bsrg 2012-2013
DESCRIPTION
Biennial report of the activities of the Biological Sciences Research Group of the Institute for Biotechnology and Bioengineering (Lisbon, Portugal), years 2012-2013.TRANSCRIPT
2012-2013
Biennial Report
BSRG | Biological Sciences Research Group
12-13 20 12-13 Biennial Report 12-13
Contents
5 Executive Summary
6 Research Programs Highlights
43 Publications
49 Communications in International Conferences
51 Communications in National Conferences
53 Other Scientific Activities
55 BSRG Members
4
Biological Sciences Research Group
5
20 12-13 Biennial Report
12-13
The outcome of the research activities of the IBB Biologi-
cal Sciences Research Group (BSRG) during the bienni-
um 2012-2013 is detailed in this report, which we gladly
present to the readers. Key research outputs that have
contributed to the consolidation and international visibility
of the BSRG activities in the fields of Molecular and Cellu-
lar Microbiology, Functional and Comparative Genomics,
and Microbial Biotechnology are described. Interdiscipli-
nary programmes involving molecular biosciences across
disciplines, from molecules to systems, were developed to
understand how biological systems orchestrate multiple
functions, envisaging the exploitation/control of their activ-
ities in Industrial, Health, Environmental and Agro-Food
Biotechnology.
Over the past decade, the group has reinforced and gen-
eralized the use of Functional and Comparative Genomics
and Bioinformatics approaches, giving a Molecular Sys-
tems Microbiology dimension to the research programmes
active during the biennium; this is highlighted under the
general thematic line “Integrative (micro)biology: post-
genomic approaches to boost biosciences research”. An-
other wide-spanning thematic line dedicated to “Microbial
Response and Resistance to Environmental Challenges”
integrates most of our research lines and projects, as
highlighted in the next pages. This thematic area aims at
understanding the complexity of cellular responses to
environmental alterations and insults, one of the major
challenges in Biology and essential for successful Bio-
technological and Biomedical applications.
During the biennium a total number of 47 research papers
were published in international peer-reviewed journals
since 2012, of which 70% were published in the first quar-
tile of scientific journals from different subject areas, 9
book chapters were co-authored and 2 patents were is-
sued. The YEASTRACT database, at the forefront of bio-
informatics tools that support gene and genomic regula-
tion analysis and systems biology studies in yeast, was
updated and upgraded. The impact of our published work
among the international scientific community was also
significant. Around 1000 of the total number of citations
gathered by the group’s publications were obtained during
this biennium.
BSRG members also continued their essential activity in
the advanced training of human resources at Instituto
Superior Técnico (IST), Universidade de Lisboa, both at
the undergraduate and postgraduate levels in all the study
cycles offered by IST at the interface between Biological
Sciences and Engineering, having launched the ULisboa
inter-schools Master’s Programme in Microbiology. They
also served in evaluation panels (e.g. Portuguese Agency
for the Evaluation and Accreditation of Higher Education
(A3ES)) and in the governing boards of scientific societies
(Microbiology and Biotechnology). Internationalization was
pursued through relevant collaborations with several for-
eign institutions, the participation in a COST action and 2
EraNets (Industrial Biotechnology and Pathogenomics),
the exchange of students and scientists, the international
dissemination of results, the participation in editorial
boards of several international journals and as guest edi-
tors of special issues, the organization and scientific com-
mittees of international conferences, congresses, and
schools, and through the participation in international ad-
visory boards and as evaluators for international funding
agencies, (e.g. European Research Council). In summary,
the mission of the BSRG to create biosciences-based
solutions to societal problems, providing useful services to
society by combining R&D activities with advanced educa-
tion was fulfilled during 2012-2013.
Executive Summary
Isabel Sá-Correia
Head of the BSRG
Biological Sciences Research Group
6
Objectives
A wide-spanning thematic line dedicated to research
on the response and resistance to environmental
challenges integrates a number of research lines and
projects highlighted in the next pages. The indicated
bibliographic references are those listed as the BSRG
publications in the period 2012-2013.
This thematic line aims at understanding the
complexity of cellular responses to environmental
alterations and insults, which is one of the major
challenges in Biology and is also crucial for
successful biotechnological and biomedical
applications. In fact, the survival and performance of
living cells depends on their ability to sense
alterations in the environment and to appropriately
respond to the new stressing situations by
remodelling genomic expression. Also, cellular
resistance to multiple drugs/xenobiotics is implicated
in the failure of many therapeutic, food-preservation
and crop protection actions but may help to improve
the productivity of biotechnological processes.
Research Topics
1. Adaptation and resistance to environmental
stresses and antifungals in yeasts
During 2012-2013, we have been contributing to the
understanding of the toxic effects caused by ethanol
and the molecular mechanisms triggered by
Saccharomyces cerevisiae at a genomic scale to
cope with this stress [24] and successfully genetically
engineered yeast cells for increased ethanol tolerance
and productivity [45]. The impact of assimilable
nitrogen in S. cerevisiae glucose uptake kinetics
during alcoholic fermentation was also examined,
providing useful guidelines for the control of nitrogen-
limited alcoholic fermentations [33]. The genetic
engineering of the transcription factor Haa1 and of the
Haa1-regulon for improved yeast tolerance to acetic
acid is also on the focus of current research. The
mechanisms underlying the intrinsic resistance to
acetic acid in the acidic food spoilage yeast
Zygosaccharomyces bailii were also examined to
guide food preservation strategies [23,31,
Palma&Roque et al., submitted]. Resistance to
natural growth inhibitors and antifungal therapy in
pathogenic Candida yeast species has also been
studied [7-9], providing new directions for the
treatment of the increasing number of drug resistant
fungal infections.
2. Multidrug/multixenobiotic resistance transporters
Multidrug/multixenobiotic resistance (MDR/MXR) is
many times the result of the action of MDR/MXR
transporters found at the membranes of all living cells.
Our longstanding research has been dedicated to the
study of the biological role and regulation of drug/
xenobiotic -pumps of the Major Facilitator Superfamily
(MFS) or the ATP Binding Cassette (ABC)
Superfamily using the eukaryotic model yeast S.
cerevisiae. This knowledge is also being explored to
functionally characterize membrane transporters from
the model plant Arabidopsis thaliana and to
understand their role in resistance to abiotic stress.
Since abiotic stress represents the most pervasive
cause of loss of plant productivity worldwide under
agricultural conditions, this thematic line attempts to
uncover novel roles for MFS transporters in plant
responses to adverse environments, paving the way
for the development of efficient strategies to improve
crop tolerance to abiotic stress [38, 39]. The
extension of the current knowledge on yeast MDR to
pathogenic yeasts is another task of this thematic line.
Mechanisms of antifungal drug resistance acquisition
in clinical isolates of C. albicans and C. glabrata have
also been examined, in particular the biological role of
several C. glabrata MDR transporters in resistance to
antifungal drugs, acetic acid and polyamines [7-9].
3. Environmental toxicology and bioremediation
One of our goals is to predict toxicological outcomes
of exposure to environmental pollutants/pesticides,
with emphasis on microbial toxicogenomics and
bioremediation of environmental pollutants. In this
context, the catabolic ability of two soil s-triazine–
degrading bacterial strains, Pseudomonas sp. ADP
and Arthrobacter aurescens TC1 is being explored
and bioassays for the assessment of the toxicity of
xenobiotics (e.g. pesticides, synthetic dyes and
others) were developed [6,47,56], based on
experimental models (the yeast S. cerevisiae and the
Response and resistance to
environmental challenges BSR
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nematode Caenorhabditis elegans). The optimization
of bioremediation strategies to decontaminate soils
polluted with s-triazine-based herbicidal formulations
is expected to allow the prevention of adjacent
freshwater compartment contamination with the
herbicides and their toxic chlorinated metabolites [6].
4. Microbial pathogenesis and host-bacteria
interactions
Chronic pulmonary infections with Burkholderia
cepacia complex (Bcc) are largely associated with a
worse prognosis and increased risk of death among
cystic fibrosis (CF) patients (Coutinho et al., 2011,
Front Cell Infect Microbiol, 1: 12). Bcc species
possess remarkable genome plasticity, providing an
invaluable advantage for adaptation to the highly
stressful CF lung environment, where bacteria must
adapt to selecting pressures resulting from
challenges of the immune system, antimicrobial
therapy, nutrient availability, oxygen limitation, etc..
The identification of the mechanisms involved in this
adaptation is crucial to define new potential drug
targets and treatment strategies. During 2012-2013
we have continued to investigate this issue, and
found further evidences supporting the crucial role of
bacterial adaptation to the evolving host
environment, highlighting the importance of
metabolic reprograming in the adaptation and
increased pathogenic potential of Bcc bacteria
occurring alongside with disease progression [25].
We have also focused on the molecular
mechanisms that trigger mucoid morphotype
variation among Bcc isolates [41], on the proteins
directing exopolysaccharide (EPS) biosynthesis, and
on EPS-mediated interaction with hosts, either in
symbiosis or pathogenesis [19,43]. Small non-coding
RNAs are increasingly recognized as powerful tools
used by bacterial pathogens to rapidly adapt their
physiology and fitness to the host environment. Work
carried out by our group envisages the identification
and functional characterization of sRNAs and their
mRNA targets, in particular those related to virulence
and resistance to stresses mimicking those faced by
the bacterium during the infection process [35-37].
Work on the nitrogen-fixing bacterium
Sinorhizobium meliloti has also been carried out
envisaging the elucidation of mechanisms involved
in symbiotic host-bacteria interactions [40].
Because live bacteria can be used in cancer
therapy and prognosis in parallel with purified
bacterial products to treat and prevent cancer growth
and metastasis, our research has also been focused
on understanding the role and use of the
Pseudomonas aeruginosa-secreted protein azurin,
as a therapeutic tool to treat poor-prognosis breast
carcinomas [3-5].
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Objectives
Genome-wide and molecular systems biology ap-
proaches were explored during 2012/13 to achieve
an integrative view on how cells interact with their
environment. The main focus of this thematic area
is the implementation and exploitation of post-
genomic approaches to gain a full understanding of
the mechanisms of adaptation and resistance to
environmental challenges in microbial systems, and
to enable their redesign and reconstruction in order
to display functions more suited to specific applica-
tions or exhibiting traits of relevance in Biotechnolo-
gy, Biomedicine and the Environment.
The research strategies employed explore
the interface between “omics” analyses and molec-
ular and cellular biology research to provide an
integrated view of gene/genomic expression, cellu-
lar signalling, and metabolic networks, allowing a
unified perspective of key scientific questions in
Biotechnology, Biomedicine and the Environment,
using microbial model systems or envisaging the
improvement/control of the physiological activity of
microorganisms, as cell factories, food spoilers or
human pathogens. This wide-spanning thematic
line is dedicated to research focusing on an inte-
grative (micro)biology standpoint and integrates a
number of our research lines and projects, as high-
lighted in the next pages and summarized below.
The indicated bibliographic references are those
listed as the BSRG publications in the period 2012-
2013.
Research topics
1. Yeast toxicogenomics
Mechanistic insights and genome-wide views on
the responses to chemicals and environmental
alterations relevant in Environmental Health, Phar-
macology and Biotechnology have been gathered
using the eukaryotic model Saccharomyces cere-
visiae. Such toxicogenomics approaches are im-
portant to assess the response to cytotoxic insults
and have the potential for predictive toxicology and
for guiding bioremediation strategies and biotech-
nological and biomedical research and applications
[16].
This knowledge of the S. cerevisiae cell
response to environmental challenges at the ge-
nome level (exploring transcriptomics, quantitative
proteomics, phosphoproteomics, metabolomics) is
essential to elucidate mechanisms of resistance
and find new targets of pharmaceutical drugs [15],
to select fermentation conditions and to engineer
more robust yeast strains able to improve the
productivity of biotechnological processes, such as
those related to assimilable nitrogen availability,
toxic concentrations of ethanol- and acetic acid-
induced stresses [23,24,33]. It is also essential to
decipher weak acid tolerance mechanisms in the
acidic food spoilage yeast species Zygosaccharo-
myces bailii [23, 31, Palma&Roque et al., submit-
ted], and to understand antifungal drug resistance
in pathogenic Candida species. The genome se-
quence of the highly acetic acid-tolerant Z. bailii
derived interspecies hybrid strain ISA1307, isolated
from a sparkling wine plant, was determined and
annotated and the adaptive response to acetic acid
in this strain was revealed by quantitative prote-
omics [23,31]. The screening of a genomic library
derived from the same strain to identify genes re-
sponsible for acetic acid resistance was also per-
formed [Palma&Roque et al., submitted].
The evolutionary history of the DHA2 (14-
spanner Drug:H+ Antiporter family 2 transporters of
the Major Facilitator Superfamily involved in multi-
drug resistance), ARN (siderophore transporters)
and GEX (glutathione:H+ antiporters) encoding
genes from 31 sequenced yeast strains of 25 differ-
ent hemiascomycetous species, was reconstructed.
A new protein family (DAG) was proposed to span
these three phylogenetic subfamilies of 14-spanner
MFS transporters [12]. The reconstruction of the
Integrative (micro)biology: post-genomic
approaches to boost biosciences research BSR
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evolutionary history of the hemiascomycete DHA1
(12-spanner Drug:H+ Antiporter family 1 transport-
ers) genes was extended to the species comprised
in the CTG phylogenetic complex to get new insights
on how these family genes have evolved in medical-
ly relevant Candida species [Dias and Sá-Correia,
submitted].
2. Genome-wide expression regulation
Our main goal is to understand how different tran-
scription regulatory networks overlap and cross talk
to influence microbial growth and the response to
environmental insults, elucidate the conditions under
which transcription factors are able to bind to pro-
moter binding sites, and explore and develop com-
putational tools to support this research. The
YEASTRACT database, a post-genomic resource at
the forefront of bioinformatics tools that support gene
and genomic regulation analysis and systems biolo-
gy studies in yeast, was updated and upgraded [44].
To get clues into the regulation of hexose transport-
ers encoding genes in response to the recovery of
glucose uptake rates following ammonium supple-
mentation of a nitrogen-limited fermentation, a bi-
clustering analysis was applied to the microarray
data from the transcriptional response of S. cere-
visiae cultivated under different nitrogen availability
conditions [33].
Genome-wide screenings led to the identifica-
tion of small non-coding regulatory RNAs in the op-
portunistic human pathogen Burkholderia cenocepa-
cia [35]. New post-transcriptional regulation mecha-
nisms involving these small non-coding RNAs and
RNA chaperones required for bacterial survival un-
der stress and full virulence in B. cenocepacia were
also described.
3. Bacterial pathogenomics
The proteome expression profiling of B. cenocepa-
cia clonal isolates with different virulence potential,
retrieved from a cystic fibrosis patient during chronic
lung infection, highlighted the involvement of pro-
teins associated with metabolic functions in in-
creased persistence and virulence potential [25]. A
book chapter describing the application of expres-
sion proteomic approaches to studies involving
Burkholderia bacteria and providing a tutorial on the
methodologies available was published [54]. Com-
parative transcriptomic analysis of the B. cepacia
tyrosine kinase bceF mutant revealed a role of this
kinase in tolerance to stress, biofilm formation and
virulence [19].
4. Host-bacteria interactions
The symbiotically relevant gene emrR of the nitrogen
-fixing bacterium Sinorhizobium meliloti was charac-
terized through an expression profiling analysis of
the deletion mutant [40]. This analysis showed a
down-regulation of genes involved in stress respons-
es and biosynthesis of Nod-factor and rhizobactin,
while genes directing the biosynthesis of polysac-
charides were up-regulated, suggesting a role for
EmrR in a regulatory network involved in the S. meli-
loti preparation for symbiosis.
The role and targets of the bacterial protein
azurin from Pseudomonas aeruginosa in a breast
cancer model was elucidated through a tran-
scriptomic profiling of the action of azurin in cells
expressing different levels of P-cadherin, a bad prog-
nosis marker in breast cancer [3].
5. Stem cell research
Quantitative proteomics has also been applied to
other systems of interest to our research unit strate-
gy. Human mesenchymal stem cells (MSC) have
been on the focus of intense clinical-oriented re-
search based on their differentiation potential and
immune-modulatory properties. However, because
ex-vivo expansion in required to reach meaningful
cell number for cellular therapy, the loss of prolifera-
tive, clonogenic and differentiation potential is a
problem associated with cell passaging. In this con-
text, we have used expression proteomics to obtain
mechanistic insights into the effect that culture condi-
tions have on human MSC proteomes, paving the
way to set up a proteome profiling strategy for quality
control to ensure safe and clinically effective expand-
ed stem cells [26].
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Objectives
The success of industrial alcoholic fermentations is
dependent on the ability of S. cerevisiae cells to cope
with the many environmental insults imposed during
the process. In this context, the accumulation of
inhibitory metabolites (e.g. ethanol, acetic acid, long
chain fatty acids) and nutrient limitation (e.g. nitrogen)
resulting directly from the use of unbalanced growth
media or, indirectly, from stress conditions affecting
transmembrane transport, are considered the main
causes of stuck (prematurely arrested) and sluggish
(slow) fermentations. The understanding of the
molecular and physiological basis of yeast response,
adaptation and resistance to stresses imposed during
alcoholic fermentation is essential to guide the design
of rational strategies to increase yeast performance in
an industrial environment. The use of yeast as a
eukaryotic model in pharmacogenomic studies to
elucidate mechanisms of drug resistance and identify
new potential drug targets is also on the focus of our
research.
Research topics
1. Impact of assimilable nitrogen availability in
glucose uptake kinetics
The expression and activity of the different S.
cerevisiae plasma membrane hexose uptake systems
(Hxt) and the kinetics of glucose uptake are
considered essential to industrial alcoholic
fermentation performance. Sugar transporters
inactivation, associated to protein synthesis arrest
and their degradation, even when glucose
concentration is still high, has been proposed as the
major limiting factor of enological fermentation arrest
upon nitrogen depletion. However, the variation of
sugar uptake kinetics throughout nitrogen-limited
sluggish fermentation and the same replenished
fermentation is poorly characterized and it was one of
the topics for research.
2. Global mechanisms of response and resistance to
bioethanol fermentation associated stresses
One of the objectives of our research is the
identification, at a genomic scale, of the genes and
mechanisms of resistance to some of the stresses
occurring during alcoholic fermentation, including high
-glucose (Teixeira et al., 2010, OMICS, 14, 201),
ethanol (Teixeira et al., 2009, Appl Environ Microbiol,
75, 5761) or acetic acid stress (Mira et al., 2010,
Microb Cell Fact, 9, 79). To further understand the
toxic effects caused by ethanol and the molecular
mechanisms triggered by yeast to cope with this
stress, a metabolomic analysis using high
resolution 1H-NMR spectroscopy coupled with
multivariate statistical analysis was explored during
2012-2013 to characterize the alterations occurring in
S. cerevisiae endo- and exo-metabolome under
ethanol induced stress. The effect of the
aquaglyceroporin encoded by FPS1, previously
implicated in ethanol stress resistance, in the yeast
metabolome in the absence or presence of ethanol
stress was also on the focus of this research.
Response and resistance to chemical stress and other
environmental challenges imposed to yeast cells during
alcoholic fermentation and in pharmacogenomic studies BSR
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Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Margarida Palma, Tânia R. Cabrito, Sandra C.
dos Santos
PhD and Msc students: Artur B. Lourenço, Joana F. Guerreiro, Sílvia F. Henriques, Filipa C. Roque, Cláudia P. Godinho,
Filipe B. Silva
S cerevisiae ATCC 201388 (BY4741)expressing Haa1 fused (C-terminal) to GFP(S65T mutant)
Cells were cultivated in MMB (pH 4.0) and harvested after 30 minutes
in the presence or absence of 60 mM acetic acid
Cells were stained with DAPI to a final conc. of 5ng/μl.
Immediately fixed in formaldehyde (1%v/v) for 10min and then visualized.
GFP
DAPI
Merged
Without acetic acid Acetic acid 60mMSubcellular localization in presence/absence of acetic acid
Sub-cellular localization of Haa1 in
response to acetic acid stress
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3. Construction of genetically engineered strains with
improved robustness against high ethanol stress to
be used in high gravity fermentations
Based on the genome-wide identification of
determinants of ethanol resistance the manipulation
of the expression of specific genes in the overall
fermentative capacity is being explored. The
overexpression of the Fps1 aquaglyceroporin, led to
a high ethanol producing strain (Teixeira et al., 2009,
Appl Environ Microbiol, 75, 5761). More recently, the
overexpression of the yeast multidrug resistance
ABC transporter encoded by the PDR18 gene,
proposed to play a role in the incorporation of
ergosterol in the yeast plasma membrane, was
examined. Yeast robustness against growth
inhibitory concentrations of ethanol and bioethanol
productivity in high gravity fermentations was
augmented by increasing the expression level of
PDR18 [3].
4. Screening of natural S. cerevisiae isolates for
acetic acid tolerance
Among the fermentative yeast microbiota it is
expected to find S. cerevisiae isolates better adapted
to the different stresses imposed during alcoholic
fermentation. In this context, to search for strains
with high levels of resistance to acetic acid a
screening among natural wine yeast strains isolated
from spontaneous fermentations of grape must of
Douro wine-producing areas (248 isolates) and
various commercial wine yeasts strains (34 strains),
was performed.
5. Yeast pharmacogenomic studies
The eukaryotic model S. cerevisiae provides an
excellent tool for drug discovery and medicinal
research, contributing to the identification of new
targets and mechanisms of drug action [4]. In recent
years our research in this field has focused on the
antimalarial quinine (dos Santos et al. 2009,
Antimicrob Agents Chemother, 53: 5213-23; dos
Santos et al., 2011, Mol Genet Genomics, 286: 333-
46) and on the anticancer agent imatinib (dos Santos
et al., 2009, Omics, 13: 185-98) [5], based on the
application of genome-wide approaches at a
systems biology level, including transcriptomics,
proteomics and chemogenomics.
Main Achievements
The impact of assimilable nitrogen availability in
glucose uptake kinetics in S. cerevisiae during
alcoholic fermentation was detailed. Results
suggested that glucose transport capacity is maximal
during the early stages of fermentation and
presumably sustained by the low-affinity and high-
capacity glucose transporter Hxt1. During nitrogen-
limited sluggish fermentation, glucose uptake
capacity was reduced to approximately 20% of its
initial values, being presumably sustained by the low
-affinity glucose transporter Hxt3. Results also
suggested that sluggish fermentation recovery after
ammonium supplementation is associated to the
increase of glucose uptake capacity, related to the
de novo synthesis of glucose transporters with
different glucose affinity for glucose and capacity,
presumably of Hxt2, Hxt3, Hxt4, Hxt6 and Hxt7 [1].
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Pdr18
ATP ADP
Pdr11
ATP ADP
Aus1
ATP ADP
Exogenous ergosterolExogenous ergosterol
PM
Free ergosterolFree ergosterol
ER
Vesicular
transport
Non-vesicular
transport
Free ergosterol
Incorporated
ergosterol
Incorporated
ergosterol
Ergosterol
biosynthesis
12
Metabolomic analysis based on quantitative 1H-
NMR revealed extensive metabolomic
reprogramming and the effect of the
aquaglyceroporin Fps1 in ethanol-stressed yeast
[2].
Consistent with the previously described role of
Pdr18 in plasma membrane incorporation of
ergosterol (Cabrito et al., 2011, Biochem J, 440,
195), Pdr18 expression was further seen to reduce
the ethanol induced membrane permeabilization. A
Pdr18-overexpressing yeast strain, in which the
weak PDR18 promoter was replaced by the strong
and fermentation-inducible PDR5 promoter, was
constructed and shown to exhibit increasing
ethanol tolerance and to be able to produce, under
Very High Gravity fermentation, otherwise highly
inhibitory ethanol concentrations [3].
About 16% of the natural isolates of S.
cerevisiae obtained from spontaneous
fermentations of grape must of Douro wine-
producing areas and of the various commercial
wine yeasts strains were able to grow when
cultivated in acetic acid concentrations above 130
mM (pH 4), but only approximately 3% of the
strains tested could grow above 150 mM acetic
acid, with the most resistant isolates (about 1.4%)
growing at concentrations ranging 170-200 mM
acetic acid. The molecular physiology of these
remarkably acetic acid tolerant strains is currently
under study to understand the mechanisms
underlying such remarkable trait.
A quantitative- and phosphoproteomics study
based on 2-dimensional electrophoresis was
designed to monitor the response of yeast cells to
an inhibitory concentration of the tyrosine kinase
inhibitor imatinib [4]. The results highlighted the
importance of gluconeogenesis and glycolytic
pathways, which have recently been associated
with the mechanism of imatinib action in human cell
line studies. The remarkable conservation of the
results obtained in this study, similar to what had
been observed in our previous work with this drug,
further highlights the potential of S. cerevisiae as
model system for pharmacological studies.
Funded projects
ZygoSacAR – Mechanistic insights into acetic
acid resistance in food spoilage yeasts: from the
experimental Saccharomyces cerevisiae to
Zygosaccharomyces spp. PTDC/AGR-
ALI/102608/2008, PI: Isabel Sá-Correia.
INTACT – Integral Engineering of Acetic Acid
Tolerance in Yeast, in the frame of ERA-NET
Industrial Biotechnology, ERA-IB/0002/2010, PI:
Isabel Sá-Correia.
CONTRACT OF TECHNOLOGICAL
DEVELOPMENT, Project FNA (processes for non-
alcoholic fermentation of fruit juices)
SUMOL+COMPAL MARCAS, S.A..
Selected publications
[1] Palma M, Madeira SC, Mendes-Ferreira A, Sá-
Correia I. Impact of assimilable nitrogen availability in
glucose uptake kinetics in Saccharomyces cerevisiae
during alcoholic fermentation, Microbial Cell Factories,
11: 99, 2012.
[2] Lourenço AB, Roque FC, Teixeira MC, Ascenso
JR, Sá-Correia I. Quantitative 1H-NMR-metabolomics
reveals extensive metabolic reprogramming and the effect
of the aquaglyceroporin FPS1 in ethanol-stressed yeast
cells, PLoS One, 8(2), e55439, 2013.
[3] Teixeira MC, Godinho CP, Cabrito TR, Mira NP, Sá-
Correia I. Increased expression of the yeast multidrug
resistance ABC transporter Pdr18 leads to increased
ethanol tolerance and ethanol production in high gravity
alcoholic fermentation, Microbial Cell Factories, 11: 98,
2012.
[4] dos Santos SC, Teixeira MC, Cabrito TR, Sá-
Correia I. Yeast Toxicogenomics: genome-wide
responses to chemical stresses with impact in
Environmental Health, Pharmacology and Biotechnology,
Frontiers in Genetics, 3: 63, 2012.
[5] dos Santos SC, Mira NP, Moreira AS, Sá-Correia I.
Quantitative- and phospho-proteomic analysis of the
yeast response to the tyrosine kinase inhibitor imatinib,
OMICS: A Journal of Integrative Biology, 16(10): 537-
551, 2012.
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Adapted from ref. [5]
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Objectives
Transcriptional regulation depends on the action of
transcription factors (TFs) (activators and/or re-
pressors) that bind to specific DNA sequences in the
promoter region of target genes, thereby modulating
transcription. At a global scale, the transcript level of
a given gene results from the concerted action of
these specific TFs, operating in complex and inter-
twined regulatory networks. In the field of gene and
genomic expression, our main goals are:
1. Understand how different transcription regulatory
networks overlap and cross-talk to influence tran-
scription in yeast under optimal growth conditions and
under stress;
2. Elucidate the molecular mechanisms that control
protein-DNA binding and the action of TFs;
3. Assess the interactions established between TFs
and their target DNA sequences;
4. Development and exploitation of computational
tools to guide gene and genomic regulation studies
(YEASTRACT database) and to build suitable predic-
tive models of transcriptional regulatory networks
active in yeast, and to extrapolate them to other
yeasts and less genetically accessible eukaryotes.
Research Topics
1. Update and upgrade of Yeastract
The YEASTRACT database is an infrastructure for
research on gene and genomic regulation in Saccha-
romyces cerevisiae that was developed in collabora-
tion between our group and the KDBIO Group of IN-
ESC-ID to support the analysis of transcription regu-
latory associations (Teixeira et al., 2006, Nucleic Ac-
ids Res. 34: D446-51). Exclusively developed and
maintained in Portugal since 2006, YEASTRACT has
become an essential tool for Yeast Molecular Biolo-
gists and Systems Biology researchers worldwide,
being the major acknowledged source of regulatory
data of the Yeast Genome Database (SGD), the ma-
jor international database that provides comprehen-
sive integrated biological information for S. cere-
visiae. YEASTRACT progression has been described
in 4 articles that appeared in 4 DataBase Issues of
Nucleic Acids Research (2006, 2008, 2011, 2014).
These papers have been cited over 300 times in ISI
journals since 2006. The YEASTRACT website has
been cited many more times and has become quite
popular, being extensively used by hundreds of re-
searchers worldwide, and receiving around 2,000
monthly visits. The database was very recently updat-
ed and upgraded with new bioinformatic tools and
more detailed regulatory information to facilitate the
exploitation of the gathered material when answering
specific biological questions [1].
We are currently planning the development of
YEASTRACT+ by expanding the scope of application
of the YEASTRACT database to the S. cerevisiae
pan-genome and to other yeasts of biomedical and
biotechnological interest (e.g. Candida species, Zygo-
saccharomyces baillii), and to highlight crucial pro-
cesses underlying adaptation and evolution. This
planned integration and analysis will give unprece-
dented insights into yet unknown regulatory mecha-
nisms and boost the understanding of pathogen–host
interactions and the exploration of biotechnological
interesting species/strains. This knowledge may
deeply impact the treatment of fungal pathogens, the
methods for food spoilage control and the design of
optimized microbes as cell factories.
2. Assessing transcription factor-DNA interactions
using a nanobiotechnology-based approach
The interaction of the S. cerevisiae transcription fac-
tor Haa1 with its target DNA sequence, the Haa1-
responsive element (HRE) 5’-(G/C)(A/C)GG(G/C)G-
3’ [Mira et al., 2011, Nucleic Acids Res. 39:6896-
907], was studied making use of a nanostructured
acoustic wave biosensor developed by IBB/CBME,
from University of Algarve [4]. The biosensor devel-
oped is based on a quartz crystal microbalance
(QCM) analytical method supported by a transmis-
sion line model (TLM) algorithm. Using this new
nanobiosensor it was possible to demonstrate that
Haa1 binding to an oligonucleotide containing the
HRE motif leads to bending of the DNA molecule (by
approximately 37º) and increases the compactness of
Gene and genomic regulation: exploring
bioinformatics and nanobiotechnology tools BSR
G
Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Sandra C. dos Santos, Margarida Palma
PhD students: Catarina Costa, Joana Guerreiro, Sílvia F. Henriques, Cláudia P. Godinho
14
the protein-DNA complex. The results obtained in this
work demonstrate the suitability of the QCM to moni-
tor the specific binding of transcription factors to im-
mobilized DNA sequences and provides an analytical
methodology to study protein–DNA biophysics and
kinetics. The interaction of Haa1 with the HRE motif
will be further examined using Force Feedback Mi-
croscopy in collaboration with the CFMC group from
FCUL.
3. Transcription factor engineering for improved yeast
tolerance to acetic acid
The transcription factor (TF) Haa1 was considered
one of the important targets for regulation of specific
TF engineering in order to achieve a reprogramming
of genes controlled by Haa1. In the context of the
INTACT project, the Bremen partner identified mutat-
ed versions of Haa1 by error-prone PCR whose ex-
pression led to increased tolerance to acetic acid,
compared with the wild type Haa1. Our research
group is being examining the reasons for such behav-
iour at the level of: i) Haa1 version protein efficiency
of binding to DNA motif in the promoter regions of
target genes; ii) RNA stability of the different Haa1
versions in presence/absence of acetic acid ; iii) dif-
ferences at the level of sub-cellular localization of
Haa1 versions in yeast cells either or not exposed to
acetic acid; iv) activation of the protein content and
physiological activity of proteins encoded by selected
target genes in response to acetic acid.
4. Biclustering analysis of the transcription profiles of
yeast cells following ammonium supplementation of a
sluggish fermentation
A CCC-biclustering analysis (Contiguous Column
Coherent Biclusters) was applied to the microarray
data previously obtained from the transcriptional re-
sponse of S. cerevisiae PYCC 4072 cultivated under
different nitrogen availability conditions in synthetic
grape juice (Mendes-Ferreira et al., 2007, Appl Envi-
ron Microbiol, 73:3049-60) in order to identify which
hexose (Hxt) transporters may be responsible for glu-
cose uptake in the recovery of the glucose uptake
rates following ammonium supplementation of a slug-
gish fermentation and the involved transcription fac-
tors [2]. CCC-Biclustering is a state of the art biclus-
tering algorithm specifically developed for time series
gene expression data analysis (Madeira et al., 2010,
Trans Comput Biol Bioinform, 7:153-165).
Main Achievements
All regulatory associations stored in the
YEASTRACT database were revisited and new infor-
mation was added regarding the experimental condi-
tions tested and whether the TF is acting as an activa-
tor or repressor. Based on this information, new que-
ries were developed allowing the selection of specific
environmental conditions, experimental evidence or
positive/negative regulatory effect (see Figure). This
release also includes new computational tools that
facilitate the exploitation of the gathered data [1].
YEASTRACT is now linked to the major information
system focused on S. cerevisiae, the SGD
(www.yeastgenome.org). SGD currently displays
data curated by YEASTRACT on its Regulatory data,
providing another avenue for researchers to access
and analyze these data. Further collaborations with
other international databases (Candida Genome Da-
tabase, MIPS-CYGD, etc.) are also planned in the
context of YEASTRACT+ implementation.
A CCC-biclustering analysis provided indications
that the activation of the expression of genes encod-
ing the glucose transporters Hxt2 (during the transi-
tion period to active fermentation) and Hxt3, Hxt4,
Hxt6 and Hxt7 (during the period of active fermenta-
tion) may have a major role in the recovery of the
glucose uptake rate following ammonium supplemen-
tation and suggested a general de-repression of the
glucose-repressible HXT genes, consistent with the
co-down-regulation of the Mig1 and Rgt1 transcrip-
tional repressors [2].
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Figure 4
HXT2
2.52
1.51
0.50
-0.5-1
-1.5-2
HXT5, MIG1, RGT1, GAT1,
GLN3, GZF3, GIS1, MSN4 2.5
21.5
10.5
0-0.5
-1-1.5
-2 LF RF-8h RF-24h
Bicluster Time-points
Period 1 Period 2
Period 1 Period 2
(U) (D)
(D) (N)
No
rmali
zed
ex
pre
ssio
n v
alu
e
(Adapted from ref. [2])
15
20 12-13 Biennial Report
12-13
The transcription factor CgPdr1 of the pathogenic
yeast Candida glabrata was found to activate the
transcription of the multidrug transporter CgQDR2 in
C. glabrata cells challenged with clotrimazole or
quinidine [3].
An acoustic nanostructured biosensor developed
IBB/CBME from University of Algarve for the study
of the interaction between S. cerevisiae Haa1 tran-
scription factor and its target DNA sequence was
explored to identify Conformational and Mechanical
changes of DNA upon transcription factor binding
detected by a QCM and transmission line model [4].
Funded projects
FUNDRING - Identification of new biomarkers for
antifungal drug resistance diagnosis in Candida
glabrata: the particular role of multidrug resistance
transporters, PTDC/EBB-BIO/119356/2010, PI: Mi-
guel Teixeira
INTACT - Integral engineering of acetic acid tol-
erance in yeast, ERA-IB/0002/2010, PI at IST: Isa-
bel Sá-Correia
EXPL/FIS-NAN/1395/2013 - Biological physics
studies based on Force Feedback Microscopy, PI
at IST: Isabel Sá-Correia
PTDC/EBB-EBI/108517/2008 - QCMTF-
Biosensor platform for transcription factor DNA bind-
ing activity detection (concluded in September
2013), PI at IST: Isabel Sá-Correia
Selected Publications
[1] Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves
JP, Mira NP, dos Santos SC, Cabrito T, Palma M, Costa
C, Francisco AP, Madeira SC, Oliveira AL, Freitas AT,
Sá-Correia I, The YEASTRACT database: an upgraded
information system for the analysis of gene and genomic
transcription regulation in Saccharomyces cerevisiae,
Nucleic Acids Res, 42: D161-D166, 2014.
[2] Palma M, Madeira SC, Mendes-Ferreira A, Sá-
Correia I. Impact of assimilable nitrogen availability in
glucose uptake kinetics in Saccharomyces cerevisiae dur-
ing alcoholic fermentation, Microb Cell Fact, 11(1): 99,
2012.
[3] Costa C, Pires C, Cabrito TR, Renaudin A, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. Candida glabrata
drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) confers
imidazole drug resistance, being activated by the CgPdr1
transcription factor, Antimicrob Agents Chemother, 57
(7): 3159-67, 2013.
[4] de Carvalho J, Rodrigues RMM, Tomé B, Henriques
SF, Mira NP, Sá-Correia I, Ferreira GNM. Conformational
and Mechanical changes of DNA upon transcription factor
binding detected by a QCM and transmission line mod-
el, Analyst, 139(8): 1847-55, 2014.
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Zygosaccharomyces bailii, the most threatening spoilage yeast in food and
beverage industries: genome-wide approaches to understand its exceptional
resistance to the food preservative acetic acid and other remarkable features
Isabel Sá-Correia, Nuno P. Mira, Margarida Palma
PhD students: Joana F. Guerreiro, Filipa C. Roque; Research Assistant: Filipa D. Valada
BSR
G
Objectives
Spoilage resulting from growth and metabolic activity
of ascomycetous yeasts of the Zygosaccharomyces
species is widespread, which leads to significant
economic losses in the food industry and to a
reduction of food supplies worldwide. Z. bailii is one of
the most problematic spoilage yeasts in food and
beverage industries, particularly in acidic foods, soft
drinks, fruit juices, dairy products, salad dressings,
sauces, and some wines [1]. This yeast species ability
to cause spoilage derives from its outstanding capacity
to resist to weak acids widely used as food
preservatives, such as acetic, benzoic, propionic and
sorbic acids, even above the permitted values by
some food legislations.
Understanding the mechanisms of weak acid
resistance is central to the development and
implementation of more effective food and beverage
preservation strategies. Although Z. bailii is the
spoilage yeast that exhibits the highest level of
resistance to acetic acid, most of the scientific
contributions of our research group and others
concerning the mechanisms underlying adaptation and
resistance to acetic acid are essentially focused on the
more susceptible experimental model and spoilage
yeast Saccharomyces cerevisiae. Recent contributions
from our laboratory to the field are highlighted under
topic “Response and resistance to chemical stress and
other environmental challenges imposed to yeast cells
during alcoholic fermentation and in pharmacogenomic
studies”. Given that the mechanistic effects of acetic
acid in Z. bailii are not well understood, in particular
those underlying its remarkable resistance to this food
preservative, our goal is to contribute to accelerate
systems-level understanding of acetic acid resistance
mechanisms through the combination of different
genome-wide approaches.
The yeast species Z. bailii has also attracted
our attention as a potential new host for
biotechnological processes. In particular, it is an
attractive candidate to allow fermentation processes to
be performed under otherwise-restrictive conditions or
to be used in heterologous protein and metabolite
production (in particular organic acids) because of its
high resilience to acidic stress, high specific growth
rate, and high biomass yield. The knowledge of the
genome sequence of the ISA1307 strain during 2013
[2] is expected to inspire and guide novel
biotechnological applications of this yeast species/
strain in fermentation processes, given its robustness
traits.
Research topics
1. Sequencing and annotation of ISA1307 genome
Our research group led a project for the sequencing
and annotation of the genome of the highly acetic acid-
tolerant strain ISA1307 [2]. This strain was isolated
from a sparkling wine continuous production plant and
was formerly considered of the Z. bailii species and
used in the study of Z. bailii remarkable physiological
traits, in particular, its extreme tolerance to acetic acid
stress at low pH and its capacity to co-consume
glucose and acetic acid. This characteristic
distinguishes this species from S. cerevisiae, a
Crabtree-positive yeast in which acetic acid
metabolisation is repressed in the presence of
glucose. The analysis of the genome sequence
revealed that strain ISA1307 is, indeed, an
interspecies hybrid strain between Z. bailii and a
closely related undetermined yeast species. The
0,01
0,1
1
10
100
0 20 40 60
OD
600
nm
Time (h)
Z. bailii -derived interspecies hybrid strain ISA1307
0,01
0,1
1
10
100
0 20 40 60O
D 6
00n
m
Time (h)
S. cerevisiae BY4741
Control
60mM Acetic acid
120mM Acetic acid
0 mM Acetic acid
60mM Acetic acid
120mM Acetic acid
MMB, pH4.0
17
20 12-13 Biennial Report
12-13
sequence and annotation of the genome of ISA1307
strain are accessible at http://pedant.helmholtz-
muenchen.de/genomes.jsp?Category=fungal,
including browsing by a GBrowse instance. To allow
a comparative navigation through the genome of the
hybrid strain with the genomes of Z. rouxii CBS732,
S. cerevisiae S288c and Z. bailii CLIB213T, a
GBrowse_syn instance is accessible under http://
mips.helmholtz-muenchen.de/gbrowse2/cgi-bin/
gbrowse_syn/zbailii.
2. Adaptive response of strain ISA1307 to a sub-
lethal growth inhibitory acetic acid concentration
revealed by quantitative proteomics
The first genome-wide expression analysis reported
for an interspecies hybrid strain derived from Z. bailii,
the strain ISA1307, was the quantitative proteomic
analysis published by our group in the journal
Proteomics [3] before the knowledge of the genome
sequence. At that time, ISA1307 was considered of
the Z. bailii species. The objective of this proteomic
analysis was to elucidate the mechanisms
underlying the adaptive response and intrinsic
tolerance to sub-lethal growth inhibitory
concentrations of acetic acid in the highly acetic acid
resistant strain ISA1307, and was based on
quantitative two-dimensional gel electrophoresis (2-
DE) coupled with mass spectrometry for protein
identification. The main goal was to identify
alterations occurring in the protein content in
response to sudden exposure or balanced growth in
the presence of an inhibitory but nonlethal
concentration of acetic acid. Although a total of 111
protein spots were identified by mass spectrometry
with an altered content under the different conditions
examined, only 40% of the differently expressed
proteins could be identified, in part due to the lack of
ISA1307 genome sequence.
3. Screening of a genomic library from strain
ISA1307 to search for genes responsible for acetic
acid resistance
In order to get clues on the genes/proteins involved
in the high level of resistance to acetic acid, we
explored an ISA1307 genomic library previously
constructed that was screened to search for library
plasmids capable to rescue the high susceptibility
phenotype of the deletion S. cerevisiae
BY4741_Δhaa1 with the HAA1 gene encoding the
Haa1 transcription factor deleted. This study, now
submitted for publication [4], identified, at the
genome level, strong candidate genes/proteins
determinants of resistance to acetic acid, also by
exploring the knowledge of the genome sequence.
Main achievements
Quantitative proteomic analysis reinforced the
concept that, when cultivated in a medium containing
glucose and acetate, ISA1307 is able to co-consume
these two carbon sources, with acetate being
channelled through the TCA cycle. This ability to use
acetic acid as a carbon source even in the presence
of glucose, whereas acetate uptake and catabolism
are repressed by glucose in S. cerevisiae, is a
remarkable feature of Z. bailii that is also able to
metabolize sorbate and benzoate. After glucose
exhaustion and in the presence of acetate as the
sole carbon source, an increase in the content of
several proteins involved in gluconeogenesis and
pentose phosphate pathway was registered [3].
The sequencing and annotation of the genome of
the highly acetic acid-tolerant Z. bailii derived
interspecies hybrid strain ISA1307 led to the genome
DNA distributed through 154 scaffolds. The genome
size was found to be around 21.2 Mb,
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Adapted from ref. [2].
18
corresponding to 96% of the genome size estimated
by flow cytometry. Annotation of ISA1307 genome
includes 4385 duplicated genes (about 90% of the
total number of predicted genes) and 1155 predicted
single-copy genes [2]. The availability of the ISA1307
genome sequence also paves the way to a better
understanding of the genetic mechanisms underlying
the generation and selection of more robust hybrid
yeast strains in the stressful environment of wine
fermentations and wines.
The screening of ISA1307 genomic library allowed
the identification of candidate genes for acetic acid
resistance potentially involved in different cellular
processes: cellular transport, transport facilities and
transport routes, protein fate, protein synthesis,
carbohydrate and amino acid metabolism and
transcription [4]. This is consistent with the concept
that the global mechanism behind yeast cells
adaptation and resistance to stress conditions is
multifactorial and do not rely on a single key cellular
process.
Funded Projects
PTDC/AGR-ALI/102608/2008: ZygoSacAR-
Mechanistic insights into acetic acid resistance in
food spoilage yeasts: from the experimental model
Saccharomyces cerevisiae to Zygosaccharomyces
spp. PI: Isabel Sá-Correia
Selected publications
[1] Sá-Correia I, Guerreiro JF, Loureiro-Dias MC, Leão
C, Côrte-Real M. “Zygosaccharomyces”. In: Batt, C.A.,
Tortorello, M.L. (Eds.), Encyclopedia of Food
Microbiology, vol 3. Elsevier Ltd, Academic Press, pp. 849
–855, 2014 (ISBN: 9780123847300)
[2] Mira NP, Münsterkötter M, Valada FD, Santos J,
Palma M, Roque FC, Guerreiro JF, Rodrigues F, Sousa
MJ, Leão C, Güldener U and Sá-Correia I. The genome
sequence of the highly acetic acid tolerant
Zygosaccharomyces bailii-derived interspecies hybrid strain
ISA1307 isolated from a sparkling wine plant, DNA
Research, 1-15, 2014.
[3] Guerreiro JF, Mira NP, Sá-Correia I. Adaptive
response to acetic acid in the highly resistant yeast species
Zygosaccharomyces bailii revealed by quantitative
proteomics, Proteomics, 12, 2303-18, 2012.
[4] Palma M, Roque FC, Guerreiro JF, Queirós L, Mira
NP and Sá-Correia I. Screening of a genomic library from
the Zygosaccharomyces bailii-derived interspecies hybrid
strain ISA1307 to search for genes responsible for acetic
acid resistance, submitted.
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20 12-13 Biennial Report
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Objectives
Multidrug resistance (MDR) is implicated in the fail-
ure of many therapeutic, food-preservation and crop
protection actions, but can also be explored in the
improvement of biotechnological process productivi-
ty. MDR is often attributed to the action of multidrug
efflux pumps found in the plasma membrane of all
living cells. In this context our research aims to:
1.Carry out the functional analysis of MDR transport-
ers from the Major Facilitator Superfamily (MFS) and
ATP-Binding Cassette (ABC) superfamily in the
model eukaryote Saccharomyces cerevisiae, explor-
ing the obtained data to engineer stress resistant
yeast strains to be used as cell factories.
2.Extend the current knowledge obtained in the
yeast model to the plant model Arabidopsis thaliana,
exploring the obtained information to elucidate the
mechanisms of underlying resistance to chemical
stresses of agricultural interest involving these MDR
transporters and to engineer stress tolerant plants.
3.Extend the current knowledge to the understanding
of antifungal drug resistance in fungal pathogens, in
particular Candida species.
Research Topics
1. Functional analysis of S. cerevisiae MDR trans-
porters of the MFS
Since the release of S. cerevisiae’s genome se-
quence, our research group has been involved in the
functional analysis of new MDR transporters from
the MFS (Sá-Correia et al., 2009, Trends Mibrobiol,
17, 22-31) and ABC superfamily. Ongoing research
pursues a deeper understanding of the physiological
role of these drug efflux pumps and their contribution
to MDR. The evolution of these transporters, based
on sequence and syntenic releationships, are also
being examined, extending current knowledge to
identify and predict the function of MFS-MDR trans-
porters in ascomycetous yeasts. Furthermore, the
functional analysis and engineering of the expres-
sion of these transporters to obtain yeast strains with
an improved stress resistance and fermentation ca-
pacity is also being carried out.
2. Extrapolation of the current knowledge on MDR
transporters to the plant model Arabidopsis thaliana
The knowledge gathered in S. cerevisiae and its use
as a heterologous expression system is being used
to guide the analysis of the role of membrane trans-
porters in A. thaliana resistance to abiotic stress
conditions, in collaboration with the Plant Molecular
Biology (PMB) group, Instituto Gulbenkian de Ciên-
cia (IGC). Several plant MFS transporters are being
functionally and biochemically characterized, focus-
ing on their physiological role and involvement in
stress tolerance (Cabrito et al., 2009, Appl Microbiol
and Biotechnol, 84: 927-936) [1,2]. This joint effort
aims at the elucidation of the biological role of this
poorly characterized family of proteins and the devel-
opment of stress-resistant crops. Due to a recent
collaboration with the Genomics of Plant Stress
Drug efflux pumps in cell defense: from the yeast
model to plant cells BSR
G
Isabel Sá-Correia, Miguel C. Teixeira, Tânia R. Cabrito, Nuno P. Mira, Paulo J. Dias, Sandra
C. dos Santos
PhD and MSc students: Cláudia P. Godinho, Catarina Costa, Joana Guerreiro, Filipa C. Roque, Catarina Prata
Fluorescence microscopy of yeast cells harboring either
the cloning vector or a recombinant plasmid that over-
produces the plant model Pht1;9 fused to the green-
fluorescence protein (GFP) (adapted from ref. [2])
20
(GPlantS) group, Instituto de Tecnologia Química e
Biológica (ITQB), our group is also aiming to contribute
to the functional and biochemical characterization of
membrane transporters from rice, focusing on abiotic
stress responses, namely cold, high salinity and
drought. Given that some of these transporters are
determinants of resistance to compounds of agricultur-
al relevance, such as herbicides, agricultural fungi-
cides, toxic soil contaminant metals, among others,
they can be considered interesting candidates to im-
prove crop growth based on genetic engineering. The
successful cloning and expression of yeast TPO1 in
the plant model Arabidopsis thaliana, resulting in multi-
ple herbicide (2,4-D, barban, metolachlor and alachlor)
and metal (cadmium and aluminium) resistant plant
strains (Patent Application PT 105727, 28/10/2011),
paved the way to the exploitation of yeast MDR efflux
pumps in Plant Biotechnology.
3. Extrapolation of the current knowledge on MDR
transporters to the fungal pathogens of the Candida
genus
The emergence of antifungal drug resistance among
fungal pathogens poses a severe clinical problem.
Drug resistance often results from the action of drug
efflux pumps from the ATP-Binding Cassette and Major
Facilitator Superfamilies (MFS). However, the role of
the putative drug:H+ antiporters (DHA) from the MFS in
fungal pathogens has largely escaped characterization.
The systematic characterization of the DHA
transporters from Candida glabrata is being undertaken
and most of its 10 uncharacterized DHA1 transporters
were indeed found to be implicated in multiple drug
resistance. Altogether, the results obtained during the
ongoing systematic characterization of the forgotten
DHA transporter family in C. glabrata are expected to
improve current understanding of multidrug resistance
in fungal pathogens and to guide de design of new
tools for the diagnosis and treatment of Candida
infections.
Main achievements
The evolutionary history of S. cerevisiae DHA2 (14-
spanner Drug:H+ Antiporter family 2 transporters of the
MFS involved in multidrug resistance), ARN
(siderophore transporters) and GEX (glutathione:H+
antiporters) encoding genes from 31 sequenced yeast
strains of 25 different hemiascomycetous species, was
reconstructed. A common evolutionary root shared by
the encoded proteins was hypothesized and a new
protein family, denominated DAG, was proposed to
span these three phylogenetic subfamilies [3].
The reconstruction of the evolutionary history of the
hemiascomycete DHA1 family genes (Dias et al., 2010,
Omics, 14(6): 701-10) was extended to the species
comprised in the CTG phylogenetic complex to get new
insights on how these family genes have evolved in
medically relevant Candida species [4].
The expression of the S. cerevisiae ABC drug efflux
pump Pdr18, previously found to play a role in the
incorporation of ergosterol in the plasma membrane
(Cabrito et al., Biochem J, 440, 195-202, 2011), was
found to increase yeast ethanol tolerance and
fermentation performance. PDR18 overexpression,
leading to the production of highly inhibitory
concentrations of ethanol, in industrial yeast strains
appears to be a promising approach to improve
alcoholic fermentation performance for sustainable bio-
ethanol production [5].
Through biochemical and functional studies using
the yeast experimental model system, A. thaliana
plasma membrane transporter Pht1;9 was found to
mediate inorganic phosphate acquisition through the
plasma membrane of cells during phosphorus
starvation, being identified as a high-affinity phosphate
Representative images of a wild type and two zifl1 loss-of-function mutant plants grown under
normal water supply or exposed to drought stress (adapted from ref. [1]).
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20 12-13 Biennial Report
12-13
transporter [2].
The results obtained in the yeast model (Cabrito et
al., Appl Microbiol Biotechnol, 84, 927-936, 2009)
guided the study of ZIFL1 A. thaliana gene in the plant
model. The ZIFL1 gene was found to generate two
alternatively-spliced transcripts, ZIFL1.1 and ZIFL1.3.
ZIFL1.1 was found to be a root tonoplast-localized
transporter that confers resistance to the auxin-like
herbicide 2,4-D, being involved in auxin transport
across the plasma membrane. ZIFL1.3 was found to
be targeted to the plasma membrane of stomatal
guard cells and to regulate drought tolerance [1].
Through heterologous expression and functional
analysis in yeast, another Arabidopsis Tpo1 homolog
gene, ZIF2, was found to be involved in increasing zinc
tolerance and decreasing its intracellular accumulation
in yeast and in A. thaliana [6].
The yeast drug efflux pump Tpo1 was
heterologously expressed in A. thaliana and such
expression was found to reduce the level of growth
inhibition of the transgenic plants exposed to
agricultural pesticides and metal ions (Patent
Application – PT 105727, 28/10/2011). Transgenic
plants that express this yeast gene were found to
accumulate less 2,4-D in the root tips, compared to
plants from the parental strain, when exposed to this
herbicide. These important findings open new avenues
for the use of biotechnology to improve crop tolerance
to chemical and environmental stress.
The Candida glabrata CgQdr2, CgAqr1 and
CgTpo3 were functionally characterized within the
scope of their role in antifungal drug resistance. The
clnical significance of these findings is currently being
inspected (more details in pages 23 and 24) [7-9].
Funded Projects
MFS-stress-Major Facilitator Superfamily transport-
ers in the context of modern agriculture constraints:
exploratory studies (EXPL/AGR-PRO/1013/2013). PI
at IST: Isabel Sá-Correia
FUNDRING – Identification of new biomarkers for
antifungal drug resistance diagnosis in Candida gla-
brata: the particular role of multidrug resistance trans-
porters (PTDC/EBB-BIO/119356/2010). PI: Miguel C
Teixeira
Resistance to pesticides and other chemical stress-
es of agricultural interest: Role of plant Major Facilita-
tor Superfamily Transporters, PTDC/AGR-
AAM/102967/2008, PI at IST: Isabel Sá-Correia
Selected Publications
[1] Remy E, Cabrito TR, Baster P, Batista RA, Teixeira
MC, Friml J, Sá-Correia I, Duque P. A Major Facilitator Su-
perfamily transporter plays a dual role in polar auxin transport
and drought stress tolerance in Arabidopsis, The Plant Cell,
25, 901-26, 2013.
[2] Remy E, Cabrito TR, Baptista R, Teixeira MC, Sá-
Correia I, Duque P. The Pht1;9 transporter mediates inor-
ganic Pi acquisition by the Arabidopsis thaliana root during
phosphorus starvation, The New Phytologist, 195, 356–371,
2012.
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Phylogenetic tree of the DHA2, ARN and GEX transportes from Saccharomyces sensu stricto
group
22
[3] Dias PJ, Sá-Correia I, The drug:H+ antiporters of family 2
(DHA2), siderophore ptransporters (ARN) and gluthatione:H+
transporters (GEX) have a common evolutionary origin in
hemiascomycete yeasts, BMC Genomics 14: 901, 2013.
[4] Dias PJ, Sá-Correia I. Phylogenetic and syntetic analysis
of the 12-spanner drug:H+ antiporters family 1 (DHA1) in
pathogenic Candida species: evolution of MDR1 and FLU1
genes. Resubmitted to Genomics, 2014.
[5] Teixeira MC, Godinho CP, Cabrito TR, Mira NP, Sá-
Correia I. Increased expression of the yeast multidrug re-
sistance ABC transporter Pdr18 leads to increased ethanol
tolerance and ethanol production in high gravity alcoholic
fermentation, Microbial Cell Factories, 11, 98, 2012.
[6] Remy E, Cabrito TR, Batista RA, Hussein MAM,
Teixeira MC, Athanasiadis A, Sá-Correia I, Duque P.
Intron Retention in the 5'UTR of the Novel ZIF2 Transporter
Enhances Translation to Promote Zinc Tolerance in
Arabidopsis, PLOS Genetics, accepted.
[7] Costa C, Nunes J, Henriques A, Mira NP, Nakayama
H, Chibana H, Teixeira MC. The Candida glabrata drug:H+
antiporter CgTpo3 (ORF CAGL0I10384g): role in azole drug
resistance and polyamine homeostasis, J Antimicrob
Chemother, doi: 10.1093/jac/dku044, 2014
[8] Costa C, Pires C, Cabrito TR, Renaudin A, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. Candida glabrata
drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) confers
imidazole drug resistance, being activated by the CgPdr1
transcription factor, Antimicrob Agents Chemother, 57(7),
3159-67, 2013.
[9] Costa C, Henriques AS, Pires C, Nunes J, Ohno M,
Chibana H, Sá-Correia I, Teixeira, MC. The dual role of
Candida glabrata Drug:H+ Antiporter CgAqr1 (ORF CA-
GL0J09944g) in antifungal drug and acetic acid resistance,
Frontiers in Microbiology, 4, 170, 2013.
[10] dos Santos SC, Teixeira MC, Dias PJ, Sá-Correia I.
MFS transporters required for multidrug/multixenobiotic (MD/
MX) resistance in the model yeast: understanding their
physiological function through post-genomic approaches,
Frontiers in Physiology, 5: 180, 2014.
[11] Costa C, Dias PJ, Sá-Correia I, Teixeira MC. MFS
multidrug transportes in pathogenic fungi: do they have real
clinical impact?, submitted to Frontiers in Physiology,
“Physiological role and regulation of Multidrug/Multixenobiotic
resistance membrane trans-porters”
Issued patent
Sá-Correia I, Cabrito TR, Teixeira MC, Remy E, Duque P.
Utilização de um gene que confere resistência a
xenobióticos em plantas (Use of a gene that confers
resistance to xenobiotics in plants), Provisional national
patent number PT105727. Priority date: 28th October 2011
(Patente de invenção nacional n.º 105727; Date: 2013.06.21)
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Phylogenetic tree of the DHA1 transportes from Saccharomyces sensu stricto group
23
20 12-13 Biennial Report
12-13
Antifungal drug resistance and interaction with the
host in the human pathogens Candida glabrata and
Candida albicans BSR
G
Miguel C. Teixeira, Nuno P. Mira
PhD and MsC students: Catarina Costa, Rúben Bernardo, Carla Pires, André Henriques, Joana Nunes, Andreia Ponte,
Diana Cunha
Objectives
Infections caused by Candida species, a problem of
increasing clinical significance, are recognized as the
4th or 5th most common cause of nosocomial infec-
tions. Candida glabrata infections rank second in fre-
quency, after those caused by Candida albicans. The
frequency and relative high mortality levels (up to 45%
for C. glabrata) of these infections are generally at-
tributed to the capacity of these pathogenic yeasts to
efficiently develop multiple drug resistance (MDR). In
the particular case of C. glabrata concern is further
raised by the observation that clinical isolates are of-
ten resistant to azoles, the frontline drugs used in the
treatment and prophylaxis against fungal pathogens.
The key goals of the ongoing research in this field
include the identification of antifungal drug resistance
mechanisms, with focus on the action of multidrug
efflux pumps, aiming the identification of resistance
diagnosis biomarkers and the development of alterna-
tive therapeutic actions. The mechanisms that allow
pathogenic yeast to thrive in the human host, particu-
larly when facing inhibitory weak acid concentrations
are also being inspected.
Research Topics
1. Deciphering antifungal drug resistance mecha-
nisms: the particular role of multidrug transporters of
the DHA family
The emergence of antifungal drug resistance among
fungal pathogens poses a severe clinical problem.
Membrane proteomics is being used to gain further
understanding of the molecular mechanisms underly-
ing the ability of C. glabrata cells to cope with some of
the currently used antifungals. Drug resistance often
results from the action of drug efflux pumps from the
ATP-Binding Cassette and Major Facilitator Super-
families (MFS). However, the role of the putative
drug:H+ antiporters (DHA) from the MFS in fungal
pathogens has largely escaped characterization (more
details in the previous topic).
The systematic characterization of the DHA transport-
ers from Candida glabrata is being undertaken and
most of its 10 uncharacterized DHA1 transporters
were indeed found to be implicated in multiple drug
resistance. In collaboration with the Microbiology Lab
of the Faculty of Medicine of the University of Porto,
the relevance of these transporters in the clinical ac-
quisition of drug resistance is further being analysed.
2. Unveiling the mechanisms of weak acid resistance
in Candida species, and their role in host colonization
To successfully colonize the human host C. albicans
and C. glabrata have to cope with numerous stresses
found in the infection sites which include the presence
of a commensal microbiota. Besides competing for
nutrients, commensal bacteria produce acetic and
lactic acids at concentrations that inhibit growth, spe-
cially in acidic niches such as the vaginal tract. Using
a combination of gene-by-gene and transcriptomic
analyses it was demonstrated that C. glabrata toler-
ance to acetic and lactic acids is largely dependent on
the activity of a novel regulatory system controlled by
the transcription factor CgHaa1. This work was per-
formed in collaboration with the Conway Institute, Uni-
versity College in Dublin (CI-UCD). In the presence of
acetic acid CgHaa1 expression was also found to
increase biofilm formation and adherence of C. gla-
brata to epithelial cells. The elucidation of the mecha-
nism by which CgHaa1 influences biofilm formation
and the adhesive properties of C. glabrata, two well
known virulence traits of this pathogenic yeast, is be-
ing further scrutinized in collaboration with IBB/CEB,
University of Minho.
24
3. Development of molecular-based diagnosis tools for
the detection of systemic candidiasis
The diagnosis of systemic infections caused by C.
albicans and C. glabrata is compromised by the fact
that often the fungal burden present in blood samples
is too low to be detected. The development of more
sensitive diagnosis tools for systemic candidiasis is of
utmost importance since a delay in diagnosis is
associated with an increased risk of death of the
patients. A project aiming the development of a novel
DNA chip that could be used for diagnosis of systemic
candidiasis has been started during 2013 within the
scope of the “PanCandida” project, one of the
awardees of the Gilead Génese 2013 Program. In this
project the transcriptomes of clinical C. glabrata and C.
albicans isolates recovered from the bloodstream of
patients with diagnosed systemic candidiasis are being
compared with the transcriptome of commensal
isolates to identify a set of genes significantly over-
expressed in the systemic isolates that could serve as
a signature of systemic candidiasis and used for
diagnosis. This project is being developed in
collaboration with Centro Hospitalar de Lisboa Central
and CI-UCD.
Main achievements
The expression of CgQDR2 in C. glabrata was found
to confer resistance to imidazole antifungal drugs.
CgQdr2 was found to play a role in the extrusion of
these antifungals from pre-loaded cells. CgQDR2
transcript levels were further seen to be up-regulated
in C. glabrata cells challenged with clotrimazole in the
direct dependency of the CgPdr1 transcription factor
[1].
The Drug:H+ Antiporter CgAqr1 was identified as a
determinant of resistance to the antifungal agent
flucytosine and to reduce the intracellular accumulation
of this drug. CgAqr1 was also found to confer
resistance to acetic acid, which easily accumulates to
inhibitory levels in the vaginal tracts, suggesting that
this transporter may play a role in C. glabrata
persistent colonization in this niche [2].
The DHA CgTpo3 was found to confer resistance to
azole antifungal drugs, catalyzing their extrusion from
within preloaded C. glabrata cells. CgTpo3 was further
found to confer resistance to polyamines, contributing
to decrease its intracellular accumulation in spermine-
challenged cells. Since this compound may reach
inhibitory concentrations in the male genital tract, it is
hypothesized that CgTpo3 may play a role in C.
glabrata persistent colonization in this niche [3].
The role played by the CgHaa1-dependent signaling
system in C. glabrata response and tolerance to acetic
acid stress was characterized. Around 25% of the C.
glabrata genes up-regulated under acetic acid stress
were found to be regulated, directly or indirectly, by
CgHaa1. Several of the CgHaa1-regulated were
required for maximal C. glabrata tolerance to acetic
acid including genes involved in regulation of internal
pH and in multidrug-resistance.
Funded Projects
FUNDRING – Identification of new biomarkers for
antifungal drug resistance diagnosis in Candida glabra-
ta: the particular role of multidrug resistance transport-
ers (PTDC/EBB-BIO/119356/2010). PI: Miguel C.
Teixeira
PanCandida- Towards the development of a pange-
nomic DNA chip for the early detection of systemic
candidiasis caused by C. albicans and C. glabrata
(PGG/035/2013). PI: Nuno P. Mira
Selected Publications
[1] Costa C, Pires C, Cabrito TR, Renaudin A, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. Candida glabrata
drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) confers
imidazole drug resistance, being activated by the CgPdr1
transcription factor, Antimicrobial Agents and Chemothera-
py, 57(7), 3159-67, 2013.
[2] Costa C, Henriques AS, Pires C, Nunes J, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. The dual role of Can-
dida glabrata Drug:H+ Antiporter CgAqr1 (ORF CA-
GL0J09944g) in antifungal drug and acetic acid resistance,
Frontiers in Microbiology, 4, 170, 2013.
[3] Costa C, Nunes J, Henriques A, Mira NP, Nakayama H,
Chibana H, Teixeira MC. The Candida glabrata drug:H+ anti-
porter CgTpo3 (ORF CAGL0I10384g): role in azole drug re-
sistance and polyamine homeostasis, Journal of Antimicro-
bial Chemotherapy, 2014, doi: 10.1093/jac/dku044.
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20 12-13 Biennial Report
12-13
Objectives
The activities of the BSRG in the area of Environmen-
tal Biotechnology and Chemistry are developed in the
fields of Environmental Microbiology and Environmen-
tal Toxicology, aiming:
1. To characterize the microbial populations and
their dynamics from processes of biotechnological
interest, such as anaerobic digesters and microbial
fuel cells.
2. To improve knowledge on bacterial biodegrada-
tion of s-triazine herbicides, viewing optimization of
bioremediation strategies that will help to decontami-
nate soils polluted with s-triazine-based herbicidal
formulations (due mainly to overuse, careless dispos-
al or accidental spills). These processes may help to
prevent contamination of adjacent freshwater com-
partments with the ecotoxic herbicides and respective
chlorinated metabolites (please see highlight in page
27).
3. To develop bioassays for the assessment of the
toxicity of xenobiotics (e.g. pesticides, synthetic dyes
and others) based on the two simple models of the
eukaryotic cell, the yeast Saccharomyces cerevisiae
and the nematode Caenorhabditis elegans.
4. To identify molecular biomarkers of toxicity and
to gain insights into potential mechanisms of response
to xenobiotic compounds (e.g. pesticides) based on
global gene expression profiling in the eukaryotic
model S. cerevisiae.
Research Topics
1. Molecular characterization of microbial communi-
ties
Microbial populations from UASB reactors operated
continuous or intermittently (collaborative work with
CESAM and DEP - Univ. Aveiro), and from Microbial
Fuel Cells producing electricity from wastewaters
(collaboration with BERG—IST) are being character-
ized by molecular methods based on 16S rDNA se-
quences. FISH methodologies are being used to
quantify key bacterial members of methane-producing
microbial consortia in UASB reactors treating dairy
industry wastewaters [3, 4, and Nadais et al., 2011,
Energy, 36: 2146-2168].
2. Yeast- and C. elegans-based toxicity testing
The single-cell eukaryotic model S. cerevisiae
(endpoints: growth inhibition, and modification of ex-
pression of selected gene biomarkers) and the simple
animal model C. elegans (endpoint: inhibition of repro-
duction) have been exploited in the assessment of the
potential toxicity of xenobiotics, namely of pesticides
and of synthetic dyes. For example, the levels of tox-
icity of textile-dyes and respective reaction products,
formed during microbial and/or enzymatic biotransfor-
mation processes were compared, in collaboration
with ITQB [Mendes et al., 2011, Bioresour Technol,
102: 9852-9859], and more recently in collaboration
with ENVERG–CEBQ (in course).
Environmental Microbiology and Toxicology
BSR
G
Cristina A. Viegas, Jorge H. Leitão, Christian G. Ramos, Sílvia A. Sousa
PhD and MSc students: Fátima N. Gil, André M. Grilo, Joana R. Feliciano, Mafalda Cardoso;
Research assistant: Vera P. Silva
26
3. Xenobiotic response in S. cerevisiae based on
gene expression profiling
Genome-wide expression profiling of the yeast re-
sponse to exposure to equitoxic concentrations
(20%-growth inhibitory concentrations) of six pesti-
cides from different chemical families
(chloroacetanilide, phenylurea, chlorophenoxyacetic
acid, carbamate and anylinopyrimidine families) has
been analysed in order to identify molecular bi-
omarkers of exposure and toxicity which may be
useful for development of yeast-based bioassays for
preliminary toxicity testing or for assessment of pes-
ticide toxicity in environmental samples, and to pre-
dict novel mechanisms of response to pesticide
moderate toxicity that may be relevant for non-target
eukaryotes, is being explored (collaboration with
IGC) [Gil et al., 2011, Environ Toxicol Chem, 30:
2506-2518].
Main Achievements
A FISH methodology was established to quantify
Synthrophomonadaceae in microbial populations
from UASB reactors [3,4].
Comparison of the datasets of differentially ex-
pressed genes in yeast in response to the six pesti-
cides under study highlighted the potential of the
transcriptional profiling approach to distinguish be-
tween the toxicological effects of structurally different
pesticides in the yeast and allowed the identification
of potential biomarkers of toxicity [1,2]
A toxicity bioassay based on measurements by
reverse transcriptase RT-PCR of transcripts levels
alterations in yeast pyrimethanil-responsive genes
was established [1,2], and its feasibility to assess the
toxicity of simulated runoff samples has been exam-
ined. These samples were generated using a semi-
field simulator of soil contamination with a pyrime-
thanil commercial formulation mimicking worse-case
scenarios (prototype developed by the project part-
ners from IMAR/Univ. Coimbra). Comparison of
data from the gene expression bioassay with con-
ventional ecotoxicity endpoints obtained with the
animal model C. elegans, and using other ecologi-
cally relevant standard aquatic test-organisms
(collaboration with IMAR/Univ. Coimbra), has ena-
bled us to discuss usefulness and limitations of the
yeast and the C. elegans bioassays in environmental
samples biomonitoring (in course).
Funded projects
Studies on the efficacy and scale-up of bioremedi-
ation strategies for soils contaminated with the herbi-
cide terbuthylazine, PTDC/AAC-AMB/111317/2009,
PI: Cristina A. Viegas
Development of bioassays for herbicide toxicity
based on gene expression profiling using yeast cells,
PTDC/AMB/64230/2006, PI: Cristina A. Viegas.
Enzymatic degradation and synthesis of azo and
anthraquinonic dyes, PTDC/BIO/72108/2006, PI at
IST: Cristina A. Viegas.
Optimization and control of intermittent UASB re-
actors and microbial population dynamics, PTDC/
AMB/65025/2006, PI at IST: Jorge H. Leitão.
Selected Publications
[1] Gil FN, Gonçalves AC, Becker JD, Viegas CA. Ge-
nome-wide transcriptional response to pesticide exposure
in the model yeast Saccharomyces cerevisiae, the FEBS
journal, 279(S1):230, 2012.
[2] Gil FN, Gonçalves AC, Becker JD, Viegas CA. Pesti-
cide toxicity studies using gene expression profiling in the
model yeast Saccharomyces cerevisiae, 7th European
Conference on Pesticides and Related Organic Micropollu-
tants in the Environment & 13th Symposium on Chemistry
and Fate of Modern Pesticides – Pesticides2012, Porto 7-
10 October, Portugal (oral communication).
[3] Nadais MH, Capela I, Arroja L, Leitão JH, Grilo AM,
Couras C. Effects of operational shocks on UASB microbi-
al populations, 8th Conference on sustainable development
of energy, water and environment systems. September 22-
27, Dubrovnik, Croatia, 2013
[4] Ramos CG, Grilo AM, da Costa PJP, Nadais H,
Leitão JH. Extraction and Purification of DNA from UASB
Reactors Sludge Samples. In: DNA Binding and DNA Ex-
traction: Methods, Applications and Limitations (Chunxu
Zhou and Xia Ling Eds.). Nova Publishers. 2012. ISBN 978
-1-61470-958-9.
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20 12-13 Biennial Report
12-13
Objectives
s-Triazine herbicides (e.g. atrazine-ATZ, and ter-
buthylazine-TBA) have been used extensively world-
wide for weed control in a variety of crops, and in
non-agriculture applications. In the European Union
(EU), about 3600 tons of s-triazine herbicides have
been used each year, representing 4.3% of the total
of herbicides. ATZ is moderately mobile through soil,
and accidental spills or careless uses can lead to
contamination of sediments and aquatic systems
due to leaching and runoff events. Indeed, ATZ and
its toxic N-dealkylated metabolites are among the
most frequently detected herbicides on water com-
partments in quantities exceeding the values set up
by regulatory agencies for drinking water [1]. As a
consequence, ATZ has recently been banned in the
EU countries due to the precautionary principle,
though it is still used in the USA, Africa, Latin Ameri-
ca, Asia and Australia. In the last years, mainly in the
EU, TBA has replaced ATZ in herbicidal formula-
tions, hence gaining agricultural relevance. It has
been considered as having more environment-
friendly properties than ATZ (e.g. lower mobility and
bioavailability in soil). However, its higher sorption to
soil organic matter may increase the potential to
reach waters and sediments due to runoff/drainage
events, with special relevance for sites where care-
less disposal or accidental spills may occur, thus
raising ecological risk concerns [1].
In this project, we aim to improve knowledge
and exploit the catabolic ability of two soil s-triazine–
degrading bacterial strains, Pseudomonas sp. ADP
and Arthrobacter aurescens TC1 [1], viewing optimi-
zation of biodegradation of s-triazine herbicides in
soil, mainly focused on ATZ and TBA. Ultimate goal
is to optimize bioremediation strategies that will con-
tribute to decontaminate soils polluted with s-triazine
-based herbicidal formulations (due mainly to over-
use, careless disposal or accidental spills), thus al-
lowing to prevent contamination of adjacent freshwa-
ter compartments with the herbicides and their toxic
chlorinated metabolites.
Main Achievements
As part of a framework for rational bioremedia-
tion of ATZ-contaminated land, we have presented
evidences that a cleanup tool combining soil bio-
augmentation with the ATZ-mineralizing bacterium
Pseudomonas sp. strain ADP and biostimulation
with citrate is effective under worst-case scenarios
of soil contamination with an ATZ-commmercial
formulation (Atrazerba FL) in bench-scale soil mi-
crocosms [1, and Lima et al., 2009, Chemosphere
74:187-192]. To assess the impact of bioremedia-
tion treatments on soil habitat function and soil re-
tention function, an integrated approach has been
used with ecotoxicity tests with standard soil and
aquatic organisms to assess the potential toxicity of
soil and water samples (e.g. eluates, leachates, and
runoffs), respectively [1, and Chelinho et al., 2010,
Soils Sediments, 10:568-578] (collaboration with
IMAR/Univ. Coimbra).
During 2012, it was demonstrated that the appli-
cation of this bioremediation tool at a larger scale
and under more realistic semifield conditions, was
also clearly effective at removing the herbicide from
soil contaminated with high Atrazerba FL (mimicking
misuse applications or accidental spills) [2]. More
importantly, it contributed to reduce the potential
ecological risks of ATZ for both soil and aquatic
compartments in just 7 days [2].
Bioremediation strategies for soils contaminated
with s-triazine herbicides
Cristina A. Viegas, Arsénio M. Fialho
PhD and MSc students: Fátima N. Gil, Carla A. Mateus, Janete Gonçalves, Viviane Varela, Mafalda Cardoso;
Research assistant: Vera P. Silva
BSR
G
1
10
100
0 2 4 6 8
Pseudom
onas
sp
AD
P
x 1
07
CF
U/g
of
soil
Time (d)
28
We also presented evidences that presence in
soil of concentrations of the chloroacetanilide herbi-
cide S-metolachlor (S-MET) representative of high
doses (up to 50xRD) of a commercial formulation
containing both ATZ and S-MET as active substanc-
es (Primextra-S-Gold), did not affect neither Pseudo-
monas sp. ADP survival nor its ability to mineralize
ATZ, in soil [3]. Consistently, biodegradation experi-
ments in soil microcosms spiked with 20x or 50xRD
of this double commercial formulation revealed rapid
ATZ removal from soil. However, it was not accom-
panied by soil detoxification, as indicated by toxicity
of eluates prepared from samples of treated soil
against a microalgae species. This high toxicity may
be due to residues of S-MET and/or its metabolites
remaining in the soil, which appear to be not degrad-
ed by Pseudomonas sp. ADP [3]. These results indi-
cate that caution should be taken when considering
bioremediation strategies for land polluted with mix-
tures of pesticides, particularly when the contami-
nants that may be mixed with ATZ cannot be biode-
graded by the bioaugmentation bacteria applied.
Concerning TBA biodegradation by Pseudomonas
sp ADP, it was found to be slower and less extensive
than that of ATZ, and this appears not to be related
with significant TBA toxicity towards the bacterial
strain [Carla Mateus, MSc in Biotechnology, IST,
2012].
The efficacy of another s-triazine-biodegrading
bacterial strain Arthrobacter aurescens TC1 over
TBA biodegradation in soil have been under focus in
our more recent research. Arthrobacter aurescens
TC1 appears to be better suited than Pseudomonas
sp ADP for TBA biodegradation in soil. Work in
course aims to optimize inocula preparation in order
to obtain and preserve, in advance, high quantities of
physiologically active cells to be used for the bioaug-
mentation of TBA-contaminated soils in semifield and
field scale trials. Studies have mainly focused the
optimization of growth media, and also of cells for-
mulation and conservation methods (e.g. freeze-
drying, vermiculite-adsorption, or refrigeration at 4ºC)
[4, and Viviane Varela, MSc in Biological Engineer-
ing, IST, 2013]. In addition, the bioremediation effica-
cy of a bioremediation tool based on soil bioaugmen-
tation with Arthrobacter aurescens TC1 (refrigerated
pellet cake, 1 month-preservation) has been exam-
ined at semifield scale in soil microcosms contami-
nated with a TBA commercial formulation (10xRD),
combining chemical and ecotoxicity data.
Funded project
Studies on the efficacy and scale-up of bioremedi-
ation strategies for soils contaminated with the herbi-
cide terbuthylazine, PTDC/AAC-AMB/111317/2009,
PI: Cristina A. Viegas.
Selected Publications
[1] Viegas CA, Chelinho S, Moreira-Santos M, Costa C,
Gil FN, Silva C, Lima D, Ribeiro R, Sousa JP, Fialho AM.
Bioremediation of soils contaminated with atrazine and
other s-triazine herbicides: current state and prospects, In:
Justin A. Daniels (ed), Advances in Environmental Re-
search - Volume 6, Nova Science Publishers, Inc., NY,
USA, pp. 1-49 (ISBN 978-1-61728-163-1), 2012.
[2] Chelinho S, Moreira-Santos M, Silva C, Costa C,
Viana P, Viegas CA, Fialho AM, Ribeiro R, Sousa JP.
Semi-field testing of a bioremediation tool for atrazine con-
taminated soils: evaluating the efficacy on soil and aquatic
compartments, Environmental Toxicology and Chemis-
try, 31: 1564-1572, 2012.
[3] Viegas CA, Costa C, André S, Viana P, Ribeiro R,
Moreira-Santos M. Does S-metolachlor affects the
performance of Pseudomonas sp. Strain ADP as bioaug-
mentation bacterium for atrazine-contaminated soils?,
PLoS ONE 7(5): e37140, doi:10.1371/
journal.pone.0037140, 2012.
[4] Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.
A bactéria Arthrobacter aurescens TC1 como ferramenta
de biorremediação para ambientes contaminados com os
herbicidas terbutilazina e atrazina, In: Borrego C, Miranda
AI, Arroja L, Fidélis T, Castro EA, Gomes AP (eds), Actas
da 10ª Conferência Nacional do Ambiente (ISBN: 978-989-
98673-0-7), Departamento de Ambiente e Ordenamento da
Universidade de Aveiro, Aveiro, Portugal, 2013.
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(d
-1)
Primextra S-Gold dose (xRD)
0 20 50
Atrazerba FL dose (xRD)
29
20 12-13 Biennial Report
12-13
Objectives
Cystic Fibrosis (CF) patients have a predisposition to
recurrent and chronic respiratory infections that con-
tribute significantly to disease progression. Although
the large majority of respiratory infections among CF
patients are caused by Pseudomonas aeruginosa,
infections caused by Burkholderia cepacia complex
(Bcc) bacteria are especially threatening and feared
since they are largely associated with a worse prog-
nosis and increased risk of death, in particular with
the so called “cepacia syndrome”. These opportunistic
pathogens are inherently resistant to most antibiotics
and can adapt to adverse environmental conditions,
rendering their eradication from the CF lung almost
impossible. Our research in the field aims to contrib-
ute to the understanding of Bcc pathogenesis in CF
patients, in particular regarding the mechanisms of
persistence and adaptive strategies employed during
long-term residence in the CF lung, occurring along-
side with disease progression. Our research has fo-
cused not only on the most frequent B. cenocepacia
and B. multivorans Bcc species, but also on B. cepa-
cia, B. dolosa, B. stabilis and B. contaminans. These
species are rarely isolated worldwide and are there-
fore poorly studied. We expect to contribute to the
identification of potential determinants of virulence as
targets for new therapeutics in CF and potential tar-
gets for inactivation to enhance antimicrobial activity
and limit persistent infections by these bacteria.
Research Topics
1. Surveillance and epidemiology of Bcc bacteria
amongst a Portuguese CF population followed at
Hospital de Sta Maria
Although transient infections may occur for some pa-
tients, acquisition of Bcc typically results in chronic
infection. Our research group has maintained a 2 dec-
ade-long collaboration with the major Portuguese CF
Treatment Centre at Hospital de Santