and the pathogen could help to uncover potential biomarkers and novel pathobiology of the fungus.

Fungal Transformers: Tracking a Moving Target

D. ChildersI, G. Mol AvelarI, J. BainI, A. PradhanI, D. LarcombeI, L. ErwigI,II, N. GowIII, A. BrownI

IMRC Centre for Medical Mycology at University of Aberdeen, Aberdeen, United Kingdom, IIGlaxoSmithKline, Immunoinflammation Therapy Area, Stevenage,United Kingdom, IIIUniversity of Exeter, Exeter, United Kingdom

In human hosts, the opportunistic fungal pathogen Candida albicans primarily proliferates in nutrient poor niches, but is exposed to sugars in other niches. Carbonsource sensing regulates several fungal cellular features including, but not limited to, metabolism, cell wall elasticity, and virulence. In addition, yeast cell division exposespathogen­associated molecular pattern (PAMPs) at the cell surface that are known to be immune­stimulatory (e.g. β­glucan). While various host environmental signalsand cell wall stressors have been implicated in PAMP exposure in vitro, little is known about the molecular mechanisms that modulate PAMP exposure. We haverecently shown that lactate, an alternative carbon source present in mucosal niches and produced by activated innate immune cells, acts as a signalling molecule tomodulate β­glucan exposure. Lactate­induced β­glucan masking is driven via a non­canonical signal transduction pathway resulting in a calcineurin­independentactivation of the transcription factor Crz1. However, it remains to be elucidated whether the reduction in β­glucan exposure is the result of PAMP camouflaging by othercell wall components, PAMP modification, or a combination of both processes. Therefore, we have characterized the signalling pathways and downstream effectorsaffecting PAMP exposure in response to different carbon sources and combinations of environmental conditions that C. albicans encounters during transit through hostmucosal and systemic niches. Through proteomics, gene deletion analysis, and pharmacological and biochemical assays we have established a working model to define‘masking’ and the downstream effectors involved in modifying recognition of β­glucan by the host pattern recognition receptor, Dectin­1. We can also show microscopicchanges to the overall distribution of Dectin­1­recognised β­glucan on the cell surface in response to masking conditions as well as alterations to the interactions ofmasked cells with phagocytes. Finally, we are examining the impact of PAMP modulation and its inhibition on disease processes.

PpoA Oxylipins as Modulators of Aspergillus fumigatus Morphogenesis,Development, and Host Interaction

M. NiuI, G. FischerII, M. HenkeIII, J.W. BokI, E. OliwIV, N. KelleherIII, N. KellerI,V

IDepartment of Medical Microbiology and Immunology, University of Wisconsin­Madison, Madison, United States of America, IIDepartment of Genetics, University ofWisconsin­Madison, Madison, United States of America, IIIDepartment of Molecular Biosciences, Northwestern University, Evanston, IL, United States of America,IVDepartment of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden, VDepartment of Bacteriology, University of Wisconsin­Madison, Madison, UnitedStates of America

Oxygenated fatty acids, or oxylipins, are chemical signals widely present in animals, plants, fungi, and bacteria. While the roles of oxylipins have been characterized indepth in animal immune and plant defense systems, the biological significance of fungal oxylipins is yet to be completely understood. Here we elucidate the impact of theoxygenase PpoA and its oxylipin products, 8R­hydroxyoctadecadienoic acid (8R­HODE) and 5,8­dihydroxyoctadecadienoic acid (5,8 diHODE), on Aspergillusfumigatus development and host macrophage encounters. Characterization of the A. fumigatus ppoA deletion and overexpression mutants revealed a significantregulatory role for PpoA in asexual development, conidial metabolism and survival in macrophage encounters. We found that 5,8­diHODE inhibits spore pigmentationand germination but induces hyphal branching in a concentration­dependent manner. Transcriptomic analysis of 5,8­diHODE treated A. fumigatus showed that thisoxylipin affects A. fumigatus cellular development by differentially regulating genes involved in protein and RNA processing and primary and secondary metabolismpathways.

Implementation of a CRISPR­based system for gene regulation inCandida albicans

E. Román I, I. CománI, D. PrietoI, R. Alonso­mongeI, J. Pla AlonsoI

IUniversidad Complutense de Madrid, Madrid, Spain

CRISPR methodology is not only an efficient tool in gene edition but an attractive platform to facilitate DNA, RNA and protein interactions. We have developed andimplemented a CRISPR­based system to regulate gene expression in the clinically important yeast Candida albicans. We have generated by mutagenesis in RuvC­likeand HNH­nuclease domains an inactive allele of S. pyogenes CAS9 and express both Cas9 and dCas9 under the control of the TETOFF system. We validated theabsence of nuclease activity by comparing the ability to induce mutagenesis in ADE2. We were able to show specific repression or activation of the tester gene CAT1,encoding the cytosolic catalase, by expressing fusions of dCas9 to a transcriptional repressor (Nrg1) or an activator (Gal4). We generated strains were a 1.6 kbpupstream regulatory region of CAT1 controls expression of GFP and demonstrate the functionality of the constructs by qPCR, flow cytometry and sensitivity/resistanceto hydrogen peroxide. The expression of different sgRNAs to the promoter show that activation and repression was strongly dependent on the position of the complexin this regulatory region. We have also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of dCas9 with the RNA binding proteinMCP fused to the VP64 activator. The strategy shown here may facilitate analysis of complex regulatory traits in this fungal pathogen

Hypoxia influences Candida albicans – enterocyte interactions

N. Engert­EllenbergerI,II, R. MikolajczykI, E. GresslerI, I.D. JacobsenI,II

IHans Knöll Institute, Jena, Germany, IICenter for Sepsis Control and Care, University Hospital Jena, Jena, Germany

The opportunistic fungal pathogen Candida albicans frequently occurs as commensal in the gastrointestinal tract of humans. While it is known that intestinal epithelialcells are highly sensitive to ischemic hypoxia, the role of hypoxia­mediated damage in the translocation of C. albicans through the intestinal barrier is unclear. Thus, wecharacterised the influence of oxygen on enterocyte­C. albicans interactions in vitro.The susceptibility of enterocytes to C. albicans infection was significantly influenced by oxygen: When enterocytes were shifted from low to higher O2 (reoxygenation),C. albicans­mediated damage increased significantly. In contrast, shifting the cells to lower O2 (hypoxic shock) led to reduced damage. Subsequent reoxygenation afterexposure to hypoxic shock (1 % O2) for 2 h abolished the protective effect of hypoxic shock and increased damage of infected enterocytes.The oxygen levels used in these studies did not significantly affect tight and adherens junction proteins; however, significantly increased ROS and IL­8 levels duringreoxygenation, indicating increased cellular stress, likely contributed to the enhanced susceptibility. Low oxygen and infection induced activation of HIF­1α, andinduction of HIF­1α by chemicals, partially enhanced resistance of enterocytes to infection. However, this protective effect appears to be independent of theantimicrobial peptide LL37, which was induced in all infected cells independent of oxygen. Expression of CEACAM receptors, previously shown to affect C. albicans­enterocyte interactions, was enhanced especially during reoxygenation indicating an increased host response at this condition.C. albicans was also affected by the different O2 levels during interaction with host cells: enterocytes subjected to hypoxic shock, compared to enterocytes constantlycultured at low oxygen, led to enhanced adhesion but reduced invasion and reduced hyphal growth of C. albicans, suggesting that oxygen­mediated changes ofenterocytes also directly influence the fungus. The observed minor but significant reduction in hyphal length partially explains the reduced damage observed duringhypoxic shock. Furthermore, deletion of the central carbon metabolism regulator TYE7 resulted in oxygen­specific changes in virulence. This suggests that oxygen­dependent adaptation of carbon metabolism affects Candida­mediated cell damage.

Heme­Iron Acquisition System of Candida albicans

U. RoyI, S. YaishI, Z. WeissmanI, M. PinskyI, S. MosheI, G. HorevI, F. GlaserI, D. KornitzerI

IThe Ruth and Bruce Rappaport Faculty of Medicine, Technion ­ Israel Institute of Technology, Haifa, Israel

Iron acquisition from host tissues is a particular challenge for the pathogenic microorganism due to the high­affinity iron chelation mechanism developed by the host thatlimit its bioavailability. Like many microbial pathogens, Candida albicans, a human commensal microorganism that can cause life­threatening systemic infections inimmunocompromised individuals, has therefore evolved a mechanism for extracting iron from hemoglobin, the largest iron reservoir in the host. This pathway, conservedin many ascomycota, includes a relay network of secreted and GPI­anchored extracellular proteins containing a CFEM domain, that can extract heme from hemoglobinoutside the cell and transfer the heme across the cell envelope from one CFEM protein to the next, followed by delivery to the endocytic pathway. The CFEMhemophores bind the planar heme molecule on a flat hydrophobic platform located on top of the CFEM domain, with a flexible N­terminal loop securing the heme inplace. A unique aspartic acid residue serves as the axial ligand and makes the heme­iron coordination redox­sensitive. We are using molecular dynamics simulations tomodel the binding and transfer of heme and the protein­protein interactions along the CFEM protein cascade. In addition, to identify the transmembrane heme receptorthat mediates the connection between the extracellular CFEM cascade and the endocytic step, we used phylogenetic profiling. This revealed a new class of plasmamembrane proteins that are essential for heme uptake. Sequence similarity with ferric reductases is consistent with these proteins having heme reductase activity. Inaddition, subcellular localization of the protein, combined with its role in promoting uptake of Zn⁺² mesoporphyrin, a fluorescent heme analog that is redox­inert, suggeststhat this transmembrane protein also functions as a heme receptor mediating its internalization. The molecular details of the fungal heme­iron acquisition pathwaydistinguish it from bacterial heme acquisition systems, indicating that fungi have evolved a novel solution to overcome iron scarcity.

Endogenous morphogenetic signals in Candida albicans

M. PinskyI, Z. WeissmanI, D. AvitanI, H. Bar­YosefI, D. KornitzerI

ITechnion ­ IIT, Haifa, Israel

C. albicans is a human commensal fungus that is able, under a range of conditions, to perform a regulated switch to filamentous morphology, characterized byemergence of a germ tube from the yeast cell followed by mold­like growth of branching hyphae. This transition from yeast to hyphal growth has attracted particularattention, as it has been linked to the virulence of C. albicans as an opportunistic human pathogen. Linear signal transduction pathways have been identified under manyconditions that mediate, upon imposition of external stimuli, the induction of the hyphal transcription program via a number of transcription factors. Among the numeroushyphal­specific genes (HSGs) induced under hyphal growth conditions, a few essential effectors that can be distinguished, chief among them the Cdk1 cyclin Hgc1,which mediates phosphorylation of several key targets in the hyphal morphogenesis apparatus. Ectopic expression of Hgc1 is sufficient to drive hyphal morphogenesis.However, in addition to external stimuli, hyphal morphogenesis can also be induced by internal signals such as partial inhibition of proliferation, which may reflect an inputfrom cellular physiology into the hyphal morphogenetic switch. One candidate for a sensor of cellular physiology is the Cdk1 cyclin Cln3, which is essential for yeast­likeproliferation but inhibits hyphal morphogenesis and HSG expression. In addition, when the hyphal morphogenesis mechanism is blocked by pharmacologicaldepolymerization of filamentous actin, or by pharmacological or genetic inhibition of endocytosis, then HSG expression is reduced as well. Conversely, when the rate ofendocytosis is increased, then both hyphal elongation and HSG expression are increased. This suggests that HSG expression not only drives hyphal elongation but isalso affected by it via internal feedback mechanisms, generating a regulatory loop between the hyphal extension mechanism and the HSG expression program. Tocharacterize endogenous signaling mechanisms, we are analyzing the genetic requirements for ectopic induction of hyphal morphogenesis by overexpression of theeffector Hgc1. We find that this induction still requires the presence of several key transcription factors, consistent with an effect of hyphal extension on HSG expression.The influence of the hyphal extension apparatus on the HSG expression program suggests that, like many development programs, hyphal morphogenesis is maintainedby reinforcing feedback mechanisms.

Novel Mycoviruse of Talaromyces marneffei Enhances Host Virulence byAltering Gene Expression

X. LIUI, L. Susanna K. P.I, L. George C. S.I, C. Franklin W. N. I, F. Rachel Y. Y. I, C. James J. I, Y. Kwok­Yung I, W. Patrick C. Y.I

IThe University of Hong Kong, Hong Kong, Hong Kong

Mycoviruses are found in a wide variety of fungi, from plant pathogenic fungi to human pathogenic fungi. Most mycoviruses cause cryptic infections, while some alter thephenotypes and physiology of their hosts, such as irregular growth, abnormal pigmentation and altered sexual reproduction. The most well­known mycovirus is theCHV1 in Cryphonectrica parasitica and FgV1 in Fusarium graminearum, which both induced hypovirulence of their fungal hosts. However, most of those studiesfocus on plant pathogenic fungi, their biological role and the potential mechanisms by which they may alter fungal virulence, especially in human­pathogenic fungi, islargely unknown. Here, for the first time, in thermal dimorphic fungi, we report the discovery of a novel partitivirus, Talaromyces marneffei partitivirus­1 (TmPV1), inTalaromyces marneffei, which is the most important opportunistic human pathogenic thermal dimorphic fungus in Southeast Asia. It is regarded as one of the world’stop ten most feared fungi, causing highly fatal systemic infection in HIV­infected and immunocompromised patients. TmPV1 was detected in 12.7% (7of 55) clinical T.marneffei isolates. TmPV1 were then revealed as double­stranded RNA (dsRNA) viruses with 2 segments coding RNA­dependent RNA polymerase (RdRp) andcapsid protein. They were showed to belong to a same and novel species of the genus Gammapartitivirus of the family Partitiviridae by phylogenetic analysis. Thepresence of isometric, nonenveloped viral particles with a diameter of 30 to 45 nm was confirmed in TmPV1­infected T. marneffei by transmission electronmicroscopy. qRT­PCR showed that the viral load of TmPV1 in the yeast phase of the infected T. marneffei was much higher (2 to 8 folds) than in the mycelial phase. TmPV1 enhanced the virulence of T. marneffei after T. marneffei was infected with purified TmPV1 particles, in mice model. Mouse challenged with these isogenicTmPV1­infected T. marneffei showed significantly shortened lifetime (P< 0.0001), a higher fungal burden in organs, and more severe inflammation in the lung than micechallenged with isogenic TmPV1­free T. marneffei. Transcriptomic analysis showed that TmPV1 upregulated the expression of some potential virulence factors whiledownregulated the expression of RNA interference (RNAi)­related genes, which may be involved in antiviral defense.

Development and evaluation of a multiplex qPCR assay to detect the sixmajor Candida species causing blood stream infections

A. ArastehfarI, W. FangI,II, F. DaneshniaI, M. LacknerIII, F. HagenI, C. Lass­ FlörlIII, T. BoekhoutI,IV

IWesterdijk Institute, Utrecht, Netherlands, IIDepartment of Dermatology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China,Shanghai, China, IIIDivision of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria, Innsbruck, Austria, IVInstitute of biodiversityand ecosystem dynamics, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands

IntroductionCandida species are considered as one of the major causes of bloodstream infections and are attributable with a mortality rate of ≥40%. C. albicans, C. dubliniensis¸C. glabrata, C. krusei, C. parapsilosis and C. tropicalis are responsible for ~90% of candidemia cases. Prompt identification of Candida species directly fromclinical samples will aid in timely administration of proper antifungal therapy and hence will improve patient outcome. We designed a species­specific, sensitive multiplexqPCR that can identify the major entities in one reaction from spiked serum samples.Materials and methodsTarget regions (rDNA) were retrieved from NCBI, primers and probes were designed on the most stable regions, and to eliminate the cross­reactivity, they werelocated in the regions that were polymorphic with non­target species. One primer­probe set was designed to detect C. albicans, C. dubliniensis, C. parapsilosis andC. tropicalis, while C. glabrata and C. krusei were targeted by two separate primer­probe sets. Initially, our assay was challenged by a specificity test set thatencompassed a diverse range of clinically important yeast species (non­target Candida species, Geotrichum, Malassezia, Saccharomyces, and Trichosporon),filamentous fungi (most predominant species of Aspergillus, Exophiala, Mucor, and Scedosporium), bacterial species (most prevalent species of Bacillus,Enterococcus, Escherichia, and Staphylococcus), and human DNA followed by sensitivity and efficiency assessments. Limit of detection (LOD) was determined forserum samples spiked with the known quantity of the DNA of target species.ResultsOur multiplex qPCR was highly specific and cross­reactivity with non­target yeasts, filamentous fungi, bacterial species, and human DNA were not observed. LOD andlimit of quantification were 0.1 and 1 genome DNA samples of target species. In spiked serum samples (SSS), LOD was 10 genomes.ConclusionResults of in vitro tests are promising, as our multiplex qPCR assay fulfills all basic requirements of specificity and sensitivity. Further evaluations on samples derivedfrom candidemia patients are needed to estimate test performance in the clinical settings.

Exploring the transcriptional landscape of Candida auris in response tomacrophages

P. Miramon *I, A.W. Pountain *I, M.C. Lorenz *I

IThe University of Texas Health Science Center, Houston/Texas, United States of America

Emerging fungal pathogens represent a serious health concern not only in susceptible populations but also in the general public. In the past decade, several reports ofCandida auris have been documented all over the world. This yeast is closely related to the pathogenic but uncommon species Candida lusitaniae (and more distantlyrelated to C. albicans). Despite this, it is particularly aggressive since it can cause invasive bloodstream infections with a poor prognosis, due in part to an intrinsicresistance to azoles and amphotericin B, two of the most commonly used antifungal drugs in the clinical setting. Thus, understanding the interactions of the host whenchallenged with C. auris is of paramount importance.

We assessed the fitness of a collection of C. auris isolates (comprising members of the four different clades reported to date) and related species (C. lusitaniae, C.haemulonii, C. doubushaemulonii) during in vitro growth in host­relevant conditions. Initially, we observed a wide range of phenotypic variance on glucose. Thisprompted us to test the ability of these isolates to grow on alternative carbon sources, since previous reports from our lab and others strongly suggest that thesecompounds play a pivotal role during interactions with the host. Interestingly, when compared the different C. auris isolates to C. lusitaniae, most of them sustained amore robust growth on amino acids. This phenomenon correlates also with the ability to neutralize the acidic extracellular environment, as previously described for C.albicans.

Transcriptional profiling of C. auris in response to phagocytosis by bone marrow derived macrophages showed conserved trends seen in other Candida spp.: inductionof alternative carbon utilization and oxidative stress response, whereas protein synthesis is halted. Additionally, genes involved in antifungal drug responses (ABCtransporters, MFS and transcription factors) and sterol metabolism were also induced in response to co­incubation with macrophages. Recent reports have shown genefamily expansions of siderophores and oligopeptide transporters, some of which were also induced in our experiments. Validation of these induced genes is in progressusing a C. auris adaptation of CRISPR gene editing technology.

* The authors marked with an asterisk equally contributed to the work.

A functional annotation of Aspergillus fumigatus transcription factors

N. van RhijnI, T. FurukawaI, M. BromleyI, E. BignellI

IUniversity of Manchester, Manchester, United Kingdom

Aspergillus fumigatus causes 90% of all Aspergillus­related infections. It has been demonstrated that virulence of A. fumigatusis multifactorial and several virulencefactors have been identified. In particular, the ability of the fungus to respond to various host­imposed stresses has repeatedly been proven to be essential forpathogenicity. Therefore, regulation of genes by transcription factors plays a critical role in establishing infection. However, we have a limited understanding of themolecular basis of A. fumigatus responses to environmental stress.

In this study, we carried out systematic phenotyping of a transcription factor knockout (TFKO) collection to define and characterize adaptation to host imposedstresses. A high throughput analytical pipeline was developed in a liquid microculture 96­well plate format to analyze relative fitness under infection­related stressconditions of 479 A. fumigatus TFKO mutants. Fitness was assessed in the presence of iron starvation or hypoxia, at elevated temperature and in response to alkalinestress. This resulted in the identification of 11 previously uncharacterized transcription factors.

A highly interconnected network for transcription factors regulating stress adaptation was identified, notably involving 16 of the 22 transcription factors previouslyreported to drive murine pathogenicity.

Hypothesising that tolerance of host­imposed stresses might explain the dominance of A. fumigatus as a pathogen compared to lesser pathogenic Aspergillus spp., weapplied the high throughput parallel fitness analysis to a panel of 40 Aspergillus isolates, representing 5 species and accrued from diverse regions of the globe.Concordant with our hypothesis, fitness in response to host­imposed stress was found to correlate directly with virulence amongst species in the genus.

Roles of the YEATS domain histone acylation reader Taf14 in Candidaalbicans

Q. WANGI, J.V. GaurI, N. VidanI, Y. WANGI, A. TravenI

IDepartment of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia

Chromatin regulation has been reported to contribute to fungal pathogenesis, as inactivating mutations of histone acetyltransferases (HATs) or histone deacetylases(HDATs) cause decreased virulence of the human fungal pathogen Candida albicans. In addition to acetylation, these enzymes are able to modify histones with otheracyl marks, such as histone crotonylation (HKcr), a recently discovered active mark that, similarly to acetylation, leads to gene transcription. Histone crotonylation is“read” by the YEATS domain, which is highly conserved from humans to yeast and found in subunits of chromatin remodelling complexes and general transcriptionfactors. Although histone crotonylation is presumed to be controlled by metabolic and developmental signals, its cellular roles are poorly understood, and the functionsof the YEATS proteins have been studied very little outside of humans and the model yeast Saccharomyces cerevisiae. We performed a comprehensive bioinformaticanalysis of YEATS domain proteins in fungi, including human fungal pathogens, which revealed that the YEATS domain is conserved, and pathogens encode 1 or 2YEATS proteins. Our characterisation of the C. albicans YEATS proteins Taf14 and Yaf9 revealed important roles in growth, stress response and virulence­relatedprocess, and our data suggests that Taf14 has both YEATS­dependent and YEAST­independent roles.

Evaluation of the in vitro antifungal activity of the Astronium urundeuvaplant extract against Candida species

B. Vidal BonifacioI,II, T. VilaIII, P. Bento da SilvaI, I. SilvaI, E. de Oliveira LopesI, L. Perez de SouzaI, W. VilegasIV, F. PavanI, J.L. Lopez­RibotII, T. BauabI

ISao Paulo State University (UNESP), Araraquara, Brazil, IIThe University of Texas at San Antonio (UTSA), San Antonio, United States of America, IIIUniversity ofMaryland, Baltimore, United States of America, IVSao Paulo State University (UNESP), São Vicente, Brazil

The increasing number of fungal infections together with the emergence of species and strains with enhanced resistance to conventional antifungals highlight the need fornew approaches to the discovery of novel antifungals. The plant extract from Astronium urundeuva Engl. (Anacardiaceae), a traditional medicinal plant widely found inSouth America, was tested against C. albicans SC5314 and C. glabrata ATCC 2001. In vitro experiments evaluated the plant extract´s antifungal activity againstplanktonic cells and biofilms (both preformed biofilms and inhibition of biofilm formation), and the XTT­reduction assay was used to measure the metabolic activity ofcontrol and treated samples. Subsequently, we evaluated its ability to induce the development of resistance, which was performed by serial passage experiments over an8 week period where aliquots of cultures were transferred daily into a fresh culture containing established concentrations of the extract or fluconazole. We alsoperformed cytotoxicity assays with three different cell lines (VERO, MRC5 and HaCaT) and an alternative model of Artemia salina L. Results against planktonic cellsdemonstrated the antifungal activity of the plant extract against both Candida species, although the extract was particularly potent against C. glabrata (MIC9031.25µg/mL) in comparison to C. albicans (MIC90 125­250µg/mL). Although preformed C. albicans biofilms displayed high levels of resistance to the extract (MIC501000µg/mL), it interestingly inhibited biofilm formation at the same concentration (MIC90 125µg/mL) as growth of planktonic cells. Similarly, the extract was also moreeffective in preventing C. glabrata biofilm formation, as compared to its activity against preformed biofilms formed by this species. In contrast to fluconazole, whichresulted in the development of resistance in both C. albicans and C. glabrata (MIC90 greater than 128µg/mL after 56 days), neither species became resistant to theplant extract after repeated exposure (MIC90 maintained at 250 and 31.25µg/mL, respectively, from week 1 to 8). Cytotoxicity assays (IC50) showed very lowtoxicity of the extract for all cell lines tested, and lack of toxicity was also confirmed in the alternative Artemia salina L. assay. In conclusion, the A. urundeuva extractdisplays promising antifungal activity and merits further investigation as an alternative therapy for treating infections caused by Candida species.

Improving CRISPR­Cas9 gene editing in Candida parapsilosis

L. LombardiI, L. SalzbergI, E. Ó CinnéideI, C. O’BrienI, G. ButlerI

IUniversity college dublin, Dublin, Ireland

We recently developed a plasmid­based CRISPR­Cas9 gene editing system in C. parapsilosis. We have now modified the system so that only one cloning step isrequired; sgRNAs are self­cleaved between tRNA and a ribozyme. We have used this system to systematically edit approximately 170 genes. We also used the pCP­tRNA system to incorporate heterozygous mutations based on previous work in mammalian cells. The C. parapsilosis strain CLIB214 was transformed with pCP­tRNA­CP101060, expressing Cas9 and an sgRNA targeting CPAR2_101060, and either one of two repair templates RTs (strategy 1), or a mix of two RTs (strategy2). Strategy 1 exploits the observation that in mammalian cells the efficiency of homology driven recombination (HDR) decreases with increasing distance from the Cas9cut site. We designed RTs carrying a codon change that disrupts the PAM (Ile154Gly substitution), and also contained one synonymous C>G SNP either 10 bp or 19bp upstream of the cut site. From 7 transformants obtained with the first RT, all contained the amino acid change in both alleles, and they were all homozygous (either Cor G) at the locus ­10 bp from the cut site. Using the second RT, all transformants again contained the amino acid change in both alleles, but 1 out of 6 colonies washeterozygous for the additional SNP, 19 bp from the cut site. The mutation­to­cut site distance can therefore be exploited to generate heterozygous mutations. Instrategy 2, two RTs were supplied simultaneously, both designed to introduce SNPs to disrupt either the PAM or the protospacer, and one RT designed to alsointroduce a stop codon immediately upstream of the PAM. Incorporation of both RTs should generate a functional heterozygote, in which one allele has a stop codonand one does not. PCR and sequence analysis showed that both RTs were incorporated in 2 out of 16 colonies tested. In conclusion, our results showed that twodifferent strategies can be used in combination with the pCP­tRNA plasmid to introduce heterozygous mutations in C. parapsilosis.

Gut microbiota­mediated colonization resistance against Candidaalbicans

S. VielreicherI, W. Krüger *I, I.D. JacobsenI

IMicrobial Immunology, Leibniz Institute for Natural Product Research and Infection Biology ­ Hans Knöll Institute (HKI), Jena, Germany

Candida albicans is a common member of the human microbiome and present on different mucosal surfaces of healthy individuals. Changes in gut environment, forexample due to antibiotic treatment, can lead to enhanced fungal proliferation, and in combination with immunosuppression result in superficial or systemic infections.Disseminated infections by C. albicans have become a major health risk factor over the last decades especially in intensive care units. The fact that antibiotics are amajor risk factor and recent research highlights the critical role of the bacterial microbiota in controlling C. albicans colonization and translocation. The underlyingmechanisms, as well as the types and features of bacteria involved in colonization resistance, are however only incompletely understood.

Aim of the project is to address the role of the microbiota during colonization and translocation of C. albicans. To determine which members of the microbiota influenceC. albicans colonization, and to elucidate whether diverse microbiota populations mediating colonization resistance share distinct features, we will determinecolonization resistance of C57/BL6 mouse colonies with different microbiome compositions. To identify suitable colonies, we have collected feces from differentacademic and commercial mouse facilities. Quantitative analysis of overall bacterial and fungal burdens by qPCR of gDNA isolated from feces demonstratedconsiderable variation in fungal burden between the facilities. ITS and 16S sequencing has been performed and the results are currently analyzed bioinformatically.Colonization experiments with these mice are under way.

Thereby, this project will provide new insights into the interaction network between C. albicans and the other members of the gut microbiome.

* The authors marked with an asterisk equally contributed to the work.

Mutational landscapes in in­vitro evolved echinocandin resistant Candidaglabrata

E. KsiezopolskaI, R. BeyerII, C. SchüllerII, T. GabaldonI

ICentre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain., Barcelona, Spain, IIUniversityof Natural Resources and Life Sciences, Vienna (BOKU), DAGZ, (Department of Applied Genetics and Cell Biology),Bioactive Microbial Metabolites Facility,Vienna, Austria

Invasive fungal infections are a major cause of global morbidity and mortality, and Candida species are the most significant contributors. Nowadays, Candida albicansis still the most common infecting species, yet there is an epidemiological shift towards non­Candida albicans Candida species like Candida glabrata or Candidaparapsilosis. Currently, there are only four approved antifungal drug families to treat humans, and, similarly to antibiotic resistance among bacteria, the number of strainsresistant to one or several antifungal drugs is increasing. This issue is particularly problematic in C. glabrata, which is intrinsically less susceptible to azoles and easilyacquires resistance to azoles and/or echinocandins. Although resistance towards echinocandins has been attributed to mutations in the drug target genes: FKS1 andFKS2, we still have a very poor understanding of whether alternative resistance­conferring mutations exist and what mutational paths lead to the acquisition ofresistance. In order to gain further insights of how resistance evolves in C. glabrata, we performed an experimental evolution experiment. First, susceptible strains wereexposed to increasing concentrations of anidulafungin (ANI, echinocandin), fluconazole (FLZ, azole) or both combined (ANI+FLZ). Secondly, evolved strainsexhibiting decreased susceptibility to FLZ were subsequently evolved in increasing concentrations of ANI (FLZinANI). As preliminary results, we would like to presentthe data on susceptibility tests and the increase of minimal inhibitory concentrations (MIC) of the drugs for the evolved samples. Most strains reached stable resistance(as measured with EUCAST protocol), although differences were observed between strains from different genetic clades. Sequencing of the mutational hotspots ofFKS1 and FKS2 genes revealed that most of the strains acquired non­synonymous mutations in these regions. Our results define a catalogue of possible resistance­conferring mutations in these hotspots together with their relative frequency in a large number of parallel adaptation experiments. Further, whole genome sequencing ofselected strains will help us define the mutational paths that lead to the acquisition of resistance.

Targeting the secretory pathway with a novel anti­cryptococcal molecule

S. BeattieI, T. MuranteII, D. KrysanI

ICarver College of Medicine, University of Iowa, Iowa City, IA, United States of America, IIUniversity of Rochester, Rochester, NY, United States of America

Studies have shown that fungal clearance from the cerebrospinal fluid through the use of fungicidal drug cocktails is associated with better outcome for cryptococcalmeningitis patients. Therefore, we are focused on identifying and developing anti­cryptococcal drugs that are fungicidal rather than fungistatic. Previously, we identified anovel anti­cryptococcal molecular scaffold using the adenylate kinase assay to screen for fungi­lytic drug activity targeted to the cell wall. These compounds contain abenzothiourea (BTU) scaffold, which is integral to the fungicidal activity of the molecules, and inhibits the activation of the cell wall integrity pathway (CWIP) in responseto cell wall stress. The BTU scaffold is highly specific for Cryptococcus and has almost no activity against Candida or Saccharomyces. In addition to inhibition of theCWIP, BTU inhibits major virulence factors of C. neoformans, including capsule and melanin production, growth at 37°C and is active against phagocytosed yeast.Our hypothesis is that the fungicidal activity of BTU is a result of secretory pathway inhibition. This potential mode of action is supported by the observation that theeffect of BTU on melanization is due to decreased localization of the key melanization enzyme, laccase, at the plasma membrane, and is not due to inhibition laccaseenzyme activity. By TEM analysis, BTU­treated cells accumulate late Golgi sized vesicles (80­200 nm). Further supporting our hypothesis, acid phosphatase secretionwas inhibited by BTU with an IC50 similar to its MIC while a BTU analog with no antifungal activity did not inhibit acid phosphatase secretion. While work is ongoing toidentify the molecular target of BTU, our data indicate that secretory pathway inhibition is the likely mode of action for BTU. The fungicidal activity of the BTUs alongwith their ability to inhibit virulence factors make the scaffold particularly attractive for further development as an anti­cryptococcal therapy.

The cell polarity GTPase, Rsr1, plays unexpected roles in mitotic exit,nuclear migration and vacuole fusion in Candida albicans

T. BedekovicI, A. BrandI

IUniversity of Aberdeen, Aberdeen, United Kingdom

The formation of invasive hyphal filaments contributes to fatal levels of sepsis and organ failure in systemic disease caused by Candida albicans. Hypha­mediatedtissue damage depends on directional growth responses that are regulated by the small N­Ras­like GTPase, Rsr1. In S. cerevisiae, Rsr1 is a K­Ras and specifies theyeast bud site where it is activated by its guanine­nucleotide exchange factor (GEF), Bud5, and de­activated by its GTPase activating protein (GAP), Bud2. However,its role and regulation in C. albicans hyphae are less well understood. We generated mutant strains expressing a single YFP­tagged copy of Rsr1 in its inactive GDP­locked form (Rsr1K16N) or constitutively active GTP­locked form (Rsr1G12V). Here we show that YFP­Rsr1K16N and YFP­Rsr1G12V are differentially localised.Phenotypic analyses of these strains suggest that Rsr1 couples the cell cycle with polarised growth as the null mutant is multinucleate and forms double or aberrant septa.Thus, the function of the C. albicans N‑Ras­like GTPase, Rsr1, extends beyond regulating directional growth through Cdc42 and is involved in regulating thepositioning of additional factors important for cell division and organelle inheritance.

Dectin­1 signaling in polarizing different subsets of CD4+ T cells.

S. OhI, D. UnderhillI

ICedars­Sinai Medical Center, Los Angeles, California, United States of America

Dendritic cells bridge innate and adaptive immunity by directing differentiation of naïve T­helper cell into specific lineages of effector T cells through presenting antigen,stimulating co­stimulatory molecules and creating an appropriate cytokine milieu. Activated dendritic cells can induce different subsets of T helper cells including Th1,Th2, Th17, Th9 lineages. Little is known about the factors contributing to Th9 polarization or whether commensal fungi regulate the Th9 response, and in this study, wehave examined the mechanisms by which dendritic cells sense C. albicans and direct Th9 polarization. We have observed that DCs stimulated with the hyphal C.albicans induce stronger Th9 polarization than the yeast form, and that DCs stimulated with hyphae polarize Th9 responses without requiring commonly­used Th9polarizing culture conditions (i.e. supplementing TGF­β and IL­4). Like previously­characterized ability of C. albicans­stimulated DCs to induce Th17 cells, the abilityof yeast and hyphae to induce Th9 cells is dependent on activation of Dectin­1, a receptor for b­glucan in fungal cell walls that is essential for protection against manyfungal infections. Polymorphisms in the genes for Dectin­1 and CARD9, an adaptor protein required for Dectin­1 signaling, are associated with increased susceptibilityto fungal infection. We hypothesized that the size difference between hyphal and yeast forms of C. albicans may contribute to their different capacities to polarize Th9cells. To test this, we stimulated DCs with different sizes of beads (3, 6, 15, 25, and 45 µm) coated with β­glucan prior to co­culture with naïve T helper cells. DCsstimulated with larger beads (≥15 µm) stimulated significantly more Dectin­1­dependent Th9 T cell polarization compared to DCs stimulated with smaller beads. Thisresult implies that DCs may recognize the sizes of microbes and that this might be an important factor in defining the nature of the immune response. In ongoing studieswe are examining the mechanisms of Dectin­1 stimulated polarization of Th9 subtypes as well as the pathogenesis of Th9 driven disease. These studies may be helpful indeveloping novel therapies for fungal infections as well as for manipulating host responses to commensal fungi that may influence immune responses in allergy,inflammatory bowel disease, and cancer therapy.

Developing a novel anti­fungal strategy by targeting an epigenetic readerdomain against Candida glabrata

K. WEII,II, N. DupperIII, Y. ZhouIII, M. ChamplebouxIV, F. MiettonI, C. GarnaudV, M. CornetV, C. E. McKennaIII, J. GovinII, C. PetosaI

IInstitute de Biologie Structurale, CNRS/CEA/UGA, 71 Avenue des Martyrs, Grenoble, France, IIInstitute for Advanced Biosciences, Université Grenoble Alpes,Inserm, Grenoble, France, IIIUniversity of Southern California, Department of Chemistry, Los Angeles, United States of America, IVInstitut de Biosciences etBiotechnologies de Grenoble, Laboratoire Biologie à Grande Échelle, Université Grenoble Alpes, CEA, Inserm, Grenoble, France, VLaboratoire de Parasitologie­Mycologie, Institut de Biologie et Pathologie, CHU Grenoble Alpes, Grenoble, France

Candida glabrata is a pathogenic yeast associated with a high rate of morbidity and mortality in immunocompromised patients. An alarming rise in drug­resistant strainsover the past decade has led to an urgent need for novel therapeutic strategies. This project concerns a potential new antifungal strategy that targets the protein Bdf1, amember of the BET family of transcription factors. BET proteins associate with chromatin through their two bromodomains (BD1 and BD2), epigenetic "reader"modules that recognize acetylated lysine residues in histones. The overall goal of this project is to validate Bdf1 bromodomains (BDs) as an antifungal drug target in C.glabrata. Specific objectives are to: (i) verify that Bdf1 BD function is essential in C. glabrata; (ii) identify and characterize compounds that selectively inhibit Bdf1BDs; and (iii) demonstrate their potential for translational development into a novel class of antifungal drugs using a mouse model of invasive candidiasis.Thus far, I have shown that the two BDs of Bdf1 are essential for the growth of C. glabrata in vitro. I determined crystal structures of Bdf1 BD1 and BD2, whichreveal that their ligand binding pockets are stereochemically distinct from those of human BDs. I developed a homogeneous time­resolved FRET (HTRF) assay toidentify Bdf1 BD inhibitors and used it to screen a library of >100,000 compounds. This identified several compounds that selectively inhibit the fungal BDs comparedto the human homologs. Current work is aimed at characterizing these inhibitors (and related analogs) in biochemical, biophysical, and fungal growth inhibition assaysand at determining crystal structures of inhibitor­bound BDs. The most promising inhibitors will be then tested for antifungal activity in a mouse model of invasivecandidiasis.Successful completion of this work will establish proof of principle that BET inhibition is a valid antifungal therapeutic strategy in C. glabrata.

Out of Control – Lack of the Histone Chaperone Hir1 Drives Candidaalbicans Hypervirulence

S. JenullI, F. ZwolanekI, M. RiedelbergerI, M. TschernerI, F.G. S. SilaoII, P.O. LjungdahlII, K. KuchlerI

IMedical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria, IIDepartment of Molecular Biosciences, The Wenner­GrenInstitute, Stockholm University, Stockholm, Sweden

Candida albicans is an extraordinary example of how a commensal fungal pathogen can sense and integrate host signals to allow for rapid adaptation and survivalwithin distinct host niches. Subtle transcriptional changes can be linked to altered chromatin organization, which profoundly affects fungal responses to changingenvironments. For instance, the C. albicans yeast­to­hyphae transition involves dynamic transcriptional modulation and chromatin re­modeling. We have shown that theHIR histone chaperone complex, which facilitates replication­independent histone deposition onto chromatin, affects transcriptional regulation of morphogenesis genes.Ablation of Hir1 decreases the sensitivity to morphogenetic signals, resulting in impaired hyphal formation. HIR1­deficient cells show markedly decreased transcriptionalamplitudes of both repressing and activating genes during hyphal initiation. Here, we show that in addition, loss of HIR1 dramatically enhances extracellular proteolyticactivities during limited nitrogen availability, which is caused by a hyperinduction of secreted proteases. These data suggest that Hir1 modulates the fine­tuning oftranscriptional responses to distinct environmental triggers. Thus, we hypothesized that defective HIR­mediated chromatin assembly may transcriptionally deregulatevirulence genes, thereby affecting C. albicans fitness in vivo. Strikingly, hir1Δ/Δ cells display a dramatic hypervirulence phenotype in a mouse model of disseminatedcandidiasis. Hypervirulence of hir1Δ/Δ is accompanied by a dramatic increase in fungal kidney burdens, indicating that the loss of HIR1 facilitates fungal growth in vivo.Most unexpectedly, neutrophils, in contrast to monocytes or macrophages, are not efficiently recruited to kidneys infected with hir1Δ/Δ cells. Neutrophils might be“blind” towards the pathogenic signals originating from hir1Δ/Δ cells which accounts for the robust fungal growth observed in vivo. We propose that hir1Δ/Δ cells alterfungal immune surveillance by selectively impairing recruitment or activation of neutrophils. Together our data suggest that the loss of Hir1­mediated chromatin dynamicsimpair fungal clearance by reducing selective inflammatory responses below threshold levels in the host.

Increased fungal specificity of novel FK506 analogs confers both in vitroand in vivo antifungal efficacy

M.J. HoyI, D. Fox IIIII, J. WheatonIII, H. LeeIV, M. MutzV, W.J. SteinbachI,VI, J. HongIV, M. CiofaniIII, J. HeitmanI

IDuke University, Department of Molecular Genetics and Microbiology, Durham, NC, United States of America, IIBeryllium Discovery, Bainbridge Island, UnitedStates of America, IIIDuke University­ Department of Immunology, Durham, NC, United States of America, IVDuke University­ Department of Chemistry, Durham,NC, United States of America, VRoche Venture Fund, San Francisco, United States of America, VIDuke University Medical Center­ Pediatrics, Durham, United Statesof America

Invasive fungal infections caused by Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans total more than 1.6 million cases per year with up to95% mortality. Although many antifungal drugs are used clinically, new antifungal drug targets are uncommon. The natural product, FK506, inhibits calcineurin which is aserine­threonine specific phosphatase and a key virulence factor in pathogenic fungi. However, FK506 is not used to treat fungal infections due to theimmunosuppressive effect of inhibiting human calcineurin function. In fact, FK506 is commonly used clinically to immunosuppress patients receiving solid organtransplants. In both fungi and mammals, FK506 binds to the immunophilin, FKBP12, and then binds calcineurin and inhibits its activity by blocking binding to substrates.Although mammalian and fungal FKBP12 are highly conserved, we have identified a key differential residue in the 80s loop proximal to the FK506 binding pocket thatcan be exploited to design FK506 analogs with increased fungal specificity.

One FK506 analog, APX879, differs from FK506 by a single chemical substitution. APX879 retains antifungal activity in vitro, and in vivo reduces fungal burden andextends animal survival in a murine inhalation model of cryptococcosis. The structure of C. neoformans calcineurin­FK506­FKBP12 suggests that the moiety modifiedin APX879 will not affect its binding to FKBP12, but instead will alter how it interacts with calcineurin. Importantly, compared to FK506, APX879 exhibits a markeddecrease in immunosuppressive activity assessed by IL­2 production in primary T cells. Additional FK506 analogs modified at the same site maintain antifungal activitysimilar to APX879 and provide a platform for probing the differences between the fungal and human ternary complexes. By utilizing a combination of structural biologyand medicinal chemistry, calcineurin inhibitors can be generated with an improved therapeutic window due to decreased action on the host.

The candidalysins are a family of cytolytic fungal toxins

J. RichardsonI, R. BrownI, A. TurkovaII, I. KabelkaII, G. CarranoI, N. KichikI, S. TangI, D. MoyesI, D. WilsonIII, B. HubeIV, R. VáchaII, J. NaglikI

IKing's College London, London, United Kingdom, IIMasaryk University, Brno, Czech Republic, IIIUniversity of Aberdeen, Aberdeen, United Kingdom, IVHansKnoell Institute, Jena, Germany

Candida albicans is a commonly encountered opportunistic fungal pathogen of humans responsible for mucosal and life­threatening systemic ailments that contribute tohigh morbidity and mortality worldwide. Infection of mucosal surfaces by C. albicans requires candidalysin, a secreted cytolytic peptide toxin that causes epithelialmembrane perturbation and activates host innate immunity. However, despite the prevalence of C. albicans in human mucosal infections, candidiasis is also caused byseveral non­albicans species of Candida fungi. Here, we performed a screen of fungal genome assemblies to identify candidalysin toxins in the fungal pathogens C.dubliniensis and C. tropicalis and characterised their activities on TR146 oral epithelial cells. While all toxins induce membrane perturbation, cause calcium influx, andactivate intracellular (p38/c­Fos) signalling and cytokine secretion, the candidalysin of C. dubliniensis and C. tropicalis exhibit increased potency when compared tothe toxin of C. albicans. Molecular dynamic simulations modelling wild type C. albicans candidalysin and its interaction with a 1,2­dioleoyl­sn­glycero­3­phosphocholine (DOPC) lipid membrane suggest that the toxin adopts a kinked conformation that forms a U­shaped pore­like structure within the hydrophobicmembrane environment. Importantly, mutational analysis of conserved amino acid residues reveals differences in candidalysin activity between species, suggesting thatthese toxins may exert their effects on epithelial cells via subtly different mechanisms. Collectively, these findings identify a conserved epithelial response to thecandidalysin family of cytolysins and reveal important differences in potency between toxins.

Hyperactivity of the zinc cluster transcription factor Rha1 of Candidaalbicans induces filamentation.

R. Parvizi OmranI, V. DumeauxI, S. Haji kazem niliI, M. HallettI, J. MorschhäuserI, M. WhitewayI

IConcordia University, montreal, Canada

Zinc cluster transcription factors are important fungal specific regulators. In the fungal pathogen Candida albicans, they control processes ranging from metabolism andstress response to mating and virulence. The role of CaORF19.1604 has been investigated for its role in regulating cell and colony morphology. Activation ofCaORF19.1604, which we have named RHA1 for regulator of hyphal activity, leads to a wrinkled colony morphology, cellular filament formation and invasivenessunder yeast growth conditions. Cells with the activated transcription factor are also sensitive to wall modifying agents such as Congo red and the echinocandinscaspofungin and anidulafungin, but show normal sensitivity to fluconazole. RNA seq analysis of the activated allele shows up­regulation of classic hyphal specific genessuch as ALS1, ALS3, ECE1, and HWP1. In addition, genes encoding other transcription factors implicated in hyphal development such as BRG1 and UME6 are up­regulated in cells with activated Rha1. Deletion of RHA1 does not significantly effect hyphal development triggered by different conditions including serum, spider,RPMI media and growth at 37°C in YPD. The cellular and colony morphology changes triggered by activated Rha1 are dependent in part on Brg1 and Ume6, asdouble mutants show reduced filamentation relative to the strain containing only the activated allele of Rha1. This suggests that Rha1 may act upstream of these hyphal­regulating TFs to control hyphal development in response to a variety of external signals.

The effects of Candida cells on oral squamous cell carcinoma cell lines

M. VadovicsI, N. IgazII, É. VeresI, D. AdameczII, R. AlföldiIII, L. NagyIII, L. PuskásIII, C. VágvölgyiI, M. KiricsiII, A. GácserI,IV

IDepartment of Microbiology, University of Szeged Interdisciplinary Excellence Centre, Szeged, Hungary, IIDepartment of Biochemistry and Molecular Biology,University of Szeged, Szeged, Hungary, IIIAvidin Ltd., Szeged, Hungary, IVMTA­SZTE "Lendület" Mycobiome Research Group, University of Szeged, Szeged,Hungary

A large number of commensal microbial species reside in the human body that have co­evolved with the human genome and adopted to the host immune system.However, defects in regulatory processes or alterations in the composition of the microbiota can lead to various diseases, including cancer. A previous study has shownthat the colonisation of Candida cells is significantly higher in patients with oral squamous cell carcinoma (OSCC) compared to healthy individuals (Berkovits et al.2016). Our hypothesis is that the dysbiotic microbiota has tumor promoting effects, and the microbes are able to promote the metastatic activity of the cancerous cells.In order to investigate the effects of yeast cells on the metastatic activity of the cancerous cells we used a metastatic (HO­1­N­1) and a non metastatic (HSC­2) OSCCcell line. Cell migration, matrix metalloproteinase (MMP), proliferation activity and 3D tumor spheroid formation of HSC­2 and HO­1­N­1 OSCC cells wereinvestigated after fungal stimuli. The migration capacity of HO­1­N­1 cells was significantly higher if we treated the cells with Candida stimuli compared to the untreatedsamples. Prominent MMP activity and larger spheroid formation were detected after 24h pre­incubation with C. albicans. Both cell lines showed increased proliferationactivity upon treatments, which clearly indicates that the presence of fungi can accelerate cancer cell proliferation. To investigate the possible molecular mechanismsunderlying the increased metastatic activity of OSCC cells after fungal or bacterial exposure, we investigated the expression level of various TAM receptors, since theyplay a role in microbial recognition through inhibition of TLR signaling and TLR­induced cytokine­receptor cascades. In addition to their roles in limiting TLR andcytokine signaling they advance cancer cell migration by assisting the epithelial­mesenchymal­transition (Rankin et al. 2016). Elevated expression Axl in OSCC cellscould be verified by Western blot following Candida treatment.These findings suggest that the presence of fungi in the oral cavity may promote cell proliferation and increased motility of oral squamous cell carcinoma cells at least inpart by the induction of TAM receptors.

The project was funded by GINOP­2.3.2­ 15­2016­ 00015 and LP2018­15/2018 and 20391­3/2018/FEKUSTRAT

Candida albicans strain­specific differences in pathogen–host interactionin the oral mucosa

C. LembergI, S. AltmeierI, S. Amorim­VazII, F.A. SchönherrI, D. SanglardII, S. LeibundGut­LandmannI

IUniversity of Zurich, Zurich, Switzerland, IIInstitute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland

Fungal growth at mucosal surfaces in healthy individuals is controlled by epithelial barrier functions and the immune system. While it is well accepted that the hostimmune status is a key determinant of C. albicans infections to develop, we are only beginning to learn about the contribution of intra­species variations in the fungus todisease development. Our previous work has revealed major differences in the interaction of distinct natural isolates of C. albicans with the host. Highly virulent isolatessuch as strain SC5314 trigger a strong epithelial response in vitro and this correlates with a rapid induction of inflammation in the oral mucosa of experimentally infectedmice followed by rapid clearance of the fungus from the host tissue. In contrast, low­virulent isolates of C. albicans, exemplified by strain 101, which trigger a verylimited epithelial response, can persist in the oral mucosa of the murine host for several months. To investigate the mechanism underlying the commensal life style of strain101, we performed a transcriptomic analysis with fungal RNA recovered from the infected murine tissue. Expression of virulence genes including those associated withepithelial adhesion, invasion and damage induction was diminished in strain 101 in comparison to strain SC5314. However, the compromised virulence program of strain101 and the consequential reduction of the inflammatory response in the host tissue does not fully explain the persistent phenotype of strain 101. In fact, the fungus alsosurvives in a highly inflammatory host environment, as revealed by co­infection experiments with the two strains. Our transcriptomic data indicate that strain 101 ismetabolically adapted and strives in the nutrient­poor stratum corneum of the oral epithelium by virtue of an enhanced expression of genes related to amino acid, glucoseand lipid metabolism. Indeed, strain 101 can cope better than strain SC5314 with N­Acetylglucosamine (GlcNAc), which is abundant on microbially­colonized surfacesof the oro­gastrointestinal tract, and it displays enhanced resistance to GlcNAc­induced intracellular stress and cell death. Together, these novel insights into the species­specific adaptation of C. albicans to the host contribute to a refined understanding of the pathogenicity determinants and will enable the rational design of novelstrategies for antifungal interventions.

Glucose enhanced oxidative stress resistance in Candida albicans

I. M. Bohovych I, D. E. LarcombeII, N. A. R. GowIII, A. J. P. Brown II

IUniversity of Nebraska­Lincoln, Lincoln, NE, United States of America, IIUniversity of Aberdeen, Aberdeen, United Kingdom, IIIUniversity of Exeter, Exeter, UnitedKingdom

Microbes often inhabit environments in which a reproducible, sequential series of conditions are frequently experienced. Under these circumstances a microbe may gaina fitness advantage if it evolves to exploit an initial stress or signal to prime an anticipatory response to a second, impending condition. This concept is called “adaptiveprediction”. Candida albicans cells colonising the colon are in an environment with very low levels of glucose. However, when the cells encounter blood or enter thebloodstream they are exposed to glucose levels of around 0.05­0.1%. Previous work has shown that glucose levels as low as 0.01% lead to multiple changes in C.albicans transcriptome that increased resistance to azoles, osmotic, and oxidative stress. This 'Glucose Enhanced Oxidative Stress Resistance' (GEXSR) phenomenonis reported to be an example of adaptive prediction that we have further studied. Our hypothesis is that, when C. albicans colonising the gut are exposed to bleeding orwhen they invade from the gut into the bloodstream, the resultant exposure to blood concentrations of glucose prepares the fungus for subsequent oxidative attack bycirculating phagocytic immune cells.We found that this GEXSR phenotype promotes resistance to neutrophil killing, as well as resistance to H2O2. This phenotype appears to be unique to Candidaalbicans as it is not observed in other Candida species (C. dubliniensis, tropicalis, guilliermondii or glabrata) or in any other fungal species tested (Yarrowialipolytica, Lodderomyces elongisporus, Kluyveromyces lactis, and Saccharomyces cerevisiae). We find that protection is dependent on signalling via the glucoserepression (AMPK), and cAMP pathways, however metabolism of glucose is not essential for the phenotype. Interestingly, the GSH/GSSG redox state of the cellappears to be important as glutathione reductase and glutaredoxin are required. Other key oxidative stress resistance mechanisms (catalase, SODs, and thioredoxins)are not essential for the GEXSR phenotype. Our results suggest that GEXSR is a specialised, adaptive response that Candida albicans has evolved to aid survivalagainst host phagocytes. The fitness advantage gained by GEXSR during invasion could contribute to the virulence of Candida albicans as a pathogen.

Characterisation of the novel C­type lectin, MelLec, in antifungalimmunity.

C. Nikolakopoulou *I, M. Stappers *I, A. ClarkI, V. AimaniandaII, D. ReidI, P. AsamaphanI, M. NeteaIII, A. CarvalhoIV, J. LatgéII, J. WillmentI, G. BrownI

IUniversity of Aberdeen, Aberdeen, United Kingdom, IIUnité des Aspergillus, Pasteur Institute, Paris, France, IIIDepartment of Internal Medicine, Radboud UniversityMedical Center, Nijmegen, Netherlands, IVLife and Health Sciences Research Institute, School of Health Sciences, Braga, Portugal

Fungi are ubiquitously found in the environment and as human commensals. In immunocompromised individuals though, they can cause serious opportunistic infections,which can be lethal if left untreated. Pattern recognition receptors (PRRs) offer protection against fungal pathogens by detecting fungi. Among the families of PRRs, C­type lectin receptors (CLRs) play a critical role in antifungal immunity. CLRs have a C­type lectin like domain (CTLD) that recognises carbohydrates found in fungal cellwalls, leading to stimulation of several innate and adaptive immune responses. We have identified a novel CLR called Melanin sensing C­type Lectin receptor (MelLec),which unlike other well­characterised CLRs, did not recognise carbohydrates. However, MelLec recognised 1, 8­dixydroxynapthalene (DHN)­melanin that is presenton the cell wall of pathogenic fungi including Aspergillus fumigatus and Foncesaea pedrosoi. We have demonstrated that several other intermediates in thebiosynthetic pathway of 1, 8­DHN melanin are also recognised by this CLR. Importantly, we have shown that mice and humans are protected against disseminated A.fumigatus infection through recognition of melanin by this receptor. Interestingly, MelLec is not expressed on myeloid cells in mice but it is found to be expressed byCD31+ endothelial cells and a sub­population of these cells that co­express EpCAM. In summary, we have demonstrated that MelLec recognises an importantcomponent of the fungal cell wall and is critical for immunity to A. fumigatus.

* The authors marked with an asterisk equally contributed to the work.

CandidaMine an integrative database for omics data from CandidaSpecies

A. HafezI,II, H. HovhannisyanI,III, L. CarretéIII, C. LlorensII, T. GabaldonIII

IUniversitat Pompeu Fabra, Barcelona, Spain, IIBiotechvana, Valencia, Spain, IIICRG, Barcelona, Spain

Background

High­throughput technologies such as next generation sequencing have become a widespread tool to study the physiology and evolution of organisms of interests.However, there is a need of centralized repositories that allow researchers to mine this wealth of information. CandidaMine is an integrative data warehouse forCandida Species genomes and transcriptomes. CandidaMine is powered by Intermine to enable advanced mining features for the development of the integrativeanalysis tools. InterMine is an open source data warehouse built specifically for the integration and analysis of complex biological data from a wide range ofheterogeneous data sources.

Results

The Main different data source for the current Version is Candida Genome database (CGD), Candida Gene order Browser CGOB, Uniport, KEGG, Sequence ReadArchive (SRA) and other published dataset studies related to Candida Species. To build CandidaMine, Candida genomes are collected from CGD and SRA, GeneHomology information are collected for CGD and CGOB, proteins dataset extracted from Uniport and Interpro databases, Functional, Phenotypes and PathwaysAnnotations are collected from CGD and KEGG databases, and Gene Expression dataset are measured on various samples obtained from SRA under differentexperiment conditions. Currently CandidaMine contains data from the four most common species causing candidiasis Candida albicans, Candida glabrata, Candidaparapsilosis, and Candida tropicalis.

Conclusions

We built a data warehouse system, CandidaMine, with a comprehensive integration of information from various resources to be used for annotations enrichment,exploration and analysis of Candida related studies. It will be regularly updated and extended with new Candida species and datasets. It is aimed to serve as adedicated resources to the Community. CandidaMine is freely available at http://opathy.uv.es:8080/candidamine

HUMAN ANTIMICROBIAL PEPTIDE RESPONSE IN FUNGAL SKININFECTIONS

D. CorzoI, C. MunroI, C. MarkII, D. MacCallum I

IUniversity of Aberdeen, Aberdeen, United Kingdom, IINHS Northern Ireland, Belfast, United Kingdom

ObjectivesThe objective of the study was to describe the human antimicrobial peptide (AMP) response during skin infections by several fungal pathogens using an ex­vivo humanskin model

MethodsAn ex­vivo model was set up using skin explants incubated at 37 oC and 5% CO2 in DMEM supplemented with inactivated foetal calf serum and 1%penicillin/streptomycin, maintaining an air­liquid interphase at the epidermis­dermis junction. Skin infections by Fusarium solani, Trichophyton rubrum, Exophialadermatitidis, Malassezia sympodialis, and Candida auris were established by direct inoculation of 1x105 conidia after damaging the skin with a needle. Histologicand microscopic evidence of infection was evaluated by silver staining and SEM. Apoptosis of skin cells was evaluated by TUNEL staining. Skin AMP gene expressionwas evaluated by qRT­PCR and their protein levels by LC/MS­MS.

Results Apoptotic cell numbers were significantly increased in T. rubrum and F. solani infection. Expression of all AMP genes increased in non­infected skin during the 10 dayincubation period. Levels of RNase7 and S100A family proteins also progressively increased. Gene expression of three important S100A family members(S100A7,8,9) decreased during T. rubrum infection. Levels of the corresponding S100A proteins also decreased, but were only significant for S100A7 (­10 foldchange,p=0.02). RNASE7 expression was three times higher (p=0.03) in this infection, but the protein level did not differ from non­infected skin. During infection with M. sympodialis, gene expression of S100A7, DEFB103B, DEFB4A and RNASE7 was significantly increased (8, 192, 6, 7­fold, p=0.03,p=0.03, p=0.003, p=0.02, respectively), but only RNase7 protein levels were significantly increased (2­fold,p=0.03) compared to non­infected skin. During C. aurisskin infection, expression of S100A8 and RNASE7 was increased (2 and 6 times respectively, p=0.02, p=0.03), but S100A8 expression was decreased in E.dermatitidis infection. Interestingly, during F. solani infection, all AMP gene expression and proteins levels were significantly decreased in skin.

Conclusion AMPs have two main functions, a direct antifungal effect and a chemotactic/triggering immune effect. In this study, the decreased S100A family gene expression andprotein levels, during T. rubrum and F. solani infections, may affect the direct damage to fungi and may result in reduced immune response in skin.

Study of the interaction between vaginal lactobacilli, Candida albicansand Candida glabrata: from physiological aspects to OMICs analyses

N. PedroI, C. LimaII, M. AlvesIII, S. PintoIV, N. MiraI

IiBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico – Department of Bioengineering, Universidade de Lisboa., Lisbon, Portugal, IIiBB,Institute for Bioengineering and Biosciences, Instituto Superior Técnico – Department of Bioengineering, Universidade de Lisboa, Lisbon, Portugal., Lisbon, Portugal,IIICentro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa., Lisbon, Portugal, IVCentro de Química Estrutural, Instituto Superior Técnico,Universidade de Lisboa, Lisbon, Portugal

To successfully colonize the vaginal niche C. glabrata and C. albicans have to face the presence of a commensal bacterial microflora that is highly enriched inlactobacilli. A reduction in the abundance of the lactobacilli population has been associated with a higher risk of developing vaginal infections, however little is knownconcerning the molecular players controlling this relevant yeast­bacteria interaction. In this work, the effect of L. gasseri, one of the most abundant species in the vaginaltract, on the physiology and virulence of C. albicans and C. glabrata was examined. Exposure of C. albicans/C. glabrata to L. gasseri in a co­culture settingreduced growth rate and cell viability of the yeast cells also attenuated relevant virulence traits, including filamentation and biofilm formation. Microscopic imaging ofmixed C. albicans/C. glabrata with L. gasseri biofilms revealed close contacts between bacteria and the yeasts, resulting in highly non­structured biofilms. In the caseof C. glabrata, a significant reduction in viability was also observed in the mixed­biofilm, compared to what was observed in single species biofilms. To gain a genome­wide picture into how C. glabrata responds to the presence of L. gasseri a transcriptomics (based on RNA­sequencing) and a large­scale phenotypic screening (usingthe available deletion mutant collection) were undertaken. The transcriptomic analysis was performed in a co­culture setting and using as a reference the genomicexpression of the two species in single­culture. Around 638 C. glabrata and 204 L. gasseri genes were differently expressed in the co­culture setting, being interestingthat the yeast genes that changed more their expression in the co­culture setting are of uncharacterized function. The phenotypic screening performed also led to theidentification of several genes as mediating survival of C. glabrata in the presence of L. gasseri, clearly reflecting the poor knowledge gathered until so far concerningthe study of this interaction. The results obtained in the OMICs analyses performed will be further discussed to better understand the Candida­vaginal bacteriainteraction and to foster the identification of new players in assuring competitiveness of C. albicans/C. glabrata in the vaginal tract, these representing important newtherapeutic targets.

Using in vitro and ex vivo models to unravel Candida auris infectionstrategies

S. AllertI, P. KämmerI, D. SchulzI, T. WolfII, S. BrunkeI, L. KasperI, B. HubeI,III

IHans­Knoell­Institute Department of Microbial Pathogenicity Mechanisms, Jena, Germany, IIHans­Knoell­Institute Department of Systems Biology and Bioinformatics,Jena, Germany, IIIFriedrich­Schiller­University ­ Institute of Microbiology, Jena, Germany

Candida species are a major cause of invasive fungal infections, with Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis being the clinically mostrelevant ones. Recently, C. auris became known as a new species affecting humans in various regions of the world, causing invasive infections with high rates of clinicaltreatment failures.As the pathogenicity mechanisms of C. auris are largely unknown, we analyzed a set of C. auris strains from different clades during interactions with various host cellsand under conditions mimicking the host environment. Although the different strains were variable in drug resistance, aggregation and other in vitro growth phenotypes,the overall behavior in our infection models was similar. C. auris strains did not cause severe damage of epithelial cells in contrast to C. albicans. Human macrophagesrapidly phagocytose C. auris cells and respond by distinct cytokine secretion. However, C. auris cells can survive inside of macrophages and show intracellularreplication comparable to C. glabrata.To mimic the initial phases of systemic Candida infections with dissemination via the bloodstream, we used an ex vivo whole blood infection model. Similar to C. albicans, C. glabrata, C. parapsilosis and C. tropicalis, various strains of C. auris were efficiently killed in human blood and showed a characteristicpattern in immune cell association, killing rates and induction of cytokines. Dual transcriptional profiling of C. albicans, C. parapsilosis, C. tropicalis or C. glabrataand blood cells during infection indicated discrete survival strategies of all four fungal species, while the transcriptional host response was rather uniform. Dualtranscriptional profiling of C. auris­infected blood is ongoing and will help us to place C. auris within the pathogenic landscape of Candida species.

Feeding on the host: how do human fungal pathogens acquire essentialtrace metals during infection?

D. WilsonI

IUniversity of Aberdeen, Aberdeen, United Kingdom

The mammalian immune system has evolved sophisticated mechanisms to withhold micronutrients such as zinc from potential invaders ­ a process known as nutritionalimmunity. In spite of this defence mechanism, pathogens still thrive and cause disease. Therefore, pathogenic microbes have, in turn, evolved mechanisms to circumventnutritional immunity. Despite the fundamental importance of this host­pathogen “tug­of­war”, its underlying mechanisms, and how they can be exploited to preventdisease remain poorly understood. We are exploring how pathogenic fungi adapt to fluctuations in zinc availability and how this essential, yet potentially toxic cation ismobilised within the fungal cell. Using Candida albicans as model fungal pathogen, we have discovered multiple assimilation strategies. For example, this fungus secretesa metal­binding protein (a “zincophore”) to scavenges zinc from host tissue. We have also developed intracellular fluorescent zinc probes and found that, following cellular internalisation, zinc is rapidly compartmentalised in zincosomes and withinthe fungal vacuole; these organellar compartmentalisation events are mediated by two distinct ZnT­type transporters. Zinc stores are then mobilised by Zip­typetransporters when cells experience subsequent nutrient restriction. This mobilisation of organellar reserves alone is sufficient to promote growth. Using models of invasivecandidiasis, we have shown that transporter management of intracellular zinc reservoirs significantly fuels the virulence of C. albicans.This novel mechanism of counteracting nutritional immunity may have considerable clinical significance. This is because the pathogenic lifecycle of C. albicans involvestranslocation of the fungus from parts of the gastrointestinal tract, an environment of high zinc, to normally sterile internal organs.

The DNA Damage Response Affects Morphogenetic Switching in theFungal Pathogen Candida auris

G. Bravo RuizI, F. LongI, N.A.R. GowII,III, A. LorenzI

IInstitute of medical sciences. University of Aberdeen, Aberdeen, United Kingdom, IIUniversity of Exeter, Exeter, United Kingdom, IIIInstitute of Medical Sciences(IMS) and MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, United Kingdom

Candida auris has been identified as a medically relevant fungus only in 2009. It is the most drug­resistant yeast known to date and its emergence and populationstructure are unusual in many respects. Because of its recent emergence, we are largely ignorant about the general biology and life cycle of C. auris. Due to theevolutionary distance to the best­studied Candida species C. albicans, inferences from research on C. albicans are not transferable to C. auris. Indeed, research byus and other laboratories indicate that these two species behave obviously different in terms of their cell biology.The ability to undergo morphogenetic switches between different growth forms (yeast and hyphae) is a key virulence factor in C. albicans. However, most of the cuescausing filamentation in C. albicans do not have this effect in C. auris. Intriguingly, our results show that most, but not all, of the 22 clinical isolates of C. auris testedproduce pseudohyphal filaments under genotoxic stress or in the absence of functional Homologous Recombination (HR). C. auris is able to produce filaments whenchallenged with genotoxic drugs, or when RAD51 or RAD57 are deleted (VPCI479/P/13 strain background). Whereas the inability to perform Non­Homologous EndJoining (NHEJ) by deleting YKU80 seems to block the formation of pseudohyphae even in the presence of DNA­damaging drugs and causes a severe growth defecteven in normal growth conditions. A possible cause for these observations is that under genotoxic stress conditions the DNA damage checkpoint is triggered, which hasbeen shown to induce filament formation in C. albicans. Recently, filamentation has been described in two Chinese isolates of C. auris. Here, NaCl­stress and passagethrough a mammalian host, primed the yeast for pseudohypha formation. This indicates that morphogenetic switching in C. auris is complex, and will likely be caused bymultiple cues.Our studies also show that deletion of key HR factors (RAD51 or RAD57) decrease resistance to various genotoxins in C. auris. Intriguingly, both rad51Δ and rad57Δshow a strongly increased frequency of sensitivity suppression in the presence of the antifungal flucytosine (5­FC) in comparison to wild type. This suggests that HR isstrongly involved in the evolution of antifungal resistance, and that its deactivation forces the emergence of 5­FC resistant colonies.

Disruption of the cotH genes of the filamentous fungus, Mucorcircinelloides by a plasmid­free CRISPR/CAS9 system

C. SzebenyiI,II, G. NagyII, E.J. TóthI,II, T. WernerI, A.G. VazI,II, C. VágvölgyiI, T. PappI,II

IUniversity of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52., H­6726, Szeged, Hungary, IIHAS­USZ “Lendület” FungalPathogenicity Mechanisms Research Group, Közép fasor 52., H­6726 Szeged, Hungary, Szeged, Hungary

Members of the order Mucorales, such as certain species of Rhizopus, Lichtheimia, Mucor and others, can be agents of devastating opportunistic human infections,known as mucormycoses. CotH proteins are widely present in Mucorales and previous studies showed the importance of one of these proteins in the pathogenicity ofthis fungal group. Construction of strains, in which the cotH genes are disrupted or overexpressed can provide a good tool to investigate their relevance in thepathogenicity and other biological mechanisms.In this study, the Cas9 enzyme and the gRNA complex were directly transferred into the Mucoral fungus Mucor circinelloides without in vitro RNP formation and theusing of plasmids to disrupt the cotH genes encoding possible spore coat proteins. In this strategy, the components of the CRISPR/Cas9 system were introducedtogether into the fungal cells by the polyethylene glycol (PEG)­mediated protoplast transformation method. The linearized deletion cassette was used as a template DNAfor the homology driven repair. This approach resulted in stable transformants, in which gene disruption occurred by the integration of the selection marker (i.e. the uracilauxotrophy complementing pyrG gene) at the expected positions. The mutation was proven for each targeted gene by PCR analysis and the lack of the appropriatetranscripts was proven by qRT­PCR. Micro­ and macromorphological characterisation of the resulting strains were carried out. Growth ability of the mutants, sporegermination and the effect of cell wall stressors Calcofluor white and Congo red on the fungal growth were tested. Spore surface morphology was examined by electronmicroscopy. Phagocytic assay was performed with the standard macrophage­like cell line J774.16. Drosophila melanogaster and Galleria mellonella was alsoinvolved to study the pathogenicity of the mutant strains.In case of the cotH5 disrupted mutants, we observed differences in the morphology of the sporangiophores by scanning electron microscope. Based on our results wesuggest that the cotH4 gene may have a role in the cell wall synthesis and cotH3 and cotH4 can be involved in pathogenicity.The study was supported by the grants UNKP­18­3 New National Excellence Program of the Ministry of Human Capacities”, LP2016­8/2016 and GINOP­2.3.2­15­2016­00035.

The phosphatidylserine flippase Drs2 has a unique role during Candidaalbicans invasive growth

M. Basante­BedoyaI, R. ArkowitzI, M. BassilanaI

IUniversity Côte d’Azur, Nice, France

Phospholipid flippases (P4­ATPases) transport lipids across the membrane bilayer to generate and maintain membrane asymmetry; these ATPases translocate lipidsfrom the external leaflet to the cytosolic leaflet of cellular membranes. There are 5 flippases in Candida albicans, including Drs2, which we showed to be critical forhyphal invasive growth (1). Indeed, a drs2 deletion mutant grew similar to the wild­type strain during budding growth, yet was deficient for invasive growth and, inparticular, unable to maintain hyphal growth. This mutant had an altered distribution of phosphatidylserine (PS), as assessed with a fluorescent reporter, and wasadditionally hypersensitive to fluconazole. Very recently, this flippase was also shown to be critical for copper sensitivity (2). To delineate the role of Drs2 during C.albicans hyphal growth, we investigated the dynamics of distribution of this flippase, as well as that of different lipids and key regulators, during initiation andmaintenance of hyphal growth. We also characterized a Drs2 point mutant, analogous to that shown to be altered for PS flipping in S. cerevisiae (3). Furthermore, weexamined the role of other flippases, such as Dnf2, in invasive hyphal growth, together with their importance in response to membrane stress. All together, our resultsindicate that Drs2 has a unique role during C. albicans hyphal growth maintenance, which appears to be particularly critical upon septum formation.

(1) Labbaoui et al., 2017, PLoS Pathogens, 13(2):e1006205. (2) Douglas & Konopka., 2019, PLoS Genetics, 15(1):e1007911. (3) Baldridge & Graham, 2012, PNAS, 109(6):E290­8.

Combinatorial chemical perturbations to abrogate azole drug resistancein the human fungal pathogen Candida albicans

N. RevieI, K. IyerI, S. LiII, Y. YashirodaII, H. HiranoII, M. YoshidaII, H. OsadaII, C. BooneIII, L. CowenI

IUniversity of Toronto, Toronto, Canada, IIRIKEN Center for Sustainable Resource Science, Wako, Japan, IIITerrence Donnelly Centre for Cellular & BiomolecularResearch, Toronto, Canada

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. The dependence on thefungistatic azole antifungals for treatment of systemic candidiasis has led to the development of multiple independent mechanisms of resistance. A promising strategy toexpand the antifungal target space and overcome resistance mechanisms is combination therapy, as combining drugs has the potential to confer enhanced efficacy, fungalselectivity, and to slow the evolution of resistance. My project aims to identify novel molecules capable of augmenting azole activity and preventing the development ofresistance in C. albicans. To probe the combinatorial chemical space, I performed a high­throughput screen with ~20,000 compounds from the RIKEN NaturalProduct Depository (NDepo) in the absence and presence of the azole antifungal, fluconazole. Hit compounds were characterized with various assays to assessmammalian cytotoxicity, fungistatic vs fungicidal activity, and evaluation of activity against azole­resistant clinical isolates and inherently drug­resistant non­albicansCandida species. To determine the mode of action of a prioritized set of these novel compounds, I performed genome­scale haploinsufficiency profiling in C. albicansusing a barcoded pool of heterozygous deletion mutants covering ~90% of the genome. The RIKEN NDepo has also been functionally annotated in the model yeastSaccharomyces cerevisiae using chemogenomic approaches, which provides an opportunity to directly assess predicted targets in evolutionarily­related fungal species.This ongoing approach has identified an antifungal that acts synergistically against C. albicans and azole­resistant C. albicans clinical isolates via activation of theunfolded protein response (UPR). Furthermore, we have identified a compound with broad spectrum activity across human fungal pathogens that may target Fas1, akey component of fatty acid synthesis in yeast. Future experiments will be aimed at elucidating the mode­of­action of these promising combination antifungal treatments.Overall, this project is poised to identify novel antifungal drug targets and expand our understanding of antifungal resistance to aid in the treatment of these life­threatening infections.

Proline catabolism is a key energy source and contributor to reactiveoxygen species homeostasis in Candida albicans

F.G.S. SilaoI, A. KühbacherII, N. UwamohoroIII, F. NogueiraIV, K. RymanI, M. WardI, T. LionIV, C. UrbanIII, S. RuppII, P.O. LjungdahlI

IDepartment of Molecular Biosciences, The Wenner­Gren Institute, Stockholm University, Stockholm, Sweden, IIDepartment of Molecular Biotechnology, FraunhoferInstitute, Stuttgart, Germany, IIIClinical Microbiology, Umeå University, Umea, Sweden, IVSt. Anna Kinderkrebsforschung e.V., CCRI ­ Children’s Cancer ResearchInstitute, Vienna, Austria

Candida albicans forms hyphae as a means to invade host tissues and escape from macrophages. Currently it is unclear how C. albicanscells support the energydemands of hyphal growth in the context of the nutrient poor microenvironment of phagosomes within engulfing macrophages. We have recently shown that ATP derivedfrom mitochondrial proline catabolism is essential to induce and support hyphal formation in macrophages. Proline utilization is sensitive to glucose in a manner consistentwith glucose catabolite repression of mitochondrial function. In mitochondria, proline is converted to glutamate in two successive reactions catalyzed by proline oxidase(Put1) and Δ1­pyrroline­5­carboxylate (P5C) dehydrogenase (Put2). Cells lacking PUT1or PUT2are unable to form invasive filaments in solid matrices (e.g., 3Dcollagen matrix and Spider medium) and are unable to escape macrophages, which results in reduced fungal cell survival when co­cultured with macrophages. Usingtime­lapse microscopy, we have observed that PUT2expression is activated shortly after phagocytosis, andthat a non­filamentingC. albicansstrain (cph1Δ/Δefg1Δ/Δ)requires proline catabolism to replicate inside macrophages. Mutant put2­/­ cells generate excessive mitochondrial superoxide in the presence of exogenous proline,presumably due to the uncoupling of proline oxidase and P5C dehydrogenase activities. The increase in superoxide levels is linked to futile P5C­proline cycling,presumably due to enhanced levels of mitochondrial P5C, which builds up as the consequence of not being oxidized to glutamate. Consistent with derepression ofmitochondrial function, inactivating PUT2is lethal when cells are cultured in synthetic proline media containing low glucose (< 0.2%) or glycerol. Strikingly, cells lackingPUT2are avirulent in Drosophilaand Reconstituted Human Epithelial (RHE) infection models, and have significantly reduced survival in the presence of neutrophils. Ourresults highlight the importance of proline catabolism in generatingenergy and reducing ROS, two important parameters for hyphal growth and macrophage evasion of C.albicans.

Human albumin increases vaginal epithelial damage caused by Candidaglabrata

M. PekmezovicI, A. KauneI, S. MogaveroI, B. HubeI,II

IHans Knöll Institut, Microbial Pathogenicity Mechanisms, Jena, Germany, IIFriedrich Schiller University, Jena, Germany

Candida glabrata is the second most common Candida species isolated from humans in many studies. Although normally a commensal on mucosal sites, C. glabratacan turn into a pathogen when host barriers or immunity are impaired, thereby causing diseases ranging from superficial to systemic infections. Pathogenicity mechanismsof C. glabrata remain unclear, in particularly because this fungus is almost avirulent in mouse models and does not cause severe damage to human cells in vitro. Byusing an in vitro vaginal epithelial cell model, we observed that extracellular proteins can dramatically change the outcome of C. glabrata–host cell interaction, forexample as bridging molecules. In this study, we investigated the role of six proteins of human body fluids and the extracellular matrix (albumin, transferrin, vitronectin,fibronectin, fibrinogen and kininogen) in mediating adhesion and damage caused by C. glabrata.One protein that, when added to the media, significantly increased C. glabrata adhesion and epithelial damage was human (but not murine) albumin. The effect wasobserved even at concentrations lower than those found in several niches in vivo (≤1mg/ml), such as serum, saliva or cervico­vaginal fluid. On the molecular level, weobserved that the presence of albumin led to transcriptional upregulation of host albumin receptor genes (measured by quantitative PCR) and increased albumin uptakeby host cells (visualized using albumin­FITC). The addition of inhibitors of phagocytosis­like processes and macropinocytosis prevented damage by C. glabrata in thepresence of albumin, while inhibitors of lipid­raft/caveolae­mediated and clathrin­mediated endocytosis had no effect and damage was still significantly high. Thissuggests that the damage caused by C. glabrata in the presence of albumin is associated with host macropinocytosis.The relevance of these findings is supported by the fact that albumin is the most abundant protein in cervico­vaginal fluids and its concentration increases during theinfection. We hypothesize that C. glabrata may use albumin to facilitate adhesion and/or induce its uptake via induced endocytosis (macropinocytosis), which may inturn activate host responses leading to damage.

Candida albicans glutamate dehydrogenase (GDH2) catalyzesenvironmental alkalization but is dispensable for survival and escapefrom macrophages

F.G. SilaoI, K. RymanI, M. WardI, N. HansmannI, P. LjungdahlI

IStockholm University, Stockholm, Sweden

Candida albicans cells depend on amino acid catabolism to obtain energy for growth and to induce hyphal formation inside phagosomes of engulfing macrophages. Ithas been proposed that deamination of amino acids by fungal cells is important for neutralizing the acidic microenvironment of phagosomes, and that alkalizationprovides the stimulus for hyphal growth. Here, we show that mitochondrial­localized NAD+­dependent glutamate dehydrogenase (GDH2), which catalyzes thedeamination of glutamate to α­ketoglutarate, is responsible for the observed alkalization of media when C. albicans cells are grown with amino acids as the source ofcarbon and nitrogen. Using CRISPR/Cas9 we have created a strain lacking GDH2 (gdh2­/­). This strain is viable and does not extrude ammonia on amino acid­basedmedia. Consistent with this finding, but in contrast to current dogma, C. albicans strains carrying inactivated alleles of DUR1,2, encoding cytosolic urea amidolyase,remain competent to alkalinize the extracellular milieu. Furthermore, we confirm that environmental alkalization by wildtype strains does not occur under conditions ofhigh glucose (2%), a finding attributable to glucose­repression of GDH2 expression and mitochondrial function. Inhibition of mitochondrial translation or oxidativephosphorylation by chloramphenicol or Antimycin A, respectively, prevents alkalization. GDH2 expression and mitochondrial function become derepressed as glucoselevels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as carbon source. At 0.2% glucose, wildtype strains, but not gdh2­/­ mutants,efficiently alkalinize the media. Unexpectedly, inactivation of GDH2 does not affect the induction of filamentous growth of C. albicans, either in vitro in filament­inducing media, or importantly, in situ in the phagosome of primary murine macrophages. Using time­lapse microscopy, we have observed that gdh2­/­ cells survive,filament and escape from macrophages at rates indistinguishable from wildtype. These results indicate that alkalization of the macrophage phagosome is not essential forC. albicans survival nor does it provide the morphogenic signal inducing hyphal growth.

Interactions of macrophages with white and opaque Candida albicans

C. GrahamI, M. LohseI, A. JohnsonI

IUniversity of California, San Francisco, San Francisco, United States of America

Candida albicans resides asymptomatically in the gastrointestinal tract as a part of most healthy people’s microbiome. In immunocompromised patients however,invasive infections can occur with mortality rates exceeding 40%. At least one third of a/α C. albicans strains isolated from patients have the ability to stochasticallyswitch between two cell types, white and opaque, which are heritable for many generations. White and opaque cells differ in their metabolism, ability to mate, responseto environmental cues, and interactions with the host innate immune system. We have previously demonstrated a difference in host phagocytic cell interactions with whiteand opaque cells, with white cells preferentially phagocytosed. To further characterize these interactions, we monitored phagocytosis of opaque cells from a library of197 transcription factor deletion strains. We identified several transcription factors, including ones that regulate white­opaque switching, that contribute to the differentialphagocytosis of the two cell types. We used time­lapse microscopy to evaluate real­time interactions between opaque cells and macrophages. In addition to decreasedphagocytosis, the opaque cells that are phagocytosed did not effectively escape the macrophage as do white cells and instead proliferated within the macrophage.Furthermore, we observed decreased cytotoxicity and inflammatory responses in macrophages co­cultured with opaque cells compared to those co­cultured with whitecells, providing further evidence that opaque cells do not kill the macrophage. These results indicate that opaque cells induce a dampened macrophage immune responsein comparison to white cells and suggests that opaque cells once phagocytosed respond very differently to that environment than do white cells.

A Taqman­based qPCR assay for the detection of Malassezia spp. fromclinical material

A. StavrouI, B. TheelenI, F. HagenI, T. DawsonII, T. BoekhoutI

IWesterdijk Institute, Utrecht, Netherlands, IIAgency for Science, Technology and Research (A*STAR), Singapore, Singapore

Malassezia species are lipid­dependent human and animal skin commensal yeasts and responsible for a variety of pathologies in humans from minor skin disorders tofungemia. Malassezia furfur and Malassezia pachydermatis are the most common species associated with blood infections in humans receiving parenteral nutrition,particularly neonates. Malassezia infections are underdiagnosed as the standard microbiology media do not contain lipids, which are essential for its growth. Here, wepresent a qPCR assay for the detection of Malassezia spp. directly from blood samples.A region of the ribosomal DNA was assessed for the development of primers/probes. This region from 83 ascomycetous and basidiomycetous yeasts and fungi causingblood infection and close relatives including the 18 described species in the genus Malassezia were aligned using MAFFT. Primer efficiency, sensitivity and specificitywas tested using pure DNA from yeast cultures. Animal blood (sheep) was spiked with cells of different concentrations of Malassezia (4000­4 genomes/ul) to reassessthe assay in the presence of high background DNA. The Molysis Complete 5 was used for depletion of background DNA. Human DNA from saliva was used in aqPCR reaction to determine cross­reactivity of the primers/probe in vitro.Efficiency and sensitivity was tested using the haploid M. furfur CBS 14141, a clinical isolate originating from a case of bloodstream infection. Efficiency is found to be96.4%. The assay detects all the Malassezia spp. associated with human blood infection, M. furfur, M. pachydermatis and M. sympodialis and up till now 7 morespecies out of the 11 tested overall. Specificity of the primers is high as for non­Malassezia species a weak positive signal is only generated at the last 5 cycles (late Cpcall). The spiked samples gave a positive signal only 3 cycles later than the pure culture DNA (25 cycles). The limit of detection for both pure culture and spikedsamples is ~1 genome of the haploid M. furfur CBS 14141. The human DNA sample never gave a positive result before 40 cycles which is promising for the use of theassay with human blood.The final aim is to validate this assay in the clinic. A fast, reliable tool for the detection of Malassezia spp. from blood will overcome the misdiagnosis of such infectionsand will significantly decrease the time of a correct diagnosis, which is of crucial importance to the timely treatment of critically­ill patients.

In vivo imaging of Candida glabrata invasive infections in mice

S. SchrevensI, D. SanglardI

ICHUV, Lausanne, Switzerland

Candida species are the most common cause of hospital acquired fungal infections. Candida albicans is a major cause of different types of diseases in many, mainlyimmunosuppressed patients. The prevalence of Candida glabrata infections, however, is increasing. C. glabrata rapidly acquires antifungal resistance to the most useddrug, fluconazole. In order to understand the progression of Candida diseases and antifungal efficacy, several animal models have been used. Most of the work hasbeen carried out with C. albicans, and recently in vivo imaging techniques have been incorporated in these studies. Utilization of such in vivo imaging techniques cansubstantially reduce the amount of required animals and allow spatio­temporal assessment of infections. With regards to bioluminescence imaging (BLI), severalluciferases have been developed mainly for C. albicans infection models. Currently, very few examples of BLI have been reported in C. glabrata. We optimized the expression of a red­shifted firefly luciferase (RS­LUC), which was previously adapted for C. albicans in two related C. glabrata clinical isolates, oneof which is azole­susceptible (DSY562) and the other azole­resistant (DSY565). We showed in mice that DSY565 is more virulent compared DSY562.RS­LUC was first codon­adapted for utilization in C. glabrata and expressed under the control of several promoters originating from S. cerevisiae (pPGK1) or C.glabrata (pENO1, pTDH3). Bioluminescence and protein expression were measured in vitro, where the pENO1 promoter showed the highest expression andluminescence in different media.Therefore, this construct was tested using the tail vein injection model for invasive C. glabrata infection in mice. BLI was performed at different time points for bothstrains and in mice infected with a C. albicans bioluminescent strain expressing a related RS­LUC. The emitted bioluminescent signals for C. glabrata were about 10­fold lower than for C. albicans infection, which is probably due to two different factors: the lower expression of the luciferase in C. glabrata and the lower CFUs in thekidneys during infection. Even though the bioluminescent signal in C. glabrata was weak, we could verify the increased virulence of the resistant isolate compared to thesusceptible isolate. Other infection models are currently being tested with these C. glabrata luminescent isolates.

Systematic interrogation of Aspergillus fumigatus cyp51A polymorphismsfor azole drug resistance

C. SasseI, E. KolanderII, A. DudakovaII, G. BrausI, U. GrossII, O. BaderII

IInstitute of Microbiology and Genetics, Goettingen, Germany, IIInstitute for Medical Microbiology, Goettingen, Germany

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging public health problem concerning critically ill patients, such as stem cell transplant recipients,cancer patients or patients under intensive care. We have previously surveyed the presence of known cyp51A polymorphisms in isolates obtained from patients as wellas the environment and found both, highly prevalent TR34/L98H rand TR46/Y121F/T289A polymorphisms as well as well as rarer substitutions whose functionalrelevance was unclear (e.g. F219C), despite correlation with drug resistance. Compilation of MIC values from the literature associated with individual substitutionsshows heterogeneous distribution between intermediate and resistance categories in several cases, indicating the necessity of molecular studies to further elucidate this.To assess the relevance of 63 individual polymorphisms known to date, we chose to express each of them in the same genetic background (AfS35­dCYP51) andmeasure the resulting MIC values according to the EUCAST protocol to different clinically and agriculturally used azole­based compounds. Deleteion of cyp51A led toa 2­4 increased sensitivity to azoles. This was alleviated in the reconstituted strain, where cyp51A was reintroduced under control of its native promotor. The receivedcyp51A deletion strain was used for the introduction of every mutated cyp51A allele which were integrated via homologous integration. To avoid side effects andspontaneous mutations a recyclable marker system was used and two independent clones of each mutated strain were checked in MIC assays. The established systemenables the investigation of cyp51A mutations in one and the same strain background in correlation with antifungal drug resistance.

How chromosomes are impacted by stress: Elucidating genome­wideLOH dynamics in Candida albicans

T. Marton *I, C. Maufrais *I, C. d'Enfert *I, M. LegrandI

IInstitut Pasteur, Paris, France

The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss­of­heterozygosity (LOH) events, which can be seenon all 8 chromosomes and affect both laboratory and clinical strains. Genomic studies in C. albicans have highlighted the occurrence of homozygosis biases for certainchromosomes, explained by the presence of lethal and/or deleterious recessive alleles on these chromosomes. LOH, which are often the consequence of DNA damagerepair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecular mechanisms attheir origins. Although LOH are frequent and ubiquitous, little is known regarding their importance in the biology of C. albicans. In diploid Saccharomyces cerevisiae,LOH facilitate the passage of recessive beneficial mutations through Haldane’s sieve allowing rapid evolutionary adaptation. This appears also true in the predominantlyclonal organism C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as recently illustrated in the case of antifungal resistanceand niche adaptation.To understand the genome­wide dynamic of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on adifferent chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneousLOH events to be detected by monitoring the loss of one of the fluorescent marker using flow cytometry. This collection was characterized through in vitro growthphenotyping and whole­genome sequencing (of note, we confirmed the mutagenic properties of transformation, as long­tract LOH and/or aneuploidies were detected in60% of the transformants tested, often occurring on chromosomes other than the one targeted by the transformation). We are assessing spontaneous LOH frequency ineach reporter strain as well as LOH frequency upon physiologically relevant stresses (pH, heat, oxidative and drug treatments). Our project will allow addressing thefollowing fundamental questions: Do all chromosomes possess the same potential of undergoing LOH events? Does LOH frequency correlate with chromosome size?Are there stress­specific hotspots for LOH? These answers will provide a significant insight in the potential coordination of LOH events, occurring at random or ascontrolled events.

* The authors marked with an asterisk equally contributed to the work.

Effects of immune dysregulation in blood from CGD patients arepathogen­specific

K. HünnigerI,II, T. Sae­OngIII, T. WolfIII, B. RöslerIV, G. PanagiotouIII, F.L. van de Veerdonk IV, O. KurzaiI

IInstitute for Hygiene and Microbiology, Julius Maximilian University of Würzburg, Würzburg, Germany, IIFungal Septomics, Leibniz Institute for Natural ProductResearch and Infection Biology ­ Hans Knoell Institute, Jena, Germany, IIISystems Biology and Bioinformatics, Leibniz Institute for Natural Product Research andInfection Biology – Hans Knoell Institute, Jena, Germany, IVDepartment of Internal Medicine, Radboud University Medical Center, Radboud Center for InfectiousDiseases, Nijmegen, Netherlands

Fungal pathogens cause severe and life­threatening infections worldwide. The majority of invasive infections occurs in immunocompromised patients and is based onacquired as well as congenital defects of innate and adaptive immune responses. In many cases, these defects affect phagocyte functions.In preliminary work we have established a human whole blood infection model to analyze the interplay of Candida albicans with host immunity, which demonstrated apredominant role for neutrophils during C. albicans infection. On the basis of these results we are currently analyzing whole blood samples from patients with ChronicGranulomatous Disease (CGD) ­ a genetically diverse group of congenital disorders that arise as a result of functional defects in NADPH oxidase. Although nodifferences in the association of C. albicans with neutrophils were detectable, activation phenotypes of control and CGD neutrophils after fungal phagocytosis differconsiderably. The specific up­regulation of CD69 and decrease in CD16 surface levels on cells in direct contact with C. albicans could not be observed for neutrophilsdeficient in NADPH oxidase. Interestingly, despite the significantly higher total monocyte number in blood from CGD patients, association of C. albicans to CGDmonocytes was markedly lower than in control experiments. Elevated plasma levels of pro­inflammatory cytokines, such as IL­6 and MIP­1α, confirmed the hyper­inflammatory state in CGD patients. Nevertheless, ex vivo infection with C. albicans and Aspergillus fumigatus resulted in an increased secretion of these cytokinescomparable to stimulation of healthy blood. However, analyzing the release of IL­8 (and IL­1β) in blood from CGD patients identified altered pathogen­specificcytokine responses. In contrast to healthy blood infections, C. albicans was not able to induce IL­8 secretion in three of four CGD patients, whereas infection with A.fumigatus resulted in a strong release of this cytokine. In addition, peripheral blood transcriptional profiling showed a massive dysregulation pattern triggered by A.fumigatus as one of the major pathogens in CGD that could be not observed for C. albicans.Using the generated experimental time­resolved data from healthy volunteers and CGD patients identified that despite the global hyper­inflammatory state in CGD bloodeffects of immune dysregulation are pathogen­specific.

Characterization of azole resistant Candida auris strains generated bylaboratory microevolution method

F. BohnerI, C. PappI, R. TóthI, A. GácserI,II

IDepartment of Microbiology, Interdisciplinary Excellence Centre, University of Szeged, Szeged, Hungary, IIMTA­SZTE “Lendület”“Mycobiome” Research Group,University of Szeged, Szeged, Hungary

Opportunistic fungal infections caused by Candida species are increasing worldwide. Candida albicans is still the most frequently isolated species among cases ofcandidiasis, but the significance of non­albicans species are rising recently. In 2009 a novel Candida species, Candida auris have been described. The emergence ofthis species was quickly recognized as a serious threat, because the high percentage of clinical C. auris strains demonstrated intrinsic resistance to fluconazole, which isthe most frequently used antifungal in cases of invasive Candida infections. Since its early occurrence, several outbreaks have been described with isolates lesssusceptible against all classes of clinically used antifungals. Further studying these outbreaks, C. auris was described as an MDR (multi drug resistant) Candida speciescapable to cause serious bloodstream infections especially in health care settings.To investigate the changes caused by the development of antifungal resistance, a laboratory microevolution method was applied. For this, two moderately fluconazolesensitive C. auris clinical isolate were treated with increasing concentration of azole class antifungals, namely fluconazole, posaconazole, voriconazole.Changes in the antifungal susceptibility of the generated azole resistant strains were determined by microdilution method using azole (fluconazole, posaconazole,voriconazole), echinocandin (caspofungin, anidulafungin, micafungin) and polyene (amphotericin­b) class antifungals. To evaluate the impact of the gained resistance tothe cell membrane biosynthesis, ergosterol profile was investigated for all generated strains. Since biofilm formation is an important virulence factor especially in case ofnosocomial pathogens, it was also defined using XTT assay. In order to reveal the connection between the detected changes and the antifungal resistance, genomicdifferences were analyzed comparing the clinical (parental) isolates with the artificially evolved strains.Connection between the virulence and the antifungal resistance were also studied both in vitro and in vivo settings. For in vitro experiments killing assay wasperformed with J774.1 macrophage­like cells and for in vivo studies the non­conventional Galleria mellonella infection model was used. This project was funded by the Hungarian Academy of Sciences “Momentum” Programme LP2018­15/2018, GINOP­ 2.3.2.­15­2016­00035, NKFIH K 123952and 20391­3/2018/FEKUSTRAT.

FUNCTIONAL CHARACTERIZATION OF A NOVELHYDROPHOBIC SURFACE BINDING PROTEIN IN MUCORALES

A.G. VazI,II, M. TakóII, C. SzebenyiI,II, E.J. TóthI,II, C. VágvölgyiII, T. PappI,II, G. NagyI

IHAS­USZ “Lendület” Fungal Pathogenicity Mechanisms Research Group, Közép fasor 52., H­6726 Szeged, Hungary, Szeged, Hungary, IIDepartment ofMicrobiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52., H­6726 Szeged, Hungary, Szeged, Hungary

Mucormycosis is a serious life­threatening disease, caused by opportunistic pathogenic fungi belonging to the order Mucorales. The poor outcome, which has beenobserved despite the current treatment options, like correction of the underlying risk factors, antifungal therapy, and aggressive surgery, arises the need to identify thepossible virulence factors of this pathogenic fungus that could pose as a potential therapeutic target.Hydrophobic surface binding protein A is a small secreted protein found in eukaryotes. HsbA proteins typically range between 171 to 275 amino acids in length. Theprotein was firstly isolated from the culture broth of Aspergillus oryzae RIB40. That protein was found to be able to recruit cutinase 1 (CutL1) to the surface ofhydrophobic solid materials (PBSA) and could promote the activity of the degradative extracellular enzymes. This protein also participates in fungal resistance to stressthat could be caused due to toxicity of some aromatic compound or reactive oxygen species released during the degradation process. The HsbA protein is functionallyuncharacterized in Mucorales and its role in the host­pathogen interactions is yet unknown.The main objective of the current study was to characterize HsbA proteins and the encoding genes in Mucor circinelloides. We found six hsbA genes in the M.circinelloides genome. By qRT­PCR analysis two genes (hsbA1 and hsbA2) was found to be highly expressed during the life cycle of the fungus. Hence, these twogenes were examined in our further studies. To determine the possible role of HsbA1 and HsbA2 in the pathogenesis of M. circinelloides, deletion and overexpressionmutants were constructed. For overexpression, the gene was placed under the regulation of the strong gpd1 promoter. To create hsbA knock out mutants, a recentlydeveloped CRISPR­Cas9 system was used. We conducted micro­ and macro morphology assays on the deletion and overexpression mutants. Our other objective wasthe heterologous expression of the HsbA proteins in Pichia pastoris and to purify the proteins. Hence, Pichia expression systems was obtained by transforming Pichiapastoris with pPICZα­hsbA vector constructs by electroporation and the positive transformants expressed HsbA1 and HsbA2 proteins in the culture supernatant,which were then purified for further analysis.The study was supported by the grants LP2016­8/2016 and GINOP­2.3.2­15­2016­00035.

The response regulator Ssk1 orchestrates stress specific changes inCandida albicans SAPK pathway architecture.

A. DayI, D. SmithI, C. Herrero­de­DiosII, A. BrownII, J. QuinnI

IInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom, IIInstitute of medical sciences. University of Aberdeen,Aberdeen, United Kingdom

The ability to adapt to diverse and often hostile environments within the host is essential for the virulence of a broad range of pathogenic fungi, including the human fungalpathogen Candida albicans. Much of our work has focussed on the C. albicans Hog1 stress activated protein kinase (SAPK), which is essential for stress­adaptationand virulence in this important pathogen. However, its structural and functional conservation with analogous human SAPKs (p38/JNK) makes the development ofantifungal drugs that will specifically target fungal SAPKs challenging. Because of this, there is much interest in identifying fungal­specific regulators of SAPKs aspotential drug targets. Such targets include two­component related phosphorelay systems which are used by fungi, but not mammals, to sense and relay stress signals toSAPK modules. In C. albicans, the phosphorelay system is comprised of three histidine kinases, a single phosphotransfer protein (Ypd1), and the response regulatorSsk1. C. albicans can tolerate loss of Ypd1 and unexpectedly we previously reported that inactivation of YPD1 actually enhances the virulence of this fungal pathogen.Thus in this study we have explored the role of Ssk1 in Hog1 regulation with a view to assessing its suitability as an antifungal target.Excitingly, we find that Ssk1 is a master regulator of Hog1. Cells lacking Ssk1 display impaired Hog1­activation and overlapping stress­sensitive phenotypes withhog1Δ cells in response to diverse stress­inducing agents with the notable exception of osmotic stress. We present data illustrating that Ssk1 functions as a coreregulator of Hog1 by acting as a scaffolding protein to promote key interactions between the Ssk2 and Pbs2 kinases within the SAPK module. This interaction ismaintained following exposure to multiple stresses with the exception of osmotic stress which drives the phosphorylation and activation of Pbs2 and triggers its releasefrom the Ssk1 scaffold. Thus in addition to delineating the function of Ssk1 as a major regulator of Hog1, such experiments have also uncovered stress­specificmechanisms of Hog1 activation. Collectively, these findings question the dogma that stress­induced activation of Hog1 depends on the activation of upstream kinaseswithin the SAPK module. Current experiments are aimed at identifying compounds that block key protein­protein interactions with the Hog1 molecule as suchcompounds could disrupt Hog1 signalling and fungal virulence.

Host­relevant alternative carbon sources independently contribute to C.albicans pathogenesis

R. WilliamsI, M. LorenzI

IUniversity of Texas Health Science Center, Houston, United States of America

The interaction between Candidaalbicansand the innate immune system is a key determinant of disease progression. Phagocytosis by macrophages induces a dynamicresponse in which this pathogen changes physiology, morphology, and metabolism, enabling it to survive and subsequently escape the phagocyte to continuedissemination. Transcriptional and genetic data indicate that phagocytosed C. albicans rapidly shifts its metabolism from glycolysis to gluconeogenesis, utilizingalternative carbon sources such as amino acids, N­acetylglucosamine (GlcNAc), and carboxylic acids. These host­relevant nutrients serve as more than energy sources;all induce major changes in fungal physiology, cell wall architecture, and stress resistance, conferring a competitive advantage in host niches. In vitro, utilization of aminoacids, GlcNAc, and carboxylic acids each result in a rapid increase in extracellular pH. This active pH neutralization is also observed within the macrophagephagosome, leading to hyphal morphogenesis and escape from the phagocyte. Mutants incapable of catabolizing alternative nutrients, such as stp2∆ (amino acids),jen1∆jen2∆(carboxylic acids), and hxt1∆ nag1∆ dac1∆(h­d∆; GlcNAc), are all impaired in their ability to neutralize the pH and survive phagocytosis. However, thesurvival defects of these mutants are more modest than anticipated. We hypothesize that utilization of each carbon source independently contributes to C. albicanspathogenesis with macrophages. In support of our hypothesis, deletion of multiple alternative carbon pathways (stp2∆jen1∆jen2∆h­d∆) display significant defects inevery macrophage and disseminated candidiasis mouse models tested. To determine whether this is due to defects in sensing the presence of these nutrients, utilization ofthem, or both, we generated sensor mutants (ngs1∆,GlcNAc; gpr1∆, monocarboxylic acids; ssy1∆, amino acids). Preliminary experiments with these mutants furthersupport our hypothesis that each alternative carbon pathway is distinct, as nutrient sensing is important for some pathways (GlcNAc), and negligible for others(carboxylic acids). Overall, these data highlight the importance of C. albicans’ metabolic flexibility. C. albicans turns to alternative carbon utilization to survivemacrophage attack and cause disseminated candidiasis.

Establishment of genotype­host correlation in Colletotrichumgloeosporioides by multi­locus microsatellite genotyping method

N. MehtaI, F. HagenII, S. SinghI, S. AamirI, A. BaghelaI

IAgharkar Research Institute, Pune, India, IIMedical Mycology Group, Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands

Colletotrichum gloeosporioides is an economically important fungal phytopathogen causing anthracnose disease in different host plants results in yield losses. C.gloeosporioides exhibit the high level of genetic and pathogenic diversity, but there is unavailability of a highly discriminatory method for genotyping. To understand thegenetic diversity and molecular epidemiology of this fungus, we have developed a novel, high resolution multi­locus microsatellite typing (MLMT) method to establishfungal genotype­host correlation. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five,CG1 (GT)n, CG2 (GT1)n, CG3 (TC)n, CG4 (CT)n, and CG5 (CT1)n were selected for further study based on their universal amplification potential, reproducibility,and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India.All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213–241, 197–227, 231–265, 209–275, and 132–188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson’s index of diversity (D)values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed thatisolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level ofcorrelation between certain genotypes and host. The MLMT method holds the potential to be fast and high throughput in nature and would be a powerful tool forstudying the genetic diversity and any possible fungal genotype­host correlation in C. gloeosporioides. This will be useful when breeding for resistance or monitoring thesensitivity of fungicides across a collection of well­defined isolates.

Mutations in hmg1, challenging the paradigm of clinical triazoleresistance in Aspergillus fumigatus

J.M. RybakI, W. GeI, N. WeiderholdII, J.E. ParkerIII, S.L. KellyIII, P.D. Rogers *I, J.R. Fortwendel *I

IUniversity of Tennessee College of Pharmacy, Memphis, TN, United States of America, IIThe University of Texas at San Antonio (UTSA), San Antonio, United Statesof America, IIISwansea University, Swansea, United Kingdom

Background: Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state associated with more than 200,000 life­threatening infectionsannually. The triazole antifungals are relied upon as both front­line and salvage therapies for the treatment of aspergillosis. However, triazole resistance among clinicalisolates of A. fumigatus is increasingly encountered worldwide, and a large proportion of clinical triazole resistance remains unexplained.

Methods: Whole genome sequencing was performed on 21 triazole­resistant A. fumigatus clinical isolates and 5 susceptible control isolates. Cas9­mediatedtransformations were used to introduce mutant hmg1 alleles into a triazole­susceptible control isolate, and to correct hmg1 mutations found in triazole­resistant clinicalisolates. Minimum inhibitory concentrations (MIC) for all clinically available triazoles were determined for all isolates by CLSI methods. Comprehensive sterol profilingwas performed for by extracting lipids from cultures of each isolate and comparing sterols with standards by gas chromatography­ mass spectrometry.

Results: Whole genome sequencing analysis revealed hmg1 mutations unique to triazole­resistant isolates in 11 of the 21 resistant isolates in this collection. Introductionof 3 different hmg1 mutations (encoding F262del, S305P, and I412S) into a triazole­susceptible control isolate resulted in a 4 to 8­fold increase in all triazole MIC.Comprehensive sterol profiling revealed accumulations of the ergosterol precursors eburicol and fecosterol in all hmg1 mutant strains, while total ergosterol contentremained unchanged as compared to the wild­type control strain. Additionally, correction of the F262del and S305P hmg1 mutations in 2 different triazole­resistantclinical isolates resulted in an 8 to 32­fold reduction in all triazole MIC, while correction of the I412S mutation in one isolate had no impact on triazole MIC.

Conclusions: These data demonstrate that mutations in the A. fumigatus HMG­CoA reductase gene, hmg1, are common among triazole­resistant clinical isolates, andthat these mutations directly contribute to triazole resistance. Additionally, mutations in hmg1 were found to be associated with accumulations of ergosterol precursors,while total ergosterol content remained unchanged. Future research is needed to further resolve the mechanism by which mutations in hmg1 mediate clinical triazoleresistance in A. fumigatus.

* The authors marked with an asterisk equally contributed to the work.

In vitro inducing conditions to promote titan­like cells formation in C.neoformans

R. Garcia­Rodas *I, N. Trevijano­Contador *I,II, H.C. de OliveiraIII,IV, J. GuilhemV, J. AriñoVI, O. Zaragoza I

IMycology Reference Laboratory. National Centre for Microbiology. ISCIII, Madrid, Spain, IIAlbert Einstein College of Medicine, New York, United States ofAmerica, IIIMycology Reference Laboratory. National Cenrte for Microbiology. ISCIII, Madrid, Spain, IVUniversidade Estadual Paulista (UNESP).CâmpusAraraquara. Faculdade de Ciências Farmacêuticas. Laboratório de Micologia Clínica., São Paulo, Brazil, VInstitut Pasteur, Paris, France, VIInstitut de Biotecnología iBiomedicina. Universitat Autonoma Barcelona, Barcelona, Spain

Cryptococcus neoformans is an opportunistic encapsulated fungal pathogen that undergoes complex morphological changes during interaction with hosts. Thesechanges were described during pulmonary murine infection and consist of capsular enlargement, and the formation of titan cells. These abnormally large cells cannot bephagocytosed and therefore they constitute a major problem for the immune system. However, research on the mechanisms involved in the formation of titan cells andtheir role during infection has been hampered by the need of using an animal model. In this work, we describe in vitro conditions that promote the transition from regularto titan­like cells.Incubation of C. neoformans in low nutrient media, supplemented with serum in a CO2 enriched atmosphere induced cryptococcal cell size enlargement. These cellswere not as large as those found in vivo. However, they reach cell body sizes of around 15 µm, and therefore we decided to call them titan­like cells. Serum wasessential for titan­like cells formation because in its absence the increase in cell size was significantly lower. Indeed, substitution of serum for phosphatydilcholine resultedin cells of enlarged size. Moreover, other factors, such as oxygen limitation and cell density seem to be key regulators in this process.Interestingly, titan­like cells were absent in cultures inoculated with high densities of cells. These findings may be explained by quorum sensing phenomenon sincesupernatant from high density cells cultures inhibit the formation of titan­like cells. Moreover, QSP1, the main quorum sensing molecule described in C. neoformans,inhibit the titan­like cells formation in a dose­dependent manner. In conclusion, in vitro generation of titan cells is an important contribution to the molecular toolboxneeded to understand the biology of this pathogenesis­associated morphotype in Cryptococcus neoformans.

* The authors marked with an asterisk equally contributed to the work.

Investigation of the zinc uptake system of the human fungal pathogenCandida parapsilosis

T. TakacsI, T. NemethI, D. WilsonII, A. GacserI,III

IDepartment of Microbiology, Interdisciplinary Excellence Centre, University of Szeged, Szeged, Hungary, IIMedical Research Council Centre for Medical Mycology atthe University of Aberdeen, Aberdeen Fungal Group, Aberdeen, United Kingdom, IIIMTA­SZTE “Lendület”“Mycobiome” Research Group, University of Szeged,Szeged, Hungary

The Candida species are among the most common causes of systemic nosocomial fungal infections worldwide. Candida albicans is the dominant ­ and consequentlythe best studied organism of the group. Over the past three decades however, the incidence of other Candida species has increased apparently. Reports show thatCandida parapsilosis is often the second or third most commonly isolated Candida species from blood cultures and is associated with outbreaks of infection inneonatal intensive care units. Both species have several virulence factors by which they can adapt to the enviroment of the host and therefore are able to cause aninfection. These factors include adhesins, as well as the ability to form biofilms, furthermore hydrolytic enzymes, such as the acidic proteinases and lipases. TheseCandida species also have the ability to obtain growth­limiting heavy metal ions from their environment such as zinc. This capability is key since host niche represents azinc­limited environment that is one way to inhibit microbial growth. Hence these patogens need to possess a zinc transport system that allows them to access to boundzinc ions from the host enviroment upon infection. In contrast with this a high zinc ion concentration can also be a way of microbial elimination as it occurs in thephagosomes of Mycobacterium tuberculosis infected macrophages. In the case of C. albicans, the way of zinc acquisition is intensively studied, but we lack anyinformation on the components playing role in the zinc homeostasis of C. parapsilosis. Thus we aimed to in silico predict potential zinc transporters in C. parapsilosis,create homozygous knock out mutants and expose them to various type of stressors and zinc limiting conditions. We have identified six genes, but only removal of twoof them (CPAR2_210740 and CPAR2_212100) resulted in difference in zinc uptake compared to the control strain. We also analyzed the kinetics of uptake of thesemutants by macrophages, their killing efficiency and investigated the zinc ion level in the phagolysosome during in vitro infection.This project was funded by the Hungarian Academy of Sciences “Momentum” Program LP2018­15/2018, by GINOP­ 2.3.2.­15­2016­00035. OTKA NKFIH K123952 and 20391­3/2018/FEKUSTRAT.

The co­chaperone Dnj1 is required for mitochondrial function andvirulence in Cryptococcus neoformans

L. HorianopoulosI, M. CazaI, G. HuI, K. SchmittII, O. VelariusII, G. BrausII, J. KronstadI

IMichael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada, IIGeorg­August­University Goettingen, Albrecht­von­Haller­Institute for Plant Sciences, Goettingen, Germany

Mitochondria are emerging as key contributors to fungal pathogenesis. In particular, manipulating mitochondrial function alters the virulence of pathogenic fungi andinfluences their susceptibility to antifungal drugs. We have identified Dnj1 as a mitochondrial co­chaperone of the J­domain family in Cryptococcus neoformans. Jdomain co­chaperones are a large, diverse group of proteins that can recruit specific targets to the more conserved chaperone machinery to prevent aggregation,promote refolding, or assist in complex assembly. We initially constructed a targeted deletion mutation to investigate the function of Dnj1 which lacks orthologs in anyspecies outside the Tremellomycetes. The dnj1Δ mutant was found to have a general growth defect, which was exacerbated at elevated temperature of 37°C comparedto 30°C. Capsule elaboration was also reduced in the mutant. Not surprisingly, based on the observed defects in major virulence factors, the dnj1∆ mutant was found tobe avirulent in a murine infection model. Consistent with its mitochondrial localization, Dnj1 was found to play major roles in mitochondrial function. The knockoutmutant is deficient in growth on alternative carbon sources and in mitochondrial membrane polarization. Furthermore, the mutant is hypersensitive to the alternativeoxidase inhibitor salicylhydroxymate and, unlike the wild­type strain, the mutant is completely insensitive to the complex III inhibitors antimycin A and myxothiazol. These results suggest that Dnj1 plays a role in complex III assembly. An observed interaction between Dnj1 and cytochrome b­c1 complex subunit 2 (Qcr2), a corecomponent of complex III, also supports an impact on complex III. To further interrogate the mechanism of Dnj1 function, we constructed a strain with a single aminoacid substitution (dnj1::DNJ1H111Q) to abolish the activity of the J domain. This strain shared the major phenotypes of the knockout mutant. These results indicate thatthe J domain activity of Dnj1 is necessary for the maintenance of functional mitochondria, ultimately contributing to growth and adaptation to the host environment. Thedivergent amino acid sequence of this co­chaperone from any human co­chaperones makes it an intriguing target for antifungal drug development. The mitochondrialdefect of this knockout also suggests that an inhibitor of Dnj1 may be used in combination with current antifungal drugs, as suggested for other drugs that targetmitochondrial function.

Methylglyoxal activation of Mrr1 leads to increased resistance to azoles inCandida lusitaniae

A. BiermannI, E. DemersI, D. HoganI

IDepartment of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States of America

In Candida species, Mrr1 is a zinc­cluster transcription factor that is of clinical interest due to its established role in regulating expression of the azole resistance geneMDR1. Although the regulation of MDR1 by Mrr1 is well­studied, particularly in C. albicans, the functional roles of many other genes in the Mrr1 regulon remain yet tobe elucidated. Here, we demonstrate that Mrr1 regulates expression of two homologous methylglyoxal reductases in C. lusitaniae, which play a role in detoxifyingexogenous methylglyoxal in vitro. Methylglyoxal is a cytotoxic byproduct of glycolysis that is produced by all living cells; it has been found to be elevated in the serumof diabetic patients, as well as at sites of infection or inflammation. In addition to the regulation of methylglyoxal reductase genes CLUG_01281 (MGD1) andCLUG_04991 (MGD2) by Mrr1, we have discovered that methylglyoxal induces a specific transcriptional response in C. lusitaniae, which appears to be dependenton Mrr1 and the redox­sensing transcription factor Cap1. Phenotypic studies indicate that increased resistance to azole antifungals is one functional consequence of thismethylglyoxal response. Given the physiological significance of methylglyoxal in human disease, we propose that the transcriptional response to methylglyoxal is clinicallyrelevant and a contributor to in vivo resistance against some antifungals in Candida species.

Role of the autophagy mechanism during infection of intestinal epithelialcells by Candida albicans.

A. Ducreux *I, P. Lapaquette *I, L. BasmaciyanI, T. ParadisI, F. DalleI

IVAlMis Unit ­ UMR PAM ­ University of Burgundy, Dijon, France

Candida albicans is an opportunistic pathogen causing infections ranging from superficial to the more life­threatening disseminated infections. In a susceptible host, C.albicans is able to reach and translocate through the gut barrier, promoting its dissemination in deeper organs. The polymorphic nature of this fungus is essential for itsvirulence since the C. albicans yeast­to­hypha transition is necessary for its effective invasion into host intestinal epithelial cells . C. albicans invades human epithelialcells by two well­documented mechanisms: the epithelial­driven endocytosis of hyphal forms and the C. albicans­driven active penetration. Host countermeasures tolimit C. albicans colonization and invasion include the secretion of anti­fungal peptides, the secretion of mucus and the recruitment of specific immune cells dedicated tomicroorganisms clearance. Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and modulating theinflammatory response. The protective role of autophagy during C. albicans infection has been already investigated in myeloid cells (macrophages and dendritic cells)however far less is known regarding the role of this process during the infection of intestinal epithelial cells. In the present study we investigated the role of the autophagicprocess during the infection of intestinal epithelial cells by C. albicans. Using immunofluorescent staining against various autophagic markers, including the LC3 protein,we demonstrated that autophagy machinery is rapidly recruited at the Candida invasion site in various intestinal epithelial cell lines. We confirmed these observations byelectron transmission microscopy that reported the presence of autophagosomal structures in the vicinity of internalized hypha. Finally, we used autophagic­deficientepithelial cell lines and a panel of C. albicans mutants to specify the precise role of autophagy during invasion of epithelial cells by the fungus.

* The authors marked with an asterisk equally contributed to the work.

Defining the role of the IL17 receptor A (IL­17RA) in fungal disease

S. GagoI, D. DenningI, P. BowyerI

IManchester Fungal Infection Group (MFIG), Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicineand Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom

More than 200 million people have asthma worldwide with over 10 million at risk on developing allergic bronchopulmonary aspergillosis. Allergic bronchopulmonaryaspergillosis is a progressive lung disease caused by colonisation of the respiratory airways by the environmental mould Aspergillus fumigatus. Although the disease isnot usually fatal, the economic costs due to patient hospitalisation and treatment are very high. Using whole exome sequencing we have discovered patients with allergicbronchopulmonary aspergillosis have multiple mutations in IL­17 receptor A (IL­17RA), a ubiquitous type I membrane glycoprotein that binds with low affinity tointerleukin 17A. It is known that the interplay between different cell subsets host and pathogen networks determines the outcome of the infection. Currently suchinteractions between cell types cannot be accurately studied in an animal model of fungal disease because precise isolation of the many cell types present is not possiblein a timely manner. We have established a system that enables dissection of the signalling events that migrate between epithelial cells and macrophages. This systemallows cell types to be self­contained but permits transmission of secreted signals between cells. We have developed using CRISPR/Cas9, 16HBE bronchial epithelialcells and THP1 macrophages genetically deficient in IL­17RA. Using RNA­seq we have observed the concerted transcriptional response against Aspergillusfumigatusis driven by inter ­ cell IL­17RA ­ dependent IFNγ signalling. Interestingly interacting IL­17RA deficient 16HBE bronchial epithelial cells and THP1macrophage cells showed a precise allergy­like phenotype characterised by IL­12, IL­23 and IL­25 dysregulation, closely matching that seen in patients with fungalallergy. Furthermore, our system provided much more consistent data than that obtained from animal models allowing us to effectively select disease markers forvalidation in well characterised patient cohorts.

Role of the autophagy mechanism during infection of intestinal epithelialcells by Candida albicans.

A. Ducreux *I, P. Lapaquette *I, L. BasmaciyanI, T. ParadisI, F. DalleI

IVAlMis Unit ­ UMR PAM ­ University of Burgundy, Dijon, France

Candida albicans is an opportunistic pathogen causing infections ranging from superficial to the more life­threatening disseminated infections. In a susceptible host, C.albicans is able to reach and translocate through the gut barrier, promoting its dissemination in deeper organs. The polymorphic nature of this fungus is essential for itsvirulence since the C. albicans yeast­to­hypha transition is necessary for its effective invasion into host intestinal epithelial cells . C. albicans invades human epithelialcells by two well­documented mechanisms: the epithelial­driven endocytosis of hyphal forms and the C. albicans­driven active penetration. Host countermeasures tolimit C. albicanscolonization and invasion include the secretion of anti­fungal peptides, the secretion of mucus and the recruitment of specific immune cells dedicated tomicroorganisms clearance. Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and modulating theinflammatory response. The protective role of autophagy during C. albicans infection has been already investigated in myeloid cells (macrophages and dendritic cells)however far less is known regarding the role of this process during the infection of intestinal epithelial cells. In the present study we investigated the role of the autophagicprocess during the infection of intestinal epithelial cells by C. albicans. Using immunofluorescent staining against various autophagic markers, including the LC3 protein,we demonstrated that autophagy machinery is rapidly recruited at the Candida invasion site in various intestinal epithelial cell lines. We confirmed these observations byelectron transmission microscopy that reported the presence of autophagosomal structures in the vicinity of internalized hypha. Finally, we used autophagic­deficientepithelial cell lines and a panel of C. albicans mutants to specify the precise role of autophagy during invasion of epithelial cells by the fungus.

* The authors marked with an asterisk equally contributed to the work.

Regulatory networks controlling pathogenesis in the fungal pathogenCandida albicans

L. van WijlickI, S. ZnaidiII, S. Bachellier­BassiI, C. d’EnfertI

IInstitut Pasteur, Paris, France, IIInstitut Pasteur de Tunis, Tunis, Tunisia

The polymorphic fungus Candida albicans inhabits the human body as a commensal. Under certain circumstances C. albicans can cause infections that range fromsuperficial­ to life­threatening systemic­infections, associated with a high mortality rate. The ability of C. albicans to infect diverse host niches is supported by a widerange of virulence factors and fitness attributes. These attributes include rapid adaptation to the changing environments of the human body, the ability to modulate thecomposition of its cell wall and most importantly morphological changes, especially the yeast­to­hypha transition. The shift from a harmless commensal to a virulentpathogen thus involves hundreds of cellular components. Therefore, understanding the virulence of this important fungal pathogen relies on global analyses.

A systematic combination of genome­wide transcript profiling (RNA­seq) with genome­wide binding analyses (ChIP­seq) of 12 stress­related transcription factors hasbeen used to establish transcriptional regulatory networks in C. albicans. Moreover, ChIP with selective isolation of chromatin­associated proteins (SICAP) combinedwith mass spectrometry was used to identify chromatin­bound partners, confirming co­regulation of shared­target genes and improving network complexity and quality.The transcription factors were either selected based on changes in expression in cells exposed to a pleiotropic stress­inducer and/or because they displayedmorphogenesis­related phenotypes. Deciphering the complex interplay of transcriptional regulatory networks components uncovered regulatory hubs – transcriptionfactors that show high connectivity within networks and can act as integrators of diverse cellular processes.

These regulatory hubs are being characterized functionally using either loss­of­function or gain­of­function mutants in in vitro and in vivo models of host­pathogeninteraction, in order to reveal their contribution to fitness and virulence of C. albicans.

Orchestration of chitin synthesis in Candida albicans

M. SpyrouI, M. LenardonII, N. GowI

IUniversity of Exeter, Exeter, United Kingdom, IIUniversity of New South Wales, Sydney, Australia

Chitin synthesis and septum formation are highly regulated processes, fundamental for cell viability, taking place during cell division. Understanding fungal septation maylead to the identification of novel drug targets for future antifungal chemotherapeutic strategies. In the pathogenic fungus Candida albicans, a primary septum consisting ofchitin is formed between two dividing cells. In C. albicans, chitin is synthesised by four chitin synthases: Chs1, Chs3, Chs2 and Chs8 and all four enzymes localise tosites of septation.

Strains expressing pairs of fluorescently tagged chitin synthases were generated and assessed by live­cell fluorescence microscopy to elucidate the temporal and spatial distribution of the chitin synthases in relation to each other. It was observed that thetiming and pattern of configuration of the chitin synthases during septation is different in yeasts and hyphae with Chs3 potentially playing a role in secondary septumformation. This information was used to generate a new model for septal chitin synthesis during septation in C. albicans yeasts and hyphae.

Proteomic analyses of GFP pull down assays identified further proteins that may be part of the septation complex. Through Chs3­GFP immunoprecipitation, it wasshown that Chs3 may interact with different sets of proteins in yeasts versus hyphae. Moreover, the localisation of the chitin synthases in the absence of CaCdc10 andCaBni4 whose homologues are involved in tethering of ScChs3 at the septation site of S. cerevisiae was assessed. This study demonstrates that the septation model regarding the regulation and tethering of the chitin synthases to septation site is different between C. albicans and S. cerevisiae yeast cells, as well as between C. albicans yeasts and hyphae.

This study has extended our knowledge of how chitin synthesis is orchestrated during septation and identified proteins that might be involved in septum formation.

Role of the autophagy mechanism during infection of intestinal epithelialcells by Candida albicans.

A. Ducreux *I, P. Lapaquette *I, L. BasmaciyanI, T. ParadisI, F. DalleI

IVAlMis Unit ­ UMR PAM ­ University of Burgundy, Dijon, France

Candida albicans is an opportunistic pathogen causing infections ranging from superficial to the more life­threatening disseminated infections. In a susceptible host, C.albicans is able to reach and translocate through the gut barrier, promoting its dissemination in deeper organs. The polymorphic nature of this fungus is essential for itsvirulence since the C. albicans yeast­to­hypha transition is necessary for its effective invasion into host intestinal epithelial cells . C. albicans invades human epithelialcells by two well­documented mechanisms: the epithelial­driven endocytosis of hyphal forms and the C. albicans­driven active penetration. Host countermeasures tolimit C. albicans colonization and invasion include the secretion of anti­fungal peptides, the secretion of mucus and the recruitment of specific immune cells dedicated tomicroorganisms clearance. Autophagy is a lysosomal degradation process that contributes to host immunity by eliminating invasive pathogens and modulating theinflammatory response. The protective role of autophagy during C. albicans infection has been already investigated in myeloid cells (macrophages and dendritic cells)however far less is known regarding the role of this process during the infection of intestinal epithelial cells. In the present study we investigated the role of the autophagicprocess during the infection of intestinal epithelial cells by C. albicans. Using immunofluorescent staining against various autophagic markers, including the LC3 protein,we demonstrated that autophagy machinery is rapidly recruited at the Candida invasion site in various intestinal epithelial cell lines. We confirmed these observations byelectron transmission microscopy that reported the presence of autophagosomal structures in the vicinity of internalized hypha. Finally, we used autophagic­deficientepithelial cell lines and a panel of C. albicans mutants to specify the precise role of autophagy during invasion of epithelial cells by the fungus.

* The authors marked with an asterisk equally contributed to the work.

Copper and fluconazole: exploiting synergies to create effective drugs

A. Gaspar­CordeiroI, A. PetronilhoI, C. LeitãoI, C. PimentelI

IITQB­NOVA, Oeiras, Portugal

For centuries and until the advent of commercially available antimicrobials, copper has been widely used to prevent and treat microbial infections. Preliminary data fromour laboratory suggests a synergistic effect between copper and the antifungal fluconazole against the non­pathogenic yeast Saccharomyces cerevisiae. Furtherreinforcing this synergistic association, we found that a mutant strain, Sc Δfet3, previously shown to accumulate high amounts of copper, is hypersensitive tofluconazole. Because invasive fungal infections caused by opportunistic Candida sp. represent the most common fungal diseases among hospitalized patients receivingimmunosuppressive or intensive antibacterial therapies, we next tested whether such a potent association was observed against Candida glabrata. This yeast, whilephylogenetically close to S. cerevisiae causes life­threatening infectious diseases with an associated mortality rate of up to 50%. Consistent with a synergistic effect ofcopper and fluconazole against Candida glabrata, by performing checkerboard assays, we observed that copper decreases the MIC50 for fluconazole, and viceversa. Differently from S. cerevisiae, however, C. glabrata Δfet3 mutant is not sensitive to copper or hypersensitive to both drugs, indicating that a differentmechanisms of copper homeostasis is operational in this organism. Based on the above findings we explored whether the coordination of copper to fluconazole mightresult in a molecule with a higher fungicidal potential. The initial steps of complex synthesis and characterization pointed towards the formation of a complex with twofluconazole molecules coordinating one Cu(II) atom, which is currently being investigated.

Modification of Candida albicans cell wall by commensal gut bacteria.

E. BuzunI

IInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom

The human gut is populated with a vast community of microbes, the microbiota, which play a crucial role in health and disease. In addition to bacteria, fungi comprise0.1% of the total gut microbiota. Some of these fungi exist as benign members, however others such Candida albicans can undergo a pathogenic switch causingdisease in immunocompromised individuals. The fungal cell wall is the first target for immune system recognition. Recent studies have suggested that Candida isdecorated with different cell wall epitopes within different physiological niches, due to the impact of carbon source, oxygen availability, and pH, on cell wall remodelling. Here we hypothesize that, in addition to physiological cues, resident gut bacteria also play a major role in fungal cell wall remodeling and immune recognition.Bacteroidetes represent one of the two dominant phyla of gut bacteria are known to be able to degrade a wide range of complex carbohydrates. Data from our lab hasshown that a common bacterium from the gut, Bacteroides thetaiotaomicron (Bt), is able to breakdown the Candida cell wall and produces an extensive repertoire ofdegradative enzymes to access the α­mannan component. More recently, we have identified novel enzymes in Bt from the glycoside hydrolase family 130 (GH130),which specifically target β­1,2­linked mannan, a unique feature of Candida mannan. We have deleted multiple fungal mannan specific loci in Bt and examined the abilityof deletion strains to utilise Candida mannan as a carbon source. These data suggest that Bt contains multiple pathways to degrade the Candida cell wall. Now wehave an extensive repertoire of Bt mutants that impact on Candida mannan degradation, we are systematically dissecting the impact of such activities on the physiologyof the fungus. Understanding the enzymatic breakdown of Candida mannan by Bacteroidetes will provide insights into how two prominent members of the gutmicrobiota interact with each other. It will also further our understanding of how the Candida cell wall is modified in the anaerobic environment of the gut, and theimportance of this in promoting immune homeostasis.

Impact of vaginal candidiasis on the women mental health: microbiotaand pathogens role

C.V. TapiaI, M. CamposI, I. MoraI, E. SepúlvedaII, P. IbarraIII, P.I. RodasIV, F. MagneV

IClinica Davila, Santiago, Chile, IILaboratorio Vidaintegra, Santiago, Chile, IIIAlatheia Medical, Santiago, Chile, IVUniversidad Andrés Bello, Concepción, Chile,VUniversidad de Chile, Santiago, Chile

The human microbiota generates a beneficial relationship with the host and has an important impact on the health of the population. Vaginal candidiasis (VC) can causestress, embarrassment, discomfort, problems in the couple and anxiety. Objective: The aim of this study was determine if there is a relationship between the detectedmicroorganisms and the mental health of the patients, focused in patients with CV.Patients and Methods: We studied 78 women (age=31.5 years in average) who attended to Cínica Dávila to perform a vaginal discharge study (vaginal culture) and/orNAAT study of sexually transmitted infections (STI) pathogens. Samples were analyzed by traditional smear and vaginal swab culture and three molecular diagnostictests, one for BV (AllplexTM Bacterial Vaginosis Assay, Seegene), one for CV (AllplexTM Candidiasis Assay, Seegene) and one for STI (Anyplex™ II STI­7,Seegene). Patients were subjected to a survey of the Center for Disease Control (CDC, USA), which evaluates four parameters of mental health: 1) Lack of interest, 2)Depression (hopeless), 3) Anxiety (nervousness) and 4) Worrying. Results: The microorganism distribution in vaginal samples by conventional culture was: commensalmicrobiota 46.8%; Candida spp (17 C. albicans and 1 C. glabrata) 29.0%; G. vaginalis 16.1%; S. agalactiae 4.8% and L. iners 3.2. By the Bacterial Vaginosis(VB) PCR assay, the distribution of microorganisms was: Atopobium vaginae (38.9%), Megasphera 1 (33.3%), Bacterial vaginosis­associated bacteria 2 (19.4%)and Mobiluncus (8.3%) were detected by real­time PCR. All these pathogens were associated to VB or CV. The detected STI agents were: U. parvum (64.7%), U.urealyticum (17.6%), M. hominis (5.9%), C. trachomatis (5.9%) and T. vaginalis (5.9%). The only one associated with Candida isolation was U. parvum.Patients with normal commensal microbiota had a higher load of Lactobacilli by PCR than those with VC, BV and STI. Patients with CV, VB and STI presented agreater proportion of anxiety and worrying than those with commensal microbiota, being Candida the one that least affected mental health in these parameters.Conclusion: This is the first study the microbiota and agents causing VC, BV or STI with the mental status of patients. Candida spp. were associated with a greateralteration of mental health parameters however, less than other vaginal alterations. Further studies are needed to elucidate the involved mechanisms.

What can Candida auris do? : A murine GI tract colonisation model.

S.C. SimantirakisI, D.M. MacCallumI

IInstitute of Medical Sciences, Aberdeen, United Kingdom

Candida auris is a newly emerged fungal pathogen that was first identified in 2009. Since its identification, it has simultaneously emerged in four continents. C. auris hasbeen found to survive in the nosocomial environment on surfaces, such as bedding and hospital equipment. It has also been characterised as a human skin and nasalcavity coloniser.However, although there have been some studies to investigate the virulence of this new pathogen, to our best knowledge, there are no studies investigating C. auris’ability to colonise the gastrointestinal tract. In this study we investigate C. auris’ ability to colonise the gut in a murine model.In the study, we evaluate the effect of inoculum level on colonisation, comparing the fungal stool burdens using three different inocula (106, 107 and 108 CFU permouse) and investigate the effect of antibiotic pre­treatment. Furthermore we compare the colonisation ability of two different Southeast Asian clade strains. From ourresults with strain A, we show that C. auris is indeed capable of colonising the mouse gut and does so much more efficiently when the mice have been pre­treated withantibiotics, a finding consistent with similar studies conducted with Candida albicans. However, although the mice did not show signs of illness, C. auris strain A wasfound to have translocated to multiple organs during the 21 day experiment. This differs from what has previously been observed for the more pathogenic species, C.albicans.The comparison of the different inocula levels showed that the 106 and 107 inocula generated similar fungal burden levels in the stool and GI tract and similar levels oftranslocation to other organs, with similar colonisation levels for both inocula. However, the highest inoculum (108 CFU) demonstrated a drop in the stool burdens withmice eventually completely clearing the fungus on the final day of the experiment.The comparison of strain A with a second strain (strain B) demonstrated that strain A colonises the mouse gut at a significantly higher level than strain B and strain B wasless successful at translocating from the gut to other organs.Our findings show that is it possible that the GI tract could serve as a reservoir of the fungus in those exposed to C. auris.

In silico design and optimisation of IFNγ­mediated antifungal therapy

N. MotsiI, T. HammedII, R. TanakaII, E. BignellI

IUniversity of Manchester, Manchester, United Kingdom, IIImperial College London, London, United Kingdom

Inhalation of A. fumigatus spores can result in a spectrum of disease ranging from pulmonary allergies to invasive disease associated with up to 90% mortality.Experimental use of cytokine immunotherapy as an adjunct to antifungal drugs for the treatment of invasive and chronic aspergillosis has proved promising in the clinicalsetting.Here, we harness the complexity of the anti­aspergillus immune response by constructing a mathematical model of first­order ODEs that describe dynamically changingmechanisms involved in the innate and adaptive immune response to A. fumigatus challenge. We use this to explore the therapeutic mode of action of recombinantIFNγ, an immunotherapeutic agent with clinically proven utility as an adjunctive antifungal agent but for which maximally effective therapeutic regimens remainunexplored.Having selected and mapped the main immune system interplay that is relevant to IFNγ mode of action in healthy immune­mediated spore clearance. An extensiveliterature review yielded an initial parameter set which accurately describes the dynamics of spore inhalation, germination and hyphal growth in the face of a competentimmune response. Using the developed model, we will test the impact of various IFNγ regimens upon the outcomes of single­ and repeated­dose fungal exposures.Finally, we will evaluate the optimum adjunctive combination of antifungal drug therapy and immunotherapy.Modelling the mechanistic action of an IFNγ immunotherapy will allow us to investigate, without the use of animals, the role of IFNγ as an adjuvant for the treatment ofaspergillosis in combination with antifungal drug regimens.

Exploring the role of mitochondria in morphogenesis in response toHsp90 inhibition in Candida albicans

S. HossainI, T. O’MearaII, A. VeriI, L. CowenI

IUniversity of Toronto, Toronto, Canada, IIUniversity of California San Francisco, San Francisco, CA, United States of America

Fungal pathogens cause life threatening infectious disease, affecting billions of people and killing at least 1.5 million per year. One of the leading human fungal pathogensis Candida albicans, which can cause superficial infections or life­threatening systemic disease in immunocompromised individuals with mortality rates of ~40%. Thereare a limited number of clinically useful antifungal drugs, which are plagued by problems of severe host toxicity or by the emergence of resistance, demanding newtherapeutic strategies. One such strategy is to target virulence factors. The ability to transition between yeast and filamentous forms is a major virulence trait of C.albicans. A central regulator of filamentation is the molecular chaperone Hsp90, such that inhibition of Hsp90 leads to induction of this morphogenetic transition. Toexplore the mechanisms governing morphogenesis, we screened the C. albicans Gene Replacement and Conditional Expression (GRACE) library to identify mutantsthat are defective in filamentation in response to several inducing cues, including Hsp90 inhibition, serum, and elevated temperature. This genetic resource, which covers~40% of the genome, consists of mutants with one allele of each gene deleted and the remaining allele is under the control of a doxycycline­repressible promoter toenable target gene repression. Strikingly, we found that genes involved in mitochondrial function were enriched as being specifically required for filamentation in responseto Hsp90 inhibition, however, the basis of this requirement remains enigmatic. Specifically, genes involved in mitochondrial transport, mitochondrial translation, andaerobic respiration were necessary for filamentation. Consistent with the genetic evidence, pharmacological inhibition of the electron transport chain using antimycin A,rotenone and CCCP impaired morphogenesis in response to Hsp90 inhibition. However, inhibition of ATP synthase using oligomycin A did not affect morphogenesis.Our efforts to define the functional connection between mitochondrial function and morphogenesis revealed that Hsp90 inhibition lowered the reducing power infilaments without reducing the energy status in cells. This work is poised to provide mechanistic insights into how mitochondria influence morphogenesis and identify newregulators important for C. albicans virulence.

Synthetic oligosaccharides related to fungal cell wall polysaccharides:tools for investigation of molecular mechanisms of fungal recognition

V. KrylovI, D. ArgunovI, B. KomarovaI, A. KarelinI, Y. TsvetkovI, E. KazakovaI, D. YashunskiiI, S. WongII, V. AimaniandaII, T. FontaineII, J. LatgéII, N. NifantievI

ILaboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991, Moscow,Russia, IIUnité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724, Paris, France

The cell wall of pathogenic fungi is highly important for the pathogenesis of fungal infections and often acts as the first cellular component which interacts with the hostimmune system. The fungal cell wall is mainly built up of different polysaccharides representing the ligands for pattern recognition receptors on immune cells. Moreover,such polysaccharides are important antigenic determinants, responsible for recognition of fungal pathogens by specific poly­ and monoclonal antibodies. Purified fungalpolysaccharides are not easily available and have complex heterogenic structure. This complicates the use of polysaccharides as the probes to investigate the molecularmechanisms of fungal recognition. An alternative to natural polysaccharides are tagged synthetic oligosaccharides of definite structure representing distinctpolysaccharide fragments.Synthetic oligosaccharides were prepared with the use of specially developed approaches including pyranoside­into­furanoside rearrangement and specific methods of1,2­cis­glycosylation. As the result, a representative library of mycoantigenic carbohydrate ligands was constructed which included the oligosaccharides related topanfungal cell wall components (β­(1→3)­glucan, chitin), and species­specific antigens: (1) α­ and β–mannans (Candida); (2) galactomannan (Aspergillus); (3) N­galactosaminoglycan and α­(1→3)­glucan (Aspergillus); (4) Galactoxylomannan (Cryptococcus neoformans). The presence of a spacer group in theseoligosaccharides permits their efficient transformation into corresponding glycoconjugates (immunogens, coating reagents, labeled molecular probes) or quantitativeimmobilization on a plate or microbeads to form a surface that mimicks fungal cell­wall.In the present communication we describe the application of synthetic oligosaccharides for the investigation the immunological role of cell wall polysaccharides includingthe induction of cytokine and chemokines production profile and their recognition by antibodies from patient’s sera. Additionally, the designed glycoarrays wereemployed for the reassessment of carbohydrate specificity of monoclonal antibodies used in diagnostic kits. Described results indicate that chemically synthesizedoligosaccharides are indispensable tools for the detailed investigation of the immune response against polysaccharide components of the fungal cell wall.This work was supported by the RSF (19­73­30017).

Candida Species Induce Differential Macrophage­mediated ImmuneResponses

D.M. da FonsecaI, A. ThompsonI, R. AndrewsI, J.S. GriffithsI, L.C. DaviesI, P.R. TaylorI, S.J. OrrII

ICardiff University, Cardiff, United Kingdom, IIDivision of Infection and Immunity, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom,Cardiff, United Kingdom

Systemic candidiasis is a potentially life­threatening bloodstream infection caused by Candida spp. Highly virulent C. albicans is the main causative agent ofcandidaemia worldwide (62% of cases), and whilst other Candida spp. with inferior virulence like C. parapsilosis induce said pathology, these non­albicans specieshave much lower incidence (7% of cases). We and others have shown that healthy mice succumb to systemic infection with highly pathogenic C. albicans whilst theycan effectively clear the low virulence species C. parapsilosis. Here we compared the immune response of bone­marrow derived macrophages (BMDMs) stimulatedwith C. albicans and C. parapsilosis by RNAseq. Interestingly, we found that C. parapsilosis induces a higher inflammatory response in BMDMs than C. albicans.Type I Interferon signalling pathway was found to be strongly upregulated in C. parapsilosis­stimulated macrophages, but not with C. albicans. We explored theimpact of administering IFN­β during C. albicans infection of BMDMs and discovered that it enhances macrophages candidacidal activity. Furthermore, we haveshown previously that C. albicans secretes a soluble molecule that actively inhibits C. parapsilosis­induced IL­27 production in macrophages, downstream of IFN­β.We think that this molecule­mediated blockade poses as a novel mechanism deployed by C. albicans to bypass part of the immune response triggered by these cellsafter recognition of the fungal pathogen. We are currently trying to elucidate both the candidacidal mechanism of IFN­β and the C. albicans­induced blockingmechanism of IL­27 in BMDMs and assess its potential as a therapeutic target for candidiasis treatment.

Iron loading impairs fluconazole efficacy against Candida glabrata

S. CaetanoI, C. AmaralI, C. LeitãoI, P. Van DijckII,III, C. PIMENTELI

IInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal, IIVIB­KU Leuven Center for Microbiology, Leuven,Belgium, IIILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium

Invasive fungal infections (IFIs) caused by the yeast Candida, also named invasive candidiasis, represent the most common fungal disease among hospitalized patientsreceiving immunosuppressive or intensive antibacterial therapies. Among them, Candida glabrata infections are considered serious clinical threats due to the inherenttolerance of this yeast to azole antifungals.Preliminary data from our laboratory indicates that iron loading conditions decrease the antifungal activity of fluconazole against Candida glabrata and its closelyrelated yeast, Saccharomyces cerevisiae, unveiling a still unexplored association between iron and antifungal therapy. This finding puts at play the idea that excessiveiron in specific tissues and cells, besides promoting infection by switching off the nutritional immunity, may affect the treatment of IFIs within clinical settings of Fe­loading. We found that in S. cerevisiae this effect is no longer observed when the yeast iron detoxification pathway is impaired by disrupting the CCC1 gene. InCandida glabrata, however, deletion of the corresponding orthologous gene, CgCCC1, did not counteract the attenuated efficacy of the antifungal promoted by highiron. We therefore put forward the idea that another yet unidentified player might be mediating Fe­loading detoxification in C. glabrata. As a first approach to addressthis hypothesis we performed a phenotypic screening and found several new promising candidate genes whose products appear to play a role in Fe­loadingdetoxification and/or fluconazole tolerance.

Role of carboxylate transporters during carbon adaptation in Candidaglabrata

R. AlvesI,II,III, B. TimmermansIII,IV, M. Van EndeIII,IV, M. HenriquesV, M. CasalI,II, P. Van DijckIII,IV, S. PaivaI,II

ICentre of Molecular and Environmental Biology (CBMA), Department of Biology, School of Sciences, University of Minho, Braga, Portugal, IIInstitute of Science andInnovation for Sustainability (IB­S), University of Minho, Campus de Gualtar, Braga, Portugal, IIILaboratory of Molecular Cell Biology, Institute of Botany andMicrobiology, KU Leuven, Kasteelpark Arenberg 31, Leuven, Belgium, IVVIB­KU Leuven Center for Microbiology, Kasteelpark Arenberg 31, Leuven, Belgium,VCentre of Biological Engineering (CEB), Department of Biological Engineering, University of Minho, Braga, Portugal

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune­compromised individuals. In order to survive and successfullyproliferate in the different host niches, C. glabrata must rapidly adapt to a diverse range of environmental stresses and assimilate the available nutrients.For instance, during gastrointestinal and vaginal colonization, where glucose is scarce, alternative carbon sources such as acetate or lactate are particularly abundant andmay support the growth and the proliferation of C. glabrata cells. Our studies have demonstrated that the presence of these alternative non­fermentable carbon sourcesinfluence biofilm formation, antifungal drug resistance and immune recognition. Additionally, there is evidence that carboxylic acid transporters have an important impacton these processes.Here, we provide a detailed view on the role of putative C. glabrata acetate transporters during carbon adaptation. Our data support the view that adaptive responsesof Candida cells to alternative carbon sources affect their virulence, through multifarious mechanisms.

Candida albicans Ece1 peptides and the dual roles of Candidalysin

A. KönigI, L. DallyI, R. MüllerI, L. KasperI, S. MogaveroI, J. NaglikII, B. HubeI

IHans Knöll Institute, Jena, Germany, IIKing's College London, London, United Kingdom

One of the key virulence attributes of Candida albicans is its ability to undergo a morphological yeast­to­hypha transition. This transition is associated with theexpression of genes which facilitate, among others, adhesion, invasion, immune evasion and host cell damage. However, hyphae per se do not damage host cells. One ofeight core filamentation genes, which are consistently expressed under hypha­inducing conditions, is ECE1, the missing link between hypha formation and damage(Wilson et al. 2016). ECE1 encodes a protein consisting of eight short peptides. One of the resulting peptides is the toxin Candidalysin, which directly causes host celldamage (Moyes et al. 2016) and facilitates fungal translocation through epithelial barriers (Allert et al. 2018). Candidalysin is not only critical for fungal pathogenicity, itis also an important signal for the human immune system. Candidalysin has been identified as the hyphal moiety that triggers the epithelial “danger response” pathway(Moyes et al. 2010) and drives protective innate type 17 cell responses during oral candidiasis (Verma et al. 2017). In an excellent example of co­evolution, the host’sdanger response has thus evolved to sense Candidalysin or damage caused by this toxin as a signal for a defensive response. However, Candidalysin is also responsiblefor immunopathology during vaginal infections (Richardson et al. 2017). Furthermore, Candidalysin induces not only non­pyroptotic cell death in macrophages, but alsopotassium efflux driven NLRP3 inflammasome activation, thus causing recruitment of immune cells to the site of infection. This suggests that Candidalysin has dualfunctions as a classical virulence factor, but also as an immunomodulatory avirulence factor.While a major contribution of Candidalysin to C. albicans pathogenicity and immune response is clear, it remains unclear why Ece1 is structured as a polyprotein ofconcatenated peptides and why Candidalysin is embedded in a polyprotein. What is the role of the seven non­Candidalysin Ece1 peptides (NCEPs) in this structure?We suggest three different, non­exclusive, possible principal functions for NCEPs: (I) an immediate role in fungal biology, (II) a role in modulating Candidalysinfunctions, or (III) a role as effectors on host cells. Indications exist for each of these roles, but our preliminary data clearly suggest that NCEPs are required in fungalbiology.

Nucleic acids enrichment from yeasts in PBMC­yeast mixtures stored inRNAlater

A.J. Rodriguez SanchezI, M. VaneechoutteI

IUniversity of Ghent, Ghent, Belgium

Acquisition of the transcriptome of Candida albicans and related species during infection is a challenge due to the low proportion of fungal RNA in clinical samples.Total RNA content from Candida in the infection site, such as the vaginal environment, constitutes less than 1 % of human RNA. This has generated poortranscriptional profiles in which few differently expressed genes have been found to be involved in the infection. To address this issue, we compared three differentmethods to enrich fungal nucleic acids by using RLT buffer (Qiagen) with β­ Mercaptoethanol, Triton X­100 and bead beating respectively, from mixtures abundant inhuman cells, stored in RNAlater (Invitrogen), a solution that provides a condition in which transcriptional profiles remain unmodified and RNases are inhibited. Thesemethods are based on selective cell lysis of human cells, leaving fungal cells intact, because their cell walls protect against the lysis. Subsequently, a centrifugation stepenables nucleic acids from lysed human cells to be discarded. Fungal cells are then lysed with RiboPure Lysis Buffer and bead beating following nucleic acids extractionwith the RiboPure Yeast Kit (Invitrogen).

Real­time PCR to quantify nucleic acids from the pathogen and the host showed more than 90% reduction of human nucleic acids when samples were treated withTriton X­100 and more than 99% with RLT buffer + β­ Mercaptoethanol, indicating that the enrichment was successful. However, bead beating did not show anyimpact on the enrichment.

In summary, this enrichment method may be used to substantially increase the number of RNA reads from C. albicans in a mixture with abundant human cells and assuch provide a better understanding of the pathogen mechanisms, and facilitate the knowledge needed for the selection of targets for vaccines development or for theacquisition of new diagnostics tools in fungal diseases.

Characterization of Candida albicans GPI­anchored proteins involved inbiofilm formation.

M. El GhalidI, G. Pehau ArnaudetI, M. DupréI, M. DuchateauI, A. BelliI, M. ChauvelI, C. d'EnfertI, S. Bachellier­BassiI

IInstitut Pasteur, Paris, France

Infections due to Candida albicans have emerged over the past three decades as a considerable threat to human health. Biofilm formation is an important virulencetrait, allowing the fungus to persist within the host either on biotic or abiotic surfaces. Previous studies in our laboratory identified genes encoding putative GPI­anchoredproteins whose overexpression impacts adherence and biofilm formation. Our goal is to understand the specific contribution of one of them, Pga59, in biofilm formation,using structural and biophysical approaches.In silico analyses on Pga59 AA sequence led us to identify 3 domains predicted to be involved in the formation of amyloid fibrils (ß­sheet aggregation domains D1­D3).The recombinant his­tagged Pga59 (rPga59) was purified under denaturing conditions and submitted to biophysical tests. A spectroscopic assay using thioflavin­Tshowed increased fluorescence intensity, characteristic of amyloid­forming proteins. Furthermore, negative­staining and cryo­EM microscopy revealed the presence ofamyloid fibrils after incubation of rPga59 at 37°C for 48h, while incubation at 4°C led to the formation of different protein conformers (ring­shapes, prefibrillar oligomersand protofibrils) characteristic of prion­like proteins. Consistent with these observations, rPga59 exhibited SDS­resistant oligomers and high molecular weightaggregates after separation in denaturing SDS­PAGE.Using the in silico analysis tool TANGO, we determined that D1 presented the highest ß­aggregation potential. The involvement of D1 in aggregation was tested bycomparing two peptides ­either with a WT sequence or with a mutation at a predicted critical position­ in their ability to form amyloid fibrils. Overexpression of the D1­mutated protein in C. albicans resulted in a reduced cell­cell aggregation when compared to strains overexpressing WT Pga59.Overall, our study demonstrated the amyloid­forming properties of Pga59. Further experiments to link the amyloid­forming domain and adhesion properties/biofilmformation of PGA59 mutants are under way. Interestingly, this amyloid­forming domain is conserved in other cell­wall proteins, highlighting a possible role in modulatingtheir function and therefore their contribution to the biology and pathogenicity of C. albicans.

Activation of the zinc cluster transcription factor Stb5 in Candidaalbicans

B. Ramírez­ZavalaI, I. KrügerI, P.D. RogersII, J. MorschhäuserI

IInstitute for Molecular Infection Biology, Julius Maximilians University, Würzburg, Germany, IIDepartment of Clinical Pharmacy, University of Tennessee Health ScienceCenter, Memphis, TN, United States of America

The family of zinc cluster transcriptional regulators is present exclusively in the fungal kingdom. The pathogenic yeast Candida albicans posseses 82 members of thisfamily. Among them, Mrr1, Tac1, and Upc2 have been found to play an important role in the development of increased fluconazole resistance in this fungus. Gain­of­function mutations in Mrr1, Tac1, and Upc2, which result in constitutive overexpression of their target genes, are frequently identified in fluconazole­resistant C.albicans isolates. In a recent study we showed that an artificially activated form of another zinc cluster transcription factor, Stb5, confers resistance to the naturalcompound beauvericin by overexpression of the efflux pump YOR1, a member of the ATP­binding cassette transporter superfamily. Beauvericin is a mycotoxin withdiverse biological effects, and it has been shown that it potentiates the effect of azole drugs against Candida species. Although Yor1 did not contribute to fluconazoleresistance when C. albicans was treated with the drug alone, Stb5­mediated YOR1 overexpression diminished the synergistic effect of the fluconazole/beauvericincombination. It is not known by which signals Stb5 is normally activated and if it can be rendered constitutively active by natural gain­of­function mutations, as is thecase for Mrr1, Tac, and Upc2. We are currently addressing these questions using different approaches, the results of which will be presented.

Exploring the interactions between clinical Candida isolates andLactobacillus species

I. ZanglI, R. BeyerI, I. PapII, C. AspöckII, B. WillingerIII, C. SchüllerI

IDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria, IIInstitute for Hygiene and Microbiology,University Hospital, St. Pölten, Austria, IIIMedical University, Vienna, Austria

The human body is host to diverse and interacting microbial communities. In the vaginal milieu Lactobacillus ssp. are thought to exhibit a probiotic effect againstCandida overgrowth. To investigate such interactions the culture setup is in various aspects far from standard culture conditions. The nonstandard environmentalparameters include pH, glucose and nutrient availability, organic acids such as lactic acid and nonplastic surfaces. Based on previous experience we not only use labstrains but also a compendium of clinical isolates from local sources. Fungal and bacterial clinical isolates were collected by the University Hospital St. Pölten and theGeneral Hospital Vienna. Quantitative phenotypic tests were inoculated and performed in a robotic setup monitoring the growth curves. As expected, growthimpairment by lactic acid was highly dependent on the pH. At neutral pH lactic acid exposure lead to reduced growth while in low pH medium lactic acid couldefficiently inhibit growth of all Candida species. We tested our strains for their ability to grown on non­fermentative carbon­sources, such as lactate and glycerol. Wefound intraspecies variations and that C. krusei isolates are the most adaptable, growing on lactate and glycerol. Lactobacillus isolates were characterized by their abilityto reduce growth of Candida species. The fungistatic effect of lactobacilli is strain specific. We found one strain to significantly reduce the growth of most Candida spp.on solid media. C. albicans and C. parapsilosis strains were more susceptible against lactobacilli in vitro whereas C. glabrata and C. krusei species were moreresistant. We found a high degree of variation in the ability of different lactobacilli isolates to influence Candida growth. We further aim at a molecular characterization ofthe interactions of these commensals.

Mistranslation potentiates antifungal drug tolerance in Candida albicans

C. BrincoI, C. OliveiraI, E. LopesI, A.R. GuimarãesI, M. SantosI, A.R. BezerraI

IiBiMED – Institute of Biomedicine, University of Aveiro, AVEIRO, Portugal

Fungal infections are an important public health concern of the twenty first century due to increasing drug resistance and small number of antifungals available. Candidaalbicans is the most prevalent fungal pathogen and exhibits a unique translational system, misincorporating Leu (3%) at the atypical Ser­CUG codon. Exposure of C.albicans cells to antifungals increases Leu misincorporation levels up to 20% and the combination of macrophages with fluconazole increases it to approximately 50%.Hypermistranslating strains have increased tolerance to azole drugs, suggesting that mistranslation produces subpopulations of C. albicans cells that are reservoirs ofdrug tolerance from where resistance may emerge. To clarify whether antifungals may select subpopulations of tolerant cells we carried out in vitro competitionexperiments with fluorescently tagged C. albicans strains. A wild­type strain was tagged with mCherry while hypermistranslating strains were tagged with GFP whichallowed strain differentiation by observation of cell fluorescence. Cultures were cultivated over extended periods of time, with constant concentrations of fluconazole, tomimic the therapeutical regimens of the drug. Results showed an increase of hypermistranslator cells during evolution that concurred with changes in the tolerancephenotype of the population. The higher incidence of hypermistranslators after 400 generations matched the appearance of microcolonies within the inhibition ellipse ofthe E test. These preliminary results suggest that antifungal therapy selects drug tolerant clones through increased mistranslation. Further studies are currently beingperformed in order to clarify whether hypermistranslators could be associated with recurrent candidiasis.

This work is supported by the Portuguese Foundation for Science and Technology, POCI­ COMPETE2020 and FEDER through grants PTDC/IMI­MIC/5350/2014and UID/BIM/04501/2013.

Nitro­oxidative stress responses in A. fumigatus

E. ShekhovaI

IMRC Centre for Medical Mycology at the University of Aberdeen, Aberdeen, United Kingdom

A. fumigatus represents one of the major human fungal pathogens, which can cause severe invasive infections. To establish a disease, A. fumigatus has to evade orovercome oxidative bursts created by immune cells. Besides oxidative stress, host environments can also provoke nitro­oxidative stress. For instance, macrophages areable to produce nitric oxide (NO) through the activity of inducible NO synthase. During phagocytosis, NO can react with NADPH­oxidase derived superoxide to forma highly reactive nitrogen intermediate, peroxynitrite (ONOO­). The mechanisms by which A. fumigatus can prevent the toxic effects of ONOO­ remain unclear. Herewe describe a novel fungal­specific protein that is required for A. fumigatus to neutralize nitro­oxidative stress. Deletion of the gene encoding this protein resulted in anincreased sensitivity of the fungus towards oxidative­ and nitrosative stress­inducing agents. Moreover, we found out that the knock out strain was killed better byimmune cells compared to the wild type. Further investigation of nitro­oxidative stress responses in A. fumigatus may provide crucial information about host­pathogeninteractions, and thereby facilitate the development of antifungal treatments.

Introduction of a rapid, cloning free CRISPR/Cas9­based method to alterthe genome of the opportunistic human fungal pathogen C. parapsilosis

T. NemethI, A. GacserI,II

IDepartment of Microbiology, Interdisciplinary Excellence Centre, University of Szeged, Szeged, Hungary, IIMTA­SZTE “Lendület”“Mycobiome” Research Group,University of Szeged, Szeged, Hungary

Candida parapsilosis is a major fungal threat for low ­, very low ­ and extremely low birth­weight neonates. Systemic infection caused by this fungus is oftenassociated with high mortality rates even in developed countries. Similarly to Candida albicans, it is also a diploid organism, belongs to the CUG clade and lackscomplete sexual cycle that hampers genetic modification. To overcome this issue a plasmid based CRISPR/Cas9 method was published recently that provided awonderful tool to alter a single nucleotide and even delete complete ORFs in this species (Lombardi et al., 2017). Here we present an alternative cloning freeCRISPR/Cas9 system that is based on the work of Nguyen and colleagues and originally was developed for C. albicans (Nguyen et al., 2017). It utilises only PCR forgRNA generation along with in vitro cassette assembly that targets HIS1 allele. The construct is flanked by FRT sites, therefore it can be removed once flippase isactivated. To maintain prototrophy selection on minimal media is needed when additional alterations are required. This approach seemed to be successful in changing orinserting nucleotide(s) at specific regions and to delete and nucleotide precisely reintegrate an ORF in C. parapsilosis. Additionally, we were able to GFP­tag the strainand overexpress a gene, and it also has the potential to GFP or HIS­tag proteins. We hope that this rapid, cloning free approach provides a useful tool for the Candidacommunity to alter C. parapsilosis genome.This project was funded by the Hungarian Academy of Sciences “Momentum” Programme LP2018­15/2018, UNKP­17­4 New National Excellence Program Of TheMinistry Of Human Capacities and EFOP­3.6 1­16­2016­00008, TN was supported by National Talent NTP­NFTÖ­18 fellowship.

Arachidonic acid increases expression of CDR1 in C. albicans, butinhibits efflux activity

O. KuloyoI, R. FourieI, J. AlbertynI, C. PohlI

IUniversity of the Free State, Bloemfontein, South Africa

Candida albicans is a commensal of humans that can cause opportunistic infections with high morbidity and mortality. This polymorphic yeast produces antifungalresistant biofilms. One mechanism associated with this increased resistance to antifungal agents, such as fluconazole, is the overexpression of ABC efflux pumps,including Cdr1p. Addition of arachidonic acid, a polyunsaturated fatty acid, to azoles has been shown to significantly increase azole susceptibility of C. albicansbiofilms. However, the underlining mechanism is not fully known. Surprisingly, transcriptome analyses of biofilms exposed to arachidonic acid alone, fluconazole aloneor a combination of fluconazole and arachidonic acid indicated a dose dependent, significant increase in CDR1 expression during early biofilm formation in the presenceof arachidonic acid. However, the efflux activity of these proteins, measured by Rhodamine 6G efflux assay, was severely diminished in the presence of arachidonic acid,even in the presence of fluconazole. In addition, in strains with fluconazole­induced resistance, this resistance to azoles was reversed with the addition of arachidonicacid. This phenomenon may be associated with a disruption in membrane/lipid raft organisation and subsequent delocalisation of Cdr1p to the cytoplasm in the presenceof arachidonic acid, as visualised using CDR1­GFP tagged cells. This not only highlights the importance of the cell membrane in the function of membrane associatedproteins such as drug efflux pumps, but also reminds us that differential expression of genes does not always correspond to differences in functionality.

Involvement of Candida glabrata trehalase enzymes during stressconditions

M. Van Ende *I,II, B. Timmermans *I,II, P. Van DijckI,II

IVIB­KU Leuven Center for Microbiology, Leuven, Belgium, IILaboratory of Molecular Cell Biology, Leuven, Belgium

Candida glabratais a commensal in most people but can cause a deadly infection in immunocompromised patients. The incidence of infections by this human fungalpathogen is on the rise. One of the reasons for this is its high resistance against a variety of stress factors encountered in the human host. These include the host immunesystem, antifungal drug treatment and the competition with the other microbiota. The trehalose metabolism has been described to be involved in this stress response andis considered to be a potential antifungal drug target as it is not present in mammals and is conserved over several pathogenic fungi. Trehalose is synthesized by trehalosesynthase and trehalose­6­phoshate phosphatase under stress conditions, where it is used to protect and preserve cellular functions. After relief of stress, trehalose isbroken down by trehalase in order to rapidly resume growth. Currently the role in stress tolerance for each of the three trehalase enzymes is unclear. Therefore, differentmutant strains were constructed to investigate the effect of different stresses such as: cell wall stresses, heat stress, oxidative stress, osmotic stress, antifungal stress, pHstress and nutrient starvation. In a collaborating project, the cellular localization of the three different trehalases is determined in order to understand their mode of action.Additionally, clinically relevant conditions (biofilm formation and in an in vivo murine model for systemic infection) are investigated for the different mutantstrains. Unravelling the role of the different trehalase enzymes will lead to new insights in pathogenesis and can possibly lead to the development of new antifungal drugsin the future.

* The authors marked with an asterisk equally contributed to the work.

The Candida glabrata CgHaa1­dependent system is required for biofilmformation, adhesion to epithelial cells and maximal virulence againstGalleria mellonela

S.B. SalazarI, S. SilvaII, D. Mil­HomensI, A.I. PimentaI, H. ChibanaIII, J. AzeredoII, A.M. FialhoI, N.P. MiraI

IiBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico – Department of Bioengineering, Universidade de Lisboa, Lisboa, Portugal, IICentre ofBiological Engineering, University of Minho, Braga, Portugal, IIIMedical Mycology Research Center, Chiba University, Chiba, Japan

To thrive in the acidic vaginal tract and avoid exclusion C. glabrata cells have to face multiple environmental challenges, including the presence of acetic and lactic acidsproduced by the commensal microbiota, availability of nutrients, among others. Response and tolerance of C. glabrata to acetic acid at a low pH (but not to lactic acid)was recently described by our group as being largely dependent on the regulatory system controlled by the transcription factor CgHaa1. In this work the involvement ofCgHaa1 in this adhesiveness phenotype and in the process of biofilm formation under acetic acid stress was further investigated. Deletion of CgHAA1 significantlyimpaired formation of biofilm by C. glabrata cells in the presence of physiologically relevant concentrations of acetic acid (at pH 4). This phenotype was linked to theCgHaa1­mediated up­regulation of CAGL0H07469g and CAGL0K10164g, two poorly characterized adhesins that were also found to be required for maximalbiofilm formation in the presence of acetic acid. Consistently, in the presence of acetic acid deletion of CAGL0H07469g and CAGL0K10164g reduced C. glabrataadhesion to vaginal epithelial cells (by 57 and 87.1%, respectively), consistent with the same adhesiveness effect that was also registered for the ΔCgHaa1 mutant. Wehave also investigated the role of the CgHaa1­regulatory system in systemic infection of Galleria mellonella. The deletion of CgHaa1 reduced 21% the mortality rateof C. glabrata­infected larvae, correlating with the reduced survival of ΔCgHaa1 in heamocytes. The previously identified CgHaa1­target genes CgBAG7,CAGL0G05632g, CAGL0E03740g, CgAWP12, CgAWP13 and CAGL0K10164g, were also found to increase virulence of C. glabrata against G. mellonella andsurvival inside heamocytes. Altogether the results suggest that the CgHaa1 may not only be relevant for vaginal virulence of C. glabrata, but also for systemic infectionprompted by this species.

Acknowledgments

Funding received by the Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2013), from PORLISBOA 2020 (project no. 007317) and from thePhD programBIOTecnico (grant PD/BD/135139/2017 to SBS) is acknowledged.

Cbk1 mediated phosphorylation regulates hyphal morphogenesis inCandida albicans at two different events.

R. WakadeI, D. Krysan I, M. WellingtonI

ICarver College of Medicine, University of Iowa, Iowa City, IA, United States of America

The ability to switch between yeast and hyphal form of the human fungal pathogen, Candida albicans, is critical to invade and thereby, establish virulence in the host.Several environmental cues trigger this transition through distinct signaling pathways. One pathway is the Regulation of Ace2 and Morphogenesis (RAM) pathway whichis highly conserved across all eukaryotes. Previous studies have shown that Cbk1, the critical serine/threonine kinase of the RAM pathway, is required for the hyphalgrowth and pathogenicity. Yet, very little is known about the importance of well­defined Cbk1 substrate motifs and we noticed that 27 transcription factors (TFs)contain Cbk1 consensus phosphorylation motifs. Thus, we hypothesised that Cbk1 might regulate distinct TFs via these phosphorylation sites. To test this hypothesis,we used CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) strategy to generate phosphodeficient mutants in the candidate Cbk1 substrate. Ourinitial results indicate that substituting serine/threonine (S/T) to alanine (A) in NRG1 and ACE2 phenocopied the classical nrg1 and ace2 mutant phenotype.Interestingly, the phosphodeficient NRG1 mutant predominantly formed filamentous under non­inducing conditions compared to that of wild­type (Wt) cells. In contrast,non­phosphorylatable ACE2 mutant showed morphological changes to the hyphal tip and formed the lateral bud. These results suggest that Cbk1 regulate two distinctsteps during the hyphal morphogenesis. First Cbk1 phosphorylation is required for Nrg1 repression of filamentation and second hyphal maintenance by activating Ace2.Ongoing studies are currently being carried out to understand the importance of Cbk1 mediated phosphorylation with the remaining TFs and its role in regulatingpathogenicity in C. albicans.

Developing a novel antifungal strategy by targeting an epigenetic readerdomain against Candida glabrata

K. WeiI, N. DupperII, Y. ZhouIII, M. ChamplebouxIV, F. MiettonV, C. GarnaudVI, M. CornetVI, C. McKennaIII, J. GovinVII, C. PetosaI

IInstitute de Biologie Structurale, CNRS/CEA/UGA, 71 Avenue des Martyrs, Grenoble, France, Grenoble, France, IIUniversity of Southern California, Department ofChemistry, Los Angeles, United States of America, Los Angeles, California, United States of America, IIIUniversity of Southern California, Department of Chemistry,Los Angeles, United States of America, Los Angeles, United States of America, IVInstitut de Biosciences et Biotechnologies de Grenoble, Laboratoire Biologie àGrande Échelle, Université Grenoble Alpes, CEA, Inserm, Grenoble, Grenoble, France, Vnstitute de Biologie Structurale, CNRS/CEA/UGA, 71 Avenue des Martyrs,Grenoble, France, Grenoble, France, VITIMC­IMAG TheREx, Université Grenoble­Alpes, Grenoble, France, VIIInstitute for Advanced Biosciences, UniversitéGrenoble Alpes, Inserm, Grenoble, France, Grenoble, France

Candida glabrata is a pathogenic yeast associated with a high rate of morbidity and mortality in immunocompromised patients. An alarming rise in drugresistantstrainsover the past decade has led to an urgent need for novel therapeutic strategies. This project concerns a potential new antifungal strategy that targets the protein Bdf1, amember of the BET family of transcription factors. BET proteins associate with chromatin through their two bromodomains (BD1 and BD2), epigenetic "reader"modules that recognize acetylated lysine residues in histones. The overall goal of this project is to validate Bdf1 bromodomains (BDs) as an antifungal drug target in C.glabrata. Specific objectives are to: (i) verify that Bdf1 BD function is essential in C. glabrata; (ii) identify and characterize compounds that selectively inhibit Bdf1BDs; and (iii) demonstrate their potential for translational development into a novel class of antifungal drugs using a mouse model of invasive candidiasis.Thus far, I have shown that the two BDs of Bdf1 are essential for the growth of C. glabrata in vitro. I determined crystal structures of Bdf1 BD1 and BD2, whichreveal that their ligand binding pockets are stereochemically distinct from those of human BDs. I developed a homogeneous timeresolvedFRET (HTRF) assay toidentify Bdf1 BD inhibitors and used it to screen a library of >100,000 compounds. This identified several compounds that selectively inhibit the fungal BDs comparedto the human homologs. Current work is aimed at characterizing these inhibitors (and related analogs) in biochemical, biophysical, and fungal growth inhibition assaysand at determining crystal structures of inhibitorboundBDs. The most promising inhibitors will be then tested for antifungal activity in a mouse model of invasivecandidiasis.Successful completion of this work will establish proof of principle that BET inhibition is a valid antifungal therapeutic strategy in C. glabrata.

The sugar metabolism of Candida albicans and its role in virulence

S. WijnantsI,II, P. Van DijckI,II

ILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium, IIVIB­KU Leuven Center for Microbiology, Leuven,Belgium

Candida albicans is an opportunistic fungal pathogen and is the most isolated Candida species. To colonize the human body this pathogen utilizesdifferent virulence factors, for example morphogenesis, adhesion and biofilm formation. Some of these virulence factors are under control of the PKA pathway. Thispathway can be activated by different external stimuli, for example glucose. The exact mechanism of how glucose has an effect on PKA is not fully understood, butCaCdc25 and CaRas play an important role in this mechanism. Over the last years it became more and more clear that central metabolism, such as glycolysis, plays animportant role during the virulence of this pathogen. For example, virulence is strongly attenuated when the activators of glycolytic enzymes are removed. Furthermore,the glycolytic pathway is upregulated during virulence conditions. We are investigating whether an abolished glycolysis also has an effect on PKA activation. For this, weaim to disrupt this pathway by blocking the first essential step, which is the conversion of glucose into glucose­6­phosphate. Based on the current knowledge about thisstep in Saccharomyces cerevisiae where ScHxk2 is the major enzyme involved, we used the protein sequence of this enzyme to identify orthologous enzymes in C.albicans. We identified four enzymes with high similarity, namely CaHxk1, CaHxk2, CaGlk1 and CaGlk4. Apart from CaHxk1, which was shown to be involved inN­acetylglucosamine metabolism, these hexose kinases have not been described before. We generated single, double, triple and quadruple deletion strains in order todetermine the substrate specificities of the different kinases as well as their catalytic activities. Furthermore, we tested these deletion strains using different virulenceassays to see which genes may play a role in virulence. Finally, we wanted to see which deletion strains had less PKA activation compared to the wild type strain uponaddition of specific sugars. These experiments will allow us to investigate the connection between glycolysis and activation of the PKA pathway in more detail, possiblyresulting in the identification of novel antifungal drug targets.

Monitoring of fluconazole and caspofungin activity against in vivoCandida glabrata biofilms by bioluminescence imaging

O. RogiersI,II, A. Persyn *I,II, M. BrockIII, G. Van De VeldeIV, M. LamkanfiV, I.D. JacobsenVI, U. HimmelreichIV, K. LagrouVII, P. Van DijckI,II, S. KucharikovaVIII

ILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium, IIVIB­KU Leuven Center for Microbiology, Leuven,Belgium, IIIUniversity of Nottingham, Nottingham, United Kingdom, IVBiomedical MRI/ MoSAIC, Department of Imaging & Pathology, KU Leuven, Leuven, Belgium,VCenter for Inflammation Research, UGent­VIB, Gent, Belgium, VILeibniz Institute for Natural Product Research and Infection Biology ­ Hans Knoell Institute, Jena,Germany, VIIDepartment of Microbiology and Immunology, Laboratory of Clinical Bacteriology and Mycology, KULeuven, Leuven, Belgium, VIIIVIB­KU LeuvenCenter for Microbiology; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium

Despite its inability to from hyphal filaments, Candida glabrata can adhere to various medical implants and forms thick biofilms. Moreover, C. glabrata biofilm­associated infections are intrinsically resistant to conventional treatment. Current in vivo C. glabrata biofilm models only provide limited information about infectiondevelopment and usually require animal sacrifice. To gain real­time information from individual BALB/c mice we developed a non­invasive imaging technique to visualizeC. glabrata biofilms in catheter fragments that were subcutaneously implanted on the back of mice. Bioluminescent C. glabrata reporter strains (lucOPT7/2/4 andlucOPT8/1/4), free of auxotrophic markers, expressing a codon­optimized firefly luciferase were generated. A murine subcutaneous model was used to follow real­timein vivo biofilm formation in the presence and absence of fluconazole and caspofungin. Fungal load in biofilms was quantified by colony forming unit counts and bybioluminescence imaging (BLI). C. glabrata biofilms formed within the first 24 h, as documented by the increased number of device­associated cells and elevatedbioluminescent signal compared to adhesion at the time of implant. The in vivo model allowed monitoring of the anti­biofilm activity of caspofungin against C.glabrata biofilms through bioluminescent imaging from day four after initiation of treatment. Contrarily, signals emitted from biofilms implanted in fluconazole­treatedmice was similar to the light emitted from control­treated mice. This study gives insights into real­time development of C. glabrata biofilms under in vivo conditions.BLI proved to be a dynamic, non­invasive and sensitive tool to monitor continuous biofilm formation and activity of antifungal agents against C. glabrata biofilmsformed on abiotic surfaces in vivo.

* The authors marked with an asterisk equally contributed to the work.

Improving Candida microscopy through codon­optimized fluorescentproteins and sub­diffraction microscopy

W. Van GenechtenI,II, L. DemuyserI,II, P. DedeckerIII, P. Van DijckI,II

ILaboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium, IIVIB­KU Leuven Center for Microbiology, Leuven,Belgium, IIILab for Nanobiology, Leuven, Belgium

Fluorescent proteins with varying colors and microscopy are indispensable tools for the life sciences research community. In this contribution, we present a palette ofCandida­optimized fluorescent proteins ranging from cyan to red. We also compare a range of reported expression optimization techniques, and find that none of thesestrategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. One of thesecodon­optimized fluorophores is a photoswitchable fluorescent protein, allowing sub­diffraction microscopy methods, such as pcSOFI, to be applied in C. albicanslocalization microscopy. This technique allows us to visualize proteins of interest that have never been seen before on an endogenous level.

Cystic fibrosis leads to hyper inflammatory NFAT­ and NFkB­drivenmacrophage responses to Aspergillus fumigatus

L. Gonzales­HuertaI, A. Bercusson *I, A. Warris *II, D. Armstrong­James *I

IImperial College London, London, United Kingdom, IIMRC Centre for Medical Mycology at University of Aberdeen, Aberdeen, United Kingdom

Cystic fibrosis confers high risk of persistent airway infection with Aspergillus fumigatus, culminating in either Aspergillus bronchitis or allergicbronchopulmonary aspergillosis in up to 30% of individuals by adulthood. In previous studies we have shown an important role of calcium­drivencalcineurin­NFAT responses in macrophage signalling responses to Aspergillus fumigatus infection. As previous studies have shown that cysticfibrosis leads to excessive store­operated calcium flux, we hypothesised that over activation of the macrophage calcineurin­NFAT pathway couldaccount for some of the hyper inflammatory response to Aspergillus fumigatus previously described for cystic fibrosis. Consistent with this, weobserved that Aspergillus fumigatus infection led to heightened NFkB and NFAT responses in CFTR­/­ bone marrow derived macrophages. Thiswas associated with increased inflammatory cytosine responses, as well as impaired fungal killing. Furthermore, calcineurin­dependentmacrophage necrosis was also increased in cystic fibrosis macrophages. Correlation of our findings in vivo in gut­corrected CFTR KO miceindicated that airway baseline macrophages were increased, and that airway challenge with Aspergillus fumigatus led to excess inflammatoryresponses as well as increased airway macrophage cell death compared to the wild type parental control mice. This was associated withincreased airway fungal burden as well as impaired solution of infection at later time points. Taken together these observations suggest that cysticfibrosis results in fundamental changes in innate signalling responses as a consequence of altered calcium homeostasis.

* The authors marked with an asterisk equally contributed to the work.

Rapid detection of Candida species resistant to echinocandins byMALDI­TOF MS within 6 hours

M. VatanshenassanI, K. SparbierI, T. BoekhoutII, C. Lass­Flörl III, J. F. Meis IV, J. BermanV, M. Kostrzewa *I

IBruker Daltonic Company, Bremen, Germany, IIInstitute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, Netherlands, IIIDivision ofHygiene and Microbiology, Medical University, Innsbruck, Austria, IVDepartment of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital(CWZ), Nijmegen, The Netherlands and Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, Netherlands, VDepartment of Molecular Microbiology &Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

The widespread use of echinocandins for treatment of candidemia has enhanced the development of antifungal­resistance. Hence, the rapid detection of resistant strainsis required. Recently, the MALDI Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA) has been described as a novel semi­quantitative technique forsusceptibility testing. Here, the susceptibility testing of C. albicans, C. glabrata and C. auris against echinocandins by MBT ASTRA is evaluated.A total of C. albicans (susceptible= 36, resistant= 22), C. glabrata (susceptible= 5, resistant= 33, intermediate= 19) and C. auris (susceptible= 44, resistant= 6) wereanalysed. In vitro antifungal susceptibility testing was performed by twofold serial dilutions of the caspofungin for C. albicans and C. glabrata, and against micafungin andanidulafungin for C. auris according to the current CLSI guideline M60 (November 2017). Twofold serial dilutions of caspofungin (0.125 to 4 µg/ml), micafungin (0.5 to32 µg/ml) and anidulafungin (0.06 µg/ml and 4 µg/ml) plus a control without antifungal were used in MBT ASTRA. After 6 hours incubation, cells were lysed andspotted in duplicate. Dried spots were overlaid with MALDI matrix containing an internal standard. Spectra were acquired in the mass range between 2 and 20 kDaand analyzed by MS­ASTRA prototype software.Caspofungin breakpoints for MBT ASTRA were found as follows: C. albicans (susceptible = 1 µg/ml and resistant > 1 µg/ml), C. glabrata (susceptible = 0.5 µg/ml andresistant > 0.5 µg/ml) at cut­off value of 0.6, respectively. For C. auris, breakpoints for anidulafungin were found at S= 4 µg/ml and resistant > 4 µg/ml and formicafungin at S= 8 µg/ml and resistant > 8 µg/ml applying a cut­off value of 0.7. A categorical agreement (CA) of 100% and 93% was calculated for C. albicans and C.glabrata, respectively. Sensitivity and specificity were 100%, 94.2% and 80% for C. albicans and C. glabrata, respectively. For C. auris, a high agreement of 98%, andsensitivity and specificity of 100% and 98% were calculated against anidulafungin, respectively. Accordingly, a categorical agreement of 96%, and sensitivity andspecificity of 100% and 95.5% were calculated against micafungin.MBT ASTRA is a new approach that facilitates rapid antifungal susceptibility testing within 6 hours, and potential to be applied for other species and antifungals inclinical laboratories.

* The authors marked with an asterisk equally contributed to the work.

Generation and isolation of Candida albicans specific llama single­domain antibodies (VHHs) by whole proteome immunization and phagedisplay

F.M. SauerI, G. Buda De CesareII, A. StavrouIII, A. KoleckaIII, C.A. MunroII, T. VerripsI, T. BoekhoutIII, E. DolkI

IQVQ Holding B.V. , Yalelaan 1, 3584 CL, Utrecht, Netherlands, IIAberdeen Fungal Group, MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen,United Kingdom, IIIWesterdijk Fungal Biodiversity Institute, Utrecht, Netherlands

Candida spp. are usually benign colonizers of human mucosa. In case of a compromised immune system they frequently turn pathogenic and cause lethal disease. Themost common opportunistic pathogen of this group is Candida albicans (C. albicans). Timely identification of the infection and pathogen is essential for survival andspecies­specific antibodies could improve or complement current diagnostics.For generation and isolation of C. albicans specific antibodies llamas were immunized with different morphological forms of C. albicans (whole proteome approach).Then llama single­domain antibody (VHH) phage display libraries were created and phage display selections were conducted on whole viable/inactivated C. albicanscells or extracts thereof. ELISA screening of C. albicans cell binding VHHs against the close relative C. dubliniensis allowed for isolation of apparently species­specific VHHs. Intraspecies binding was confirmed by ELISA with a set of clinical C. albicans isolates whose identity had been verified by MALDI­TOF MS. Nobinding could be detected to the closely related species C. gigantensis, C. bohiensis and C. buenavistaensis in ELISA.The clinical relevance and species specificity of selected VHHs was further evaluated in a blinded ELISA set­up: 95 (clinical) isolates of Candida spp. (albicans,dubliniensis, tropicalis, parapsilosis, glabrata, krusei, lusitaniae) were screened and C. albicans could be unequivocally detected.Immunfluorescence microscopy of viable cells with selected VHHs showed that antigen mainly localizes in the yeast body. Complementary hyphae specific VHHs havealso been selected from the VHH libraries and bind exclusively to hyphal elongations of filamentous C. albicans cells. Preliminary ELISA screening of cell wall mutantssuggests involvement of mannosylations in binding of most VHHs.These results indicate that whole proteome immunization followed by phage display selection is a practical approach for isolation of species­specific VHHs.

Variability of biofilm formation and macro morphology among Candidaparapsilosis clinical isolates

E. Gomez­MoleroI, A. MorenoII, J. WillisIII, I. DelapintaIV, N. TiboV, H. DekkerVI, A. DudakovaI, A. GacserV, M. WeigI, U. GroßI, G. QuindosIV, P. de GrootII, T.GabaldónIII, O. BaderI

IInstitute for Medical Microbiology, University Medical Center Göttingen, Göttingen, Germany, IIRegional Center for Biomedical Research, Albacete Science &Technology Park, University of Castilla–La Mancha, Albacete, Spain, IIIComparative Genomics Group, CRG­Centre for Genomic Regulation, Barcelona, Spain,IVDepartment of Immunology, Microbiology and Parasitology, School of Medicine and Dentistry, Universidad del País Vasco (UPV/EHU), Bilbao, Spain, VDepartmentof Microbiology; University of Szeged, Szeged, Hungary, VISwammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, NL­1098 XHAmsterdam, the Netherlands, Amsterdam, Netherlands

The human pathogenic yeast Candida parapsilosis has gained significant importance over the past decades as one of the principal causes of fungal bloodstreaminfections. Its capacity to adhere to host cells and clinically used abiotic surfaces (e.g. prostheses or catheters) is crucial during colonization processes and theestablishment of infections. Isolates of C. parapsilosis are known to be able to switch between several different colony morphologies in vitro, which are correlated withdifferent cell shapes and altered cell surface properties.Prompted by a set of clinical specimen from a single patient that yielded stable smooth­ as well as crepe­morphology lineages, we investigated the differences betweenthese 5 linages and characterized the phenotypic spectrum as well as underlying mechanisms in >300 different­morphology clinical isolates.In contrast to the initial assumption that the 5 different patient isolates were switched forms of an otherwise clonal strain, we found by genome sequencing that the patientwas colonized by at least three distinct linages of C. parapsilosis. Detailed statistical analysis placed the linages distantly across the population of C. parapsilosis.Interestingly the Candidemia­associated blood culture isolate was of smooth morphology and matched with an isolate from the patient’s nose of similar morphology;however the BC­isolate had undergone significant genomic rearrangements.Next we analyzed biofilm formation capacity and differences in morphotypes among several hundred clinical isolates which showed a multitude of phenotypiccombinations, but no correlation with site of isolation was evident. Differences in colony morphology correlated with biofilm formation capacity and agar invasion, butnot necessarily with virulence in a galleria infection model. Drug susceptibility profiles were altered between groups of biofilm­forming vs. non­forming isolates.In addition to a core cell wall proteome consisting of carbohydrate­active enzymes a set of adhesins were detected in the cell walls of selected hyperbiofilm­formingisolates by mass spectrometry. Downstream analyses showed distinct correlations of phenotypes such as altered virulence or morphology with adhesinincorporation. Incorporation of specific adhesin proteins into the cell wall of C. parapsilosis is associated with colony morphology which governs clinical parameterssuch as biofilm formation capacity and alters drug susceptibility.

C. glabrata evolution towards posaconazole resistance irrespective ofPDR1 GOF mutations

M. GalochaI, A. Silva­DiasII, P. PaisI, M. CavalheiroI, I. M. MirandaII, H. ChibanaIII, A. G. RodriguesII, M. C. TeixeiraI

IciBB­Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal, IIDepartmentof Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal CINTESIS­Center for Health Technology and Services Research, Faculty of Medicine,University of Porto, Porto, Portugal, Porto, Portugal, IIIMedical Mycology Research Center, Chiba University, Chiba, Japan, Chiba, Japan

Candida glabrata is an emerging fungal pathogen that constitutes the second most common causative agent of candidiasis worldwide. Its increasing prevalence is mostlyrelated to its ability to rapidly develop azole resistance. The central mechanism of acquired resistance to azoles is the hyperactivation of the transcription factor PDR1through the emergence of Gain Of Function (GOF) mutations, resulting in the upregulation of drug efflux pumps.Posaconazole has the broadest antifungal spectrum of available triazole antifungal medications and the development of resistance is less well understood, whencompared to other azoles. In this work, in vitro directed evolution was used to force four azole susceptible clinical isolates towards posaconazole resistance, throughprolonged exposure to serum concentrations of posaconazole. Remarkably, the PDR1 gene of each evolved posaconazole­resistant isolate was sequenced and no GOFmutations were found. To understand the origin of the resistant phenotype, the whole genome of the resistant isolates was sequenced, in the search for common relevantSNPs, when compared to the susceptible parental isolates. 12 genes were found to display non­synonymous mutations in three of the resistant isolates, when comparedto the susceptible isolates. The functional characterization of these genes is ongoing in order to understand the mechanism(s) that enabled these isolates to becomeresistant to posaconazole.Current results provide in vitro evidence that induction of azole resistance in C. glabrata, at least to posaconazole, does not necessarily lies on the emergence of PDR1GOF mutations.

The Candida albicans acetylome: concepts, challenges and therapeuticopportunities

R. ShivarathriI, M. ChauhanII, S. JenullI, M. TschernerI, K. KuchlerI, N. ChauhanIII

IDepartment for Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria, Vienna, Austria, IIPublicHealth Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States of America, IIIRutgers University,Newark, United States of America

Posttranslational modification (PTM) of proteins is a critical and ubiquitous means of regulating protein function. One of the most prominent PTMs in eukaryotes is lysineacetylation, which plays a key role in many processes, most notably regulation of gene expression by histone modification and modulation of chromatin function. Despiteimpressive recent advances in our understanding of cellular roles of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), the role of reversible lysineacetylation in infectious disease still remains poorly understood. We present data that suggest that lysine acetylation of proteins is a major mechanism that regulatesmorphogenesis in C. albicans. We performed proteomics experiments to identify acetylated proteins in C. albicans. Ten milligrams of protein from C. albicans WT strainSC5314 grown to log­phase at 300C were used for the experiment. We performed in­solution trypsin digestion and the resulting peptides were desalted andlyophilized. The acetylated peptides were enriched and analyzed by LC­MS/MS. The MS/MS spectra were searched against a C. albicans database(www.candidagenome.org) using a local MASCOT search engine (V.2.3). Using this approach, we discovered that proteins involved in diverse biological processes,including pathogenesis, were acetylated in C. albicans. In this study, we present data on the effect of acetylation on the function of DNA ligase 4 (Lig 4). Lig4 plays akey role in DNA double strand break (DSB) repair by non­homologous end joining (NHEJ). To determine the effect of acetylation of Lig4 in morphogenesis andvirulence we constructed Lig4 variants in which K654 and K655 residues were substituted either with glutamine (K654Q and K655Q), an acetyl­lysine mimic, or witharginine (K654R and K655R), an unacetylated lysine mimic. In hyphal inducing media such as spider agar, the WT and lig4∆/∆::LIG4 reconstituted strains formedhyphae. In contrast, hyphal formation was completely inhibited in the lig4∆/∆ null mutant and the K655Q point mutant. Interestingly, when residue K655 was changed toa basic amino acid (arginine, K655R) the mutant behaved similar to WT. Collectively, these data imply that acetylation of Lig4 on K655 is an important regulatorymechanism for filamentation in C. albicans.

A regulatory system that links sphingolipid homeostasis to the nutrientsensor TOR in the human fungal pathogen Candida albicans

S.D. Moreno VelásquezI, C.J. Pérez *I

IUniveristy of Würzburg, Institute for Molecular Infection Biology, Würzburg, Germany

Multiple transcriptional circuits have been found to contribute to the proliferation of Candida albicans in diverse niches of the human body. However, the molecularfunctions governed by these regulators in vivo remain underexplored. Here, we investigate the C. albicans transcriptional regulatory system RTG1/3 which, we havepreviously shown, plays key roles in mouse models of disseminated infection and gut colonization. We establish that the Rtg1/3 system links the nutrient sensor TOR(target of rapamycin) to sphingolipid homeostasis in C. albicans. Deletion of the rtg1 and rtg3 genes resulted in altered lipid composition, particularly in majorglycosphingolipid classes. The rtg1 and rtg3 deletion mutants displayed abnormal intracellular vesicle formation, a phenotype consistent with the crucial role ofsphingolipids in this process. Additionally, the rtg1 and rtg3 mutants were hypersensitive to drugs that block sphingolipid biosynthesis, but tolerant to TOR­inhibitingdrugs. Blocking sphingolipid biosynthesis promoted the nuclear localization of the C. albicans Rtg3 protein but this process was prevented upon TOR inhibition.Furthermore, we identified two TOR­dependent kinases that feed into the post­transcriptional control of the Rtg1/3 system. Finally, we show that the Rtg3 system isactivated in response to engulfment of C. albicans cells by human neutrophils. Taken together, our findings implicate sphingolipids in C. albicans pathogenesis anduncover a regulatory system that links homeostasis of this important lipid class to the nutrient sensor TOR.

* The authors marked with an asterisk equally contributed to the work.

A synthetic system that combats fungal infections

M. TschernerI, T.W. GiessenII, L. MarkeyIII, C.A. KumamotoIII, P.A. SilverII

IMedical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria, IIDepartment of Systems Biology, Harvard Medical School andWyss Institute for Biologically Inspired Engineering, Boston, United States of America, IIISackler School of Graduate Biomedical Sciences and Department ofMolecular Biology and Microbiology, Tufts University, Boston, United States of America

Due to a limited set of antifungals available and problems in early diagnosis invasive fungal infections caused by Candida species are among the most common hospital­

acquired infections with staggering mortality rates. Here, we describe an engineered system able to sense and respond to the fungal pathogen Candida albicans, the

most common cause of candidemia. In doing so, we identified hydroxyphenylacetic acid (HPA) as a novel molecule secreted by C. albicans. Furthermore, we

engineered E. coli to be able to sense HPA produced by C. albicans. Finally, we constructed a sense­and­respond system by coupling the C. albicans sensor to the

production of an inhibitor of hypha formation thereby reducing filamentation, virulence factor expression and fungal­induced epithelial damage. This system could be

used as a basis for the development of novel prophylactic approaches to prevent fungal infections.

A resilience function for the Toll pathway in host defense against systemicAspergillus fumigatus infection

R. XUI,II, C. HuangI, S. LiégeoisI,II, Z. LiI, D. FerrandonI,II

ISino­French Hoffmann Institute, Guangzhou, China, IIIBMC­CNRS, Strasbourg, France

Host defense encompasses two complementary dimensions, resistance, the immune response that results in the neutralization and killing of invading pathogens, andresilience, the homeostatic reactions that participate in enduring and repairing damages inflicted either by pathogen's virulence factors or the host's own immuneresponse.Aspergillus fumigatus is a major human opportunistic pathogen that causes high morbidity and mortality in immunodeficient patients. We are currently performing alarge­scale screen to identify Drosophila host defense genes. A first step was to better characterize the infection model, which we have optimized for our purpose. Asreported previously, A. fumigatus does kill MyD88, Toll pathway immunodeficient, flies but not wild­type flies. However, we observed that the fungal burden hardlyincreases in the mutant flies, even upon death, in contrast to other fungal infections. Some 250 injected conidia suffice to kill MyD88 flies in the absence of invasion ofmost fly tissues. We have therefore tested whether some of the many toxins known to be secreted by A. fumigatus might be involved in the pathogenesis. We shallreport that some but not all such toxins differentially kill MyD88 and not wild­type flies. Therefore, the Toll pathway is not only a major pathway in the resistance againstfungal infections but also appears to be required for resilience against such infections by protecting the host from the action of pathogen­secreted toxins. Futureexperiments will tell whether this novel function of the Toll pathway in resilience to mycotoxins has also been conserved during evolution as has been the case for itsfunction in resistance.

CRISPR­CAS9 gene editing tools in pathogenic and non­pathogenicyeasts

J. Albertyn *I, C. PohlI, R. Fourie *I, E. Bisschoff *I, J. Klinck *I, M. du Plooy *I

IUniversity of the Free State, Bloemfontein, South Africa

CRISPR­Cas9 is a revolutionary method to effectively and efficiently alter the genomic make­up of an organism. This system uses a short stretch of DNA,complementary to the gene of interest, as well as the CAS9 gene which are introduced into targeted cells. The complementary sequence, known as the guide DNA(gDNA) sequence, is transcribed into guide RNA (gRNA) and forms a complex with the Cas9 protein. When a sequence complementary to the gRNA is found in thegenome of the target cell, a single double strand break is made by the Cas9 endonuclease in this complementary sequence. We have successfully used (with smalladaptations) the CRISPR­Cas9 system developed for Candida albicans by the Hernday lab. Using this system, we deleted various genes including HST6, IPTI,ADE2, RTA3, SET3, WOR1 and others. Furthermore, we have also constructed a PMA1::GFP fusion using the Hernday system. Apart for application in C. albicans,a similar system for Candida auris was developed where the promotors and integration sites of the C. albicans system were replaced for optimal efficacy in C. auris.The system was validated through the deletion of ADE2 gene in C. auris. A wide range CRISPR­Cas9 system was also developed. Three different codon optimizedCAS9 genes (optimized for Pichia pastoris, Candida albicans and Homo sapiens) genes were tested using the wide range expression vector pKM180. Forexpression of the gRNA, a Ribozyme‐gRNA‐Ribozyme cassette was used that was regulated by the Yarrowia lipolytica TEF1 gene. This system was validated withsuccessful disruption of the ADE2 gene in Arxula adeninivorans; Debaryomyces hansenii; Kluyveromyces lactis; Ogataea polymorpha; Komagataella phaffii;Saccharomyces cerevisiae and Yarrowia lipolytica.

* The authors marked with an asterisk equally contributed to the work.

Neutral pH is not required for hyphal morphogenesis during activemodulation of environmental pH by Candida albicans

T. VogesI, E. GarbeII, P. BrandtII, D. DreischIII, O. KurzaiIV, S. VylkovaII

IFriedrich­Schiller University, Jena, Germany, IISeptomics Research Center, Jena, Germany, IIIBioControl Jena, Jena, Germany, IVUniversity Hospital Würzburg,Wuezburg, Germany

The adaptation to acidic versus neutral pH is critical for Candida albicans virulence in distinct host sites. We have shown that C. albicans co­opts amino acidcatabolism to generate and excrete ammonia, which raises extracellular pH and induces hyphal morphogenesis, a major virulence factor. The process occurs in vitro inconditions that mimic the environment within host niches frequently colonized by C. albicans, suggesting that this is a physiologically relevant process. However, sincethese investigations were performed exclusively with laboratory propagated strains, such as SC5314, the objectives of this study were to: 1) evaluate the capacity ofclinically isolated strains to neutralize the environment; 2) test whether the pH modulation potential of C. albicans clinical isolates is niche­dependent. We tested strainsisolated from human blood, oral cavity and the vaginal area (10 strains each) for growth, pH modulation and hyphal formation in liquid and solid unbuffered Medium199, pH 4.0 or in medium buffered to various pHs. Growth under niche­specific nutrient and pH conditions, such as artificial saliva and vaginal simulating fluid, wasaccessed as well. Strain SC5314 was used as a positive control. On average, the clinical strains displayed growth and pH modulation capacity comparable to theSC5314 strain. However, the ability of the clinical isolates to switch to the hyphal form appeared to be niche specific, since upon neutralization of the environment thevaginal isolates appeared mostly as yeasts, but formed normal filaments in neutral medium supplemented with serum. This suggests that hyphal morphogenesis is not aconsequence of C. albicans­driven neutralization of the environment, but rather a distinct process. Indeed, SC5314 cells initiated hyphal growth within 3 hours ofswitch to Medium 199, when the average pH was below 5.0. Transcriptional profiling of SC54314 cells collected at different stages of alkalinization showed that thetransition of cells from 4.5 to pH 5.5 was sufficient to induce the expression of many hyphae­specific genes such as ECE1, HWP1, and IHD1. Further evaluation of theprocesses involved in the hyphal switch under these conditions is undergoing. Thus, our results demonstrate that C. albicans strains isolated from various host nichesshare the ability to modulate the environmental pH, while hyphal growth is not uniform. Further, neutral pH is not required for morphogenesis under pH modulationconditions.

Identification of targets of the essential protein kinase Snf1 in Candidaalbicans

A. MottolaI, J. MorschhäuserII

IUniveristy of Würzburg, Institute for Molecular Infection Biology, Würzburg, Germany, IIInstitute for Molecular Infection Biology, Julius Maximilians University,Würzburg, Germany

Candida albicans is a typical member of the human microbiome, wherein it typically behaves as a commensal. However, particularly among immunocompromisedpeople, it can become a life­threatening pathogen. Treatments are limited due to its Eukaryotic nature and the natural evolution of drug resistance; therefore, greaterunderstanding of its mechanisms of virulence and host survival are essential to the development of novel therapies. To this end, a comprehensive deletion library of C.albicans kinases has been constructed and is currently being screened for relevant phenotypes. In particular, the Snf1 kinase pathway, which is involved in diversecellular processes including carbon metabolism and cell wall integrity, has proven to be of interest. While its importance for such activities are known, its targets are not;current work, including in vitro evolution of suppressors of mutants of the Snf1 pathway, progresses towards identifying such targets in order to clarify the role of Snf1as well as explain its essential nature in C. albicans. Additionally, combination of our kinase mutant library with a previously constructed library of artificially activatedzinc­cluster transcription factors, followed by RNA­Seq, has revealed a previously undescribed role for the transcription factor Czf1 in the maintenance of cell wallintegrity.

Low hydrostatic pressure inhibit morphogenesis in Candida albicans

G. ZoreI, P. RapteI

ISRTM University, Nanded, India

We have evaluated impact of low hydrostatic pressure (LHP) on Candida albicans morphogenesis in response to L­proline at 37ºC using an in vitro study. C.albicans showed LHP dependent modulation in morphogenesis. Most dramatic effect was observed at 1800 Pa i.e. 13.5 mmHg (less than the lowest capillaryhydrostatic pressure); wherein hyphae induction was inhibited by 99%, pseudohyphe increased to 91% without affecting percentage of budded and un­budded cells.However complete inhibition of hyphae and pseudohyphe formation was observed at 3000 Pa. LC­MS/MS analysis revealed significant modulation in level of 30proteins (53% up regulated and 47% down regulated) involved in different biological processes, in response to LHP (1800 Pa). Dog1 & LELG_01648, reported to beassociated with morphogenesis were down regulated by 8.62 and 4.55 fold respectively. It suggests that LHP inhibit hyphae induction by activating antagonistic pathway(as Dog1 is regulated by Nrg1 and Tup1) while induce pseudohyphe by down regulating Kel1 (12 fold), maintaining cell shape & Cht3 (4 fold), separating mother anddaughter cells. Up regulation of Mic19, an important component of MICOS complex maintaining crista junctions, inner membrane architecture and proper mitochondrialfunctions could be a compensatory response to LHP induced strain on mitochondrial membrane structure and function. It was supported by enhanced biosynthesis ofphosphatidyl choline required for strengthening membranes through Sam2 up regulation. Similarly, LHP affect vacuolar trafficking and proper folding of proteins, as levelof components involved in transport and proteolysis of aberrant and unfolded proteins has gone up. In addition to metabolic processes (carbohydrate, amino acid,nucleotide), gene expression, energy generation in C. albicans. This is the first report on modulation of C.albicans morphogenesis by LHP along with an insight into themechanism of action. Our results cites the significance of capillary hydrostatic pressure in maintaining C. albicans cells in yeast form during blood stream infections

Flotillin­dependent lipid rafts are required for immunity against fungalinfections

F. Schmidt *I,II, M. Röcker *I,II, A. Thywissen *I,II, H. SchmidtI,II, Z. CseresnyésI, S. GalianiIII, M. StraßburgerI, T. HeinekampI, M.T. FiggeI, C. EggelingIII, S.FillerIV, A.A. BrakhageI,II

ILeibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute (HKI), Jena, Germany, IIFriedrich­Schiller University, Jena, Germany,IIIWeatherall Institute of Molecular Medicine; University of Oxford, Oxford, United Kingdom, IVDavid Geffen School of Medicine at UCLA, Los Angeles, UnitedStates of America

Aspergillus fumigatus represents the most important air­borne fungal pathogen. In lung alveoli, resident alveolar macrophages belong to the first line of defense againstinhaled conidia. The presence of dihydroxynaphthalene (DHN) melanin on the conidial surface is crucial to inhibit phagolysosomal acidification and to cause increaseddamage to macrophages compared to non­pigmented pksP mutant conidia. Here, we discovered the importance of lipid rafts for immune cells to attack A. fumigatus.We found that reduced acidification of phagolysosomes is specific for phagolysosomes containing wild­type conidia and correlates with reduced assembly of functionalvATPase in the phagolysosomal membrane. The membrane of conidia­containing phagolysosomes is characterized by a high content of cholesterol and sphingolipids.Further classification of the lipid raft microdomains showed that they contained flotillins but not caveolins. Flotillin­enriched lipid rafts co­localized with sphingolipids onphagolysosomal membranes containing pksP conidia but not wild­type conidia. In human and mouse, two flotillins, Flot­1 and 2, are present. As chaperon, flotillinsstabilize lipid­raft microdomains and thus represent a marker protein for microdomains. To further classify the lipid­raft microdomains we improved a protocol forisolation of phagolysosomes from macrophages. This allowed to monitor the dependence of the recruitment of receptors and enzyme complexes on lipid­raftmicrodomains and also to co­localize microdomains and receptor/enzyme complexes by high­resolution fluorescence microscopy. In addition, bone marrow­derivedmacrophages (BMDMs) of Flot­1/2 knockout mice were compared with C57BL7/6 wild­type macrophages. BMDMs were co­incubated with melanized wild­typeconidia as well as pigmentless pksP conidia. Consistently, acidification of phagolysosomes containing pksP conidia was drastically reduced in Flot­1/2 knockoutBMDMs as well as vATPase assembly and phagocytosis. Infection of Flot­1/2 knockout mice with pksP conidia resulted in increased cell damage and killing comparedto wild­type mice. Taken together, these data indicate that lipid raft microdomains provide important platforms for signaling and defense against A. fumigatus.

* The authors marked with an asterisk equally contributed to the work.

Identifying the mechanisms and understanding the importance ofheteroresistance in Candida glabrata

N. LyonsI, N. Wertheimer *I, J. UsherII, J. BermanI

ITel Aviv University, Tel Aviv, Israel, IIUniversity of Exeter, Exeter, United Kingdom

C. glabrata is an important human fungal pathogen; rates of life­threatening infection are increasing, particularly in immunocompromised individuals, while multi­drugresistance is a serious concern, as C. glabrata develops resistance to both azoles and echinocandins quickly.

Antifungal drug resistance is the ability of an entire population of cells to grow in high drug concentrations. Heteroresistance (HetR) is defined as the ability of a smallsubpopulation from cells of clonal origin to grow at drug concentration far in excess of the MIC for the bulk population.

This project explores the contributions of such subpopulations to the evolution of antifungal drug resistance and virulence, and investigates the mechanisms by which theyarise, with the aim of providing insights to improve efficacy of therapeutic strategies by preventing or slowing the evolution of drug resistance.

We are exploring the mechanisms and effects of HetR through a variety of experimental approaches. Evolution experiments in vitro and infections in vivo will be usedto evaluate the effect of HetR on the evolution of resistance and on virulence. Analysis of genomic DNA sequences and transcription profiling of isogenic pairs withvariable proportion of HetR (PHet) will be used to evaluate how PHet is modulated. Analysis of cell growth, division dynamics, and lineage tracking will be used toidentify the mechanisms of the HetR state.

Heteroresistance is a largely undiscovered facet of antifungal reistance and elucidating the mechanisms behind the emergence of drug resistance is key to tackling thegrowing threat of antifungal­resistant pathogens.

* The authors marked with an asterisk equally contributed to the work.

New ergosterol functions in the Candida albicans cell: too little, toomuch, or lack of ergosterol versus resistance to drugs

J. SuchodolskiI, M. ŁukaszewiczI, A. KrasowskaI

IDepartment of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland

Two important mechanisms of Candida albicans resistance to azoles and amphotericin B result from modification of ergosterol biosynthesis and multidrug transporters’(Cdr1, Cdr2 and Mdr1) activity. Mutations in the ERG11 gene reducing or increasing ergosterol synthesis may protect C. albicans against amphotericin B. Ourhypothesis assumes that controlling the amount of ergosterol in the cell may help to overcome drug resistance. In our studies, we depleted ergosterol in cells usingERG11 gene knock­out and treatment with drugs. We found that capric acid causes overproduction of ergosterol, while lactic acid or fluconazole decreases theergosterol content. Using our model of variable ergosterol quantity, we noticed alternations in drug resistance (azoles and amphotericin B), plasma membranebiophysics, bioenergetics, cell wall structure, and filamentation of C. albicans. All conditions, which affect ergosterol content, caused mislocalization of Cdr1p from theplasmalemma and increased amphotericin B resistance. Reducing the amount of ergosterol decreased the plasmalemma potential and fluidity, as well as reduced theintracellular ATP and H+­ATPase activity. Interestingly, ergosterol depletion not only affects plasma membrane, but also cell wall. We noticed localized accumulation ofchitin and β­glucans and changes in the mannoprotein distribution pattern. In conclusion, we showed a pleiotropic effect of ergosterol on cell physiology. Thus, using ourmodel, we can evaluate synergistic or antagonistic combinations of known drugs and new compounds. While searching for a drug that promotes the activity offluconazole, we noticed its synergism with surfactin, a lipopeptide that is synthesized by the probiotic strain Bacillus subtilis. Alternations in the cell wall caused byfluconazole additionally sensitized C. albicans cells towards surfactin. Our results suggest new possibilities for better treatment strategies against C. albicans mycosis.

Siderophore transporter 1 (Sit1)­mediated uptake determinessusceptibility to the antifungal VL­2397

A. DietlI, S.M. SullivanII, M. MisslingerI, V. IvashovIII, D. TeisIII, C. DecristoforoIV, L.R. SmithII, H. HaasV

IDivision of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria, IIVical Inc., San Diego, United States of America, IIIDivision of Cell Biology,Medical University of Innsbruck, Innsbruck, Austria, IVDepartment of Nuclear Medicine, Medical University of Innsbruck, Innsbruck, Austria, VMedical University ofInnsbruck, Innsbruck, Austria

VL­2397 is an antifungal drug with a novel mechanism of action currently undergoing Phase 2 testing for invasive aspergillosis. VL­2397 is an aluminum­chelating cyclichexapeptide with a structure analogous to ferrichrome­type siderophores. Previous studies indicated that (i) a missense mutation in the siderophore­iron transporter Sit1causes resistance to VL­2397, (ii) iron starvation increases susceptibility to VL­2397, and (iii) replacement of aluminum by iron in VL­2397 decreased its antifungalactivity.To further elucidate the antifungal mode of action, an Aspergillus fumigatus strain was generated in which the endogenous sit1 gene is under control of a xylose­inducible promoter to uncouple sit1 expression from iron repression. Fluorescent protein­tagging confirmed xylose­controlled localization of Sit1 in the plasmamembrane and xylose mediated modulation of VL­2397 susceptibility indicated that Sit1­mediated uptake is essential for VL­2397 susceptibility. In agreement, deletionof the sit1gene rendered A. fumigatus resistant to VL­2397. Under xylose­induced sit1 expression, VL­2397 retained antifungal activity also after replacing aluminumwith iron, which demonstrates that VL­2397 bears antifungal activity independent of aluminum importation. Analysis of sit1 expression indicated that the reducedantifungal activity of the iron­chelated VL­2397 is caused by downregulation of sit1 expression by the imported iron. In contrast to A. fumigatus and Candidaglabrata, Saccharomyces cerevisiae displays intrinsic resistance to VL­2397. However, expression of sit1, or its homologue from C.glabrata, resulted insusceptibility to VL­2397, which suggests that the intrinsic resistance of S. cerevisiae is based on lack of uptake and that A. fumigatus, C. glabrata and S.cerevisiae may share a common intracellular target for VL­2397.

The cell wall proteome of the emerging pathogenic yeast Candida auris

M. AlvaradoI, J. Fernández­PereiraI, A. SánchezI, E. ValentinII, E. ErasoIII, P. De GrootI

IRegional Center for Biomedical Research, Castilla­La Mancha Science & Technology Park, University of Castilla–La Mancha, Albacete, Spain, IIGMCA ResearchUnit, Departamento de Microbiología y Ecología, Universidad de Valencia, Burjassot, Valencia, Spain, IIIDepartamento de Inmunología, Microbiología y Parasitología,Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), Bilbao, Spain

The emerging multidrug­resistant pathogenic yeast Candida auris causes life­threatening invasive infections and shows a capacity for hospital transmission that isuncommon in other Candida species. In our laboratories, we study the cell wall of C. auris because it plays an important role in the primary steps that underlie theestablishment of fungal infections. Here, we present a bioinformatic inventory of the cell wall biosynthetic machinery as well as mass spectrometric studies of purified cellwall preparations under various infection­relevant growth conditions such as the presence of antifungals or biofilm formation. Results are compared to the commonpathogen C. albicans. The next step in our studies is to generate knockout mutants of identified cell wall proteins for functional analysis. Candida auris is frequentlymisidentified in a clinical setting. Therefore, we also designed a rapid and easily applicable assay for PCR identification of C. auris using primers from GPI protein­encoding genes that do not have homologs in the gene databases. Our PCR method, verified using a panel of different Candida species and C. auris isolates fromdifferent continents, proves useful for easy, low­cost, and accurate identification of C. auris infections in a clinical setting.This work was supported by grant SAF2017­86188­P and PI17/01538 from the Spanish Ministry of Economy and Competitiveness (MINECO), co­financed by theEU (FEDER).

Index

Presenter, institution and email

Participants marked with * do not allow visualization of affiliation in this booklet.

Last name First name Institute Country Mail address Poster/talk

Ahmed Yasmin Newcastle University United Kingdom y.ahmed2@newcastle.ac.uk P11B

Albertyn Jacobus University of the Free State South Africa albertynJ@ufs.ac.za P153C

Allert Stefanie Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute

Germany stefanie.allert@leibniz-hki.de P87C

Altamirano Sophie University of Minnesota United States daltamir@umn.edu P54C

Alves Rosana University of Minho Portugal rosanamaalves@gmail.com P127A

Andrianopoulos Alex University of Melbourne Australia alex.a@unimelb.edu.au

Aparicio Pedro Nuno Alexandre Instituto Superior Técnico Portugal nunoapariciopedro@gmail.com P86B

Arastehfar Amir Westerdijk Institute The Netherlands a.arastehfar.nl@gmail.com P64A

Azimova Dinara University of California San Francisco United States dinara.azimova@ucsf.edu P49A

Bachellier-Bassi Sophie Institut Pasteur France bachelli@pasteur.fr P130A

Bader Oliver University Medical center Göttingen Germany obader@gwdg.de P147C

Baltussen Tim Radboudumc The Netherlands tim.baltussen@radboudumc.nl

Basante Bedoya Miguel University Côte d'Azur France mabasante@unice.fr P91A

Bassilana Martine University Côte d'Azur / CNRS France mbassila@unice.fr T7

Beattie Sarah University of Iowa United States sarah-r-beattie@uiowa.edu P72C

Bedekovic Tina University of Aberdeen United Kingdom tina.bedekovic@abdn.ac.uk P73A

Berman Judith Tel Aviv University Israel jberman@post.tau.ac.il T1

Bezerra Ana Rita iBiMED - Institute of Biomedicine Portugal armbezerra@ua.pt P133A

Bicanic Tihana St George's University of London United Kingdom tbicanic@sgul.ac.uk T30

Biermann Amy Dartmouth College United States amy.biermann.gr@dartmouth.edu P111C

Bignell Elaine University of Manchester United Kingdom elaine.bignell@manchester.ac.uk T8

Bohner Flora University of Szeged Hungary flora.bohner@gmail.com P102C

Böttcher Bettina ZIK Septomics Germany bettina.boettcher@hki-jena.de P52A

Brakhage Axel Leibniz Institute for Natural Product Research and Infection Biology

Germany axel.brakhage@hki-jena.de T25

Brandt Philipp ZIK Septomics, Friedrich-Schiller-University Jena Germany Philipp.Brandt@uni-jena.de P50B

Bravo Gustavo University of Aberdeen United Kingdom gustavo.bravoruiz@abdn.ac.uk P89B

Brown Hannah Duke University United States heb20@duke.edu P3C

Brunke Sascha Hans Knoell Institute Germany sascha.brunke@hki-jena.de P42C

Buda De Cesare Giuseppe University of Aberdeen United Kingdom giuseppe.budadecesare@abdn.ac.uk P5B

Butler Geraldine University College Dublin Ireland gbutler@ucd.ie

Buzun Ekaterina Newcastle University United Kingdom e.buzun@newcastle.ac.uk P119B

Carolus Hans VIB KU Leuven Center for Microbiology Belgium hans.carolus@kuleuven.vib.be

Chapuis Ambre University of Aberdeen United Kingdom r01afc16@abdn.ac.uk P29B

Chauhan Neeraj Rutgers University United States chauhan1@njms.rutgers.edu P149B

Childers Delma University of Aberdeen United Kingdom delma.childers@abdn.ac.uk P57C

Cohen Allison University of California at San Francisco United States allison.cohen@ucsf.edu P46A

Cornet Muriel Grenoble Alpes University France mcornet@chu-grenoble.fr P140B

Corzo Dora University of Aberdeen United Kingdom r01dec17@abdn.ac.uk P85A

Crawford Aaron University of Aberdeen United Kingdom a.crawford@abdn.ac.uk P23B

Cuomo Christina Broad Institute United States cuomo@broadinstitute.org T4

d'Enfert Christophe Institut Pasteur France christophe.denfert@pasteur.fr T2

Dalle Frederic University of Burgundy France frederic.dalle@u-bourgogne.fr P114C

Day Alison Newcastle University United Kingdom a.m.day@ncl.ac.uk P104B

de Groot Piet University of Castilla-La Mancha Spain piet.degroot@uclm.es P161B

de Pinho Mixao Verónica Centro de Regulacion Genomica Spain veronica.mixao@crg.eu P22A

Demers Elora Dartmouth College United States elora.g.demers.gr@dartmouth.edu P37A

Demuyser Liesbeth VIB KU Leuven Center for Microbiology Belgium liesbeth.demuyser@kuleuven.vib.be P2B

Desanti Guillaume University of Birmingham, UK United Kingdom g.desanti@bham.ac.uk P39C

Doering Tamara Washington University School of Medicine United States doering@wustl.edu T23

Ducreux Amandine University of Burgondy France amandine.ducreux@u-bourgogne.fr P117C

Edwards John LA Biomedical Research Institute at Harbor/UCLA United States jedwards@labiomed.org T31

Engert-Ellenberger Nicole Hans-Knöll-Institute Jena Germany engert.nicole@gmx.de P60C

Esquivel Brooke University of Missouri Kansas City United States bdp3k9@mail.umkc.edu P14B

Ferling Iuliia Leibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute

Germany iuliia.ferling@leibniz-hki.de P36C

Gabaldon Toni Centre for Genomic Regulation Spain tgabaldon@crg.es T3

Gago Sara University of Manchester United Kingdom sara.gago-2@manchester.ac.uk P113B

Galocha Mónica Instituto Superior Técnico, Universidade de Lisboa, Lisboa Portugal monicagalocha@gmail.com P148A

Garbe Enrico ZIK Septomics Jena Germany enrico.garbe@hki-jena.de P55A

Garcia-Rodas Rocio Instituto de Salud Carlos III Spain rocio.rodas@gmail.com P108C

Gaspar-Cordeiro Ana Carolina ITQB NOVA Portugal carolinagcordeiro@gmail.com P118A

Gonzales-Huerta Luis Imperial College London United Kingdom lucho.g@gmail.com P144C

Graham Carrie University of California, San Francisco United States carrie.graham2@ucsf.edu P96C

Gresnigt Mark Hans Knöll Institute Germany markgresnigt@gmail.com P27C

Grinstein Sergio The Hospital for Sick Children Canada sergio.grinstein@sickkids.ca T15

Gusa Asiya Duke University United States asiya.gusa@duke.edu P4A

Haas Hubertus Medical University of Innsbruck Austria hubertus.haas@i-med.ac.at P160A

Hafez Ahmed Biotechvana Spain ah.hafez@gmail.com P84C

Heaney Helen University of Aberdeen United Kingdom r01hh17@abdn.ac.uk P7A

Heitman Joseph Duke University United States * T5

Hickman Meleah Emory University United States mhickm3@emory.edu P45C

Hillman Falk Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute

Germany falk.hillmann@leibniz-hki.de P30C

Horianopoulos Linda University of British Columbia Canada horianol@msl.ubc.ca P110B

Hossain Saif University of Toronto Canada saif.hossain@mail.utoronto.ca P123C

Hovhannisyan Hrant Centre For Genomic Regulation Spain grant.hovhannisyan@gmail.com P34A

Hoy Michael Duke University United States michael.hoy@duke.edu P77B

Huang Guanghua Fudan University and Institute of Microbiology, CAS China huanggh@im.ac.cn T24

Hube Bernhard Hans Knoell Institute Germany bernhard.hube@hki-jena.de P128B

Hünniger Kerstin

Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena

Germany kerstin.huenniger@leibniz-hki.de P101B

Iliev Iliyan Weill Cornell Medicine United States ili2001@med.cornell.edu T16

Iracane Elise University College Dublin Ireland elise.iracane@ucd.ie P47B

Iyer Kali University of Toronto Canada kali.iyer@mail.utoronto.ca P41B

Jabra-Rizk Mary Ann University of Maryland Baltimore United States mrizk@umaryland.edu

Jacobsen Ilse D. Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute

Germany ilse.jacobsen@hki-jena.de

Janbon Guilhem Institut Pasteur France janbon@pasteur.fr T12

Jenull Sabrina Medical University of Vienna Austria sabrina.jenull@meduniwien.ac.at P76A

Kannan Abhilash CHUV Switzerland abhilifescizurich@gmail.com P13A

Kornitzer Daniel Technion Faculty of Medicine Israel danielk@technion.ac.il P62B

Kowalski Caitlin Geisel School of Medicine United States caitlin.h.kowalski.GR@dartmouth.edu P51C

Krom Bastiaan Academic Centre for Dentistry Amsterdam The Netherlands b.krom@acta.nl T17

Kronstad Jim University of British Columbia Canada kronstad@msl.ubc.ca T9

Krylov Vadim N.D. Zelinsky Institute of Organic Chemistry RAS Russia vadimkrilov@yandex.ru P124A

Krysan Damian University of Iowa United States damian-krysan@uiowa.edu

Ksiezopolska Ewa Centre for Genomic Regulation Spain ewa.ksiezopolska@gmail.com P71B

Kuchler Karl Medical University Vienna Austria karl.kuchler@meduniwien.ac.at T11

Kurzai Oliver University of Würzburg Germany okurzai@hygiene.uni-wuerzburg.de T20

Larcombe Daniel University of Aberdeen United Kingdom delarcombe@gmail.com P82A

Legrand Melanie Institut Pasteur France mlegrand@pasteur.fr P100A

LeibundGut-Landmann Salomé University of Zurich Switzerland salome.leibundgut-landmann@uzh.ch T22

Lemberg Christina University of Zurich Switzerland christina.lemberg@uzh.ch P81C

Liu Xueyan University of Hong Kong China xueyanliu126@gmail.com P63C

Ljungdahl Per O. Stockholm University Sweden per.ljungdahl@su.se P93C

Lombardi Lisa University College Dublin Ireland lisa.lombardi@ucd.ie P69C

Lopez-Ribot Jose UTSA United States jose.lopezribot@utsa.edu T29

Lorenz Michael Univ. of Texas McGovern Medical School United States michael.lorenz@uth.tmc.edu

Lukaszewicz Marcin Uniwersytet Wrocławski Poland marcin.lukaszewicz@uwr.edu.pl P159C

Lyons Naomi Tel Aviv University Israel naomislyons@gmail.com P158B

Marra Emanuele Takis Srl Italy marra@takisbiotech.it

Martinez de San Vicente Kontxi University Zurich Switzerland kontxi.martinez@vetvir.uzh.ch P28A

Marton Timea Institut Pasteur France timea.marton@pasteur.fr P16A

Matos da Fonseca Diogo Cardiff University United Kingdom matosdafonsecad@cardiff.ac.uk P125B

May Robin University of Birmingham United Kingdom r.c.may@bham.ac.uk T27

Mehta Nikita Agharkar Research Institute, G. G. Agarkar Road, Pune India nikitamehta@aripune.org P106A

Miramon Pedro UTHealth United States pedro.miramonmartinez@uth.tmc.edu P65B

Mitchell Aaron Carnegie Mellon University United States apm1@andrew.cmu.edu T10

Moreno Velasquez Sergio David Institute for Molecular Infection Biology Germany sergio.moreno-velasquez@uni-wuerzburg.de P150C

Morschhäuser Joachim University of Würzburg Germany joachim.morschhaeuser@uni-wuerzburg.de

Motsi Natasha University of Manchester United Kingdom natasha.motsi@postgrad.manchester.ac.uk P122B

Mottola Austin University of Würzburg Germany austin.mottola@uni-wuerzburg.de P155B

Munro Carol University of Aberdeen United Kingdom c.a.munro@abdn.ac.uk

Naglik Julian King's College London United Kingdom julian.naglik@kcl.ac.uk

Nemeth Tibor University of Szeged Hungary narvaltm@gmail.com P135C

Nikolakopoulou Christina University of Aberdeen United Kingdom r02cn17@abdn.ac.uk P83B

Niu Mengyao University of Wisconsin-Madison United States mniu5@wisc.edu P58A

Nogueira Filomena CCRI Austria filomena.nogueira@ccri.at P32B

O'brien Caoimhe UCD Conway Institute Ireland caoimhe.o-brien@ucdconnect.ie P10A

Oh Seeun Cedars-Sinai Medical Center United States seeun.oh@cshs.org P74B

Oliveira Carla iBiMED - Institute for Biomedicine Portugal cbritesoliveira@ua.pt P38B

Olivera Pacheco João University College Dublin Ireland jmopacheco@gmail.com P44B

Orr Selinda Cardiff University United Kingdom orrs@cardiff.ac.uk T19

Ortiz Sébastien University of Wisconsin, Madison United States sortiz2@wisc.edu P31A

Paradis Tracy University of Burgundy France tracy.paradis@u-bourgogne.fr P112A

Parvizi Raha Concordia University Canada raha.parvizi.o@gmail.com P79A

Pekmezovic Marina Hans-Knöll Institute Germany marina.pekmezovic@hki-jena.de P94A

Perez Christian University Wuerzburg Germany christian.perez@uni-wuerzburg.de T16

Perez Hansen Antonio Medical University of Innsbruck Austria antonio.hansen@i-med.ac.at P25A

Perlin David Hackensack Center for Discovery and Innovation USA perlinds@njms.rutgers.edu T28

Pimentel Catarina ITQB NOVA Portugal pimentel@itqb.unl.pt P126C

Pohl Carolina University of the Free State South Africa pohlch@ufs.ac.za P136A

Priest Shelby Duke University United States shelbyjpriest@gmail.com P19A

Puerner Charles University Côte d'Azur France cpuerner@unice.fr P21C

Quinn Jan Newcastle University United Kingdom janet.quinn@ncl.ac.uk T26

Quintin Jessica Institut Pasteur France jessica.quintin@pasteur.fr

Raffa Nicholas University of Wisconsin- Madison United States nraffa@wisc.edu P53B

Ramírez-Zavala Bernardo University of Würzburg Germany b.ramirez@uni-wuerzburg.de P131B

Reuter–Weissenberger Philipp Insitute for Molecular Infection Biology Germany philipp.reuter-weissenberger@uni-

wuerzburg.de P15C

Revie Nicole University of Toronto Canada nicole.revie@mail.utoronto.ca P92B

Richardson Jonathan King's College London United Kingdom jonathan.richardson@kcl.ac.uk P78C

Röcker Marie Leibniz Institute for Natural Product Research and Infection Biology. Hans-Knöll-Institute

Germany marie.roecker@leibniz-hki.de P157A

Rodriguez Rosa UC San Francisco United States rosa.rodriguez2@ucsf.edu P33C

Rodriguez Sanchez Antonio Jose Ghent University Belgium antoniojrodri@hotmail.com P129C

Rogiers Ona VIB KU Leuven Center for Microbiology Belgium ona.rogiers@kuleuven.be P142A

Román Elvira Universidad Complutense de Madrid Spain elvirarg@farm.ucm.es P59B

Rosowski Emily Clemson University United States erosows@clemson.edu P17B

Roy Udita The Ruth and Bruce Rappaport Faculty of Medicine, Technion - I.I.T.

Israel udita@campus.technion.ac.il P61A

Ruben Sophia Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute

Germany sophia.ruben@leibniz-hki.de P35B

Rybak Jeffrey M. University of Tennessee College of Pharmacy United States jrybak@uthsc.edu P107B

Salazar Sara University of Lisbon, IST Portugal sara.barbosa.salazar@gmail.com P138C

Sanglard Dominique University of Lausanne and University Hospital Switzerland *

Sasse Christoph University Germany csasse1@gwdg.de P99C

Sauer Frank-Martin QVQ BV The Netherlands f.sauer@qvquality.com P146B

Schrevens Sanne CHUV Switzerland schrevenssanne@gmail.com P98B

Scott Jennifer University of Manchester United Kingdom jennifer.scott-12@postgrad.manchester.ac.uk P24C

Selmecki Anna Creighton University Medical School United States annaselmecki@creighton.edu T6

Seoane Paula University of Birmingham United Kingdom PIS528@bham.ac.uk P9C

Sephton Clark Poppy University of Birmingham United Kingdom psephtonclark@gmail.com P12C

Shekhova Elena MRC Centre for Medical Mycology at the University of Aberdeen

United Kingdom shekhova.elena@yandex.com P134B

Silao Fitz Gerald Stockholm University Sweden fitzgerald.silao@su.se P95B

Simantirakis Stylianos Charalampos University of Aberdeen United Kingdom r01scs17@abdn.ac.uk P121A

Siscar Lewin Sofia Hans Knoell Institute Germany sofia.lewin@hki-jena.de P40A

Spyrou Maria University of Exeter United Kingdom m.spyrou@exeter.ac.uk P116B

Stavrou Aimilia Westerdijk Fungal Biodiversity Institute The Netherlands a.stavrou@westerdijkinstitute.nl P97A

Szebenyi Csilla University of Szeged Hungary szebecsilla@gmail.com P90C

Takács Tamás University of Szeged, Department of Microbiolog Hungary ttakacs.ttamas@gmail.com P109A

Tapia Cecilia Clínica Dávila Chile cvtapiap@gmail.com P120C

Telzrow Calla Duke University United States calla.telzrow@duke.edu P43A

Thornton Christopher University of Exeter United Kingdom c.r.thornton@exeter.ac.uk T13

Tóth Eszter Judit University of Szeged Hungary scedobipo@gmail.com P18C

Traven Ana Monash University Australia ana.traven@monash.edu T18

Tscherner Michael Medical University of Vienna Austria michael.tscherner@univie.ac.at P151A

Tuck Laura University of Edinburgh United Kingdom Laura.Tuck@ed.ac.uk P48C

Vadovics Mate University of Szeged Hungary vadovicsmate@gmail.com P80B

Van Dijck Patrick VIB KU Leuven Center for Microbiology Belgium patrick.vandijck@kuleuven.vib.be

Van Dyck Katrien VIB KU Leuven Center for Microbiology Belgium katrien.vandyck@kuleuven.vib.be P8B

Van Ende Mieke VIB KU Leuven Center for Microbiology Belgium mieke.vanende@kuleuven.vib.be P137B

Van Genechten Wouter KU Leuven Belgium wouter.vangenechten@kuleuven.vib.be P143B

van Rhijn Norman University of Manchester United Kingdom norman.vanrhijn@manchester.ac.uk P66C

van Wijlick Lasse Institut Pasteur France lasse.vanwijlick@pasteur.fr P115A

Vande Velde Greetje KU Leuven Belgium greetje.vandevelde@kuleuven.be

Vandoren Leni VIB KU Leuven Center for Microbiology Belgium leni.vandoren@kuleuven.vib.be

Vangoethem Nico VIB KU Leuven Center for Microbiology Belgium nico.vangoethem@kuleuven.vib.be

Vatanshenassan Mansoureh Bruker Daltonic Company Germany m.vatanshenassan@bruker.com P145A

Vaz Amanda Grace University of Szeged Hungary amanda.grace6vaz@gmail.com P103A

Vidal Bonifacio Bruna University of Texas at San Antonio United States brunavidalb@gmail.com P68B

Vielreicher Sarah Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute

Germany sarah.vielreicher@leibniz-hki.de P70A

Vila Taissa University of Maryland, Baltimore United States tvila@umaryland.edu P20B

Vylkova Slavena Septomics Research Center Germany slavena.vylkova@hki-jena.de P154A

Wakade Rohan University of Iowa United States rohan-wakade@uiowa.edu P139A

Walker Louise University of Aberdeen United Kingdom louise.walker@abdn.ac.uk P26B

Wall Gina University of Texas at San Antonio United States gina.wall@utsa.edu P6C

Wallace Edward University of Edinburgh United Kingdom edward.wallace@ed.ac.uk P1A

WANG Qi Monash University Australia bella.wang@monash.edu P67A

WEI Kaiyao CNRS - Institut de biologie structurale France kaiyao.wei@ibs.fr P75C

Wijnants Stefanie VIB KU Leuven Center for Microbiology Belgium stefanie.wijnants@kuleuven.vib.be P141C

Williams Robert University of Texas McGovern Medical School United States robert.williams@uth.tmc.edu P105C

Wilson Duncan University of Aberdeen United Kingdom duncan.wilson@abdn.ac.uk P88A

XU Rui IBMC France xurui850822@yahoo.com P152B

Zangl Isabella University of Natural Sciences, Vienna Austria isabella.zangl@boku.ac.at P132C

Zimmermann Ann-Kathrin Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute

Germany ann-kathrin.zimmermann@hki-jena.de P56B

Zore Gajanan SRTM University, Nanded India gbzsrtmun@gmail.com P156C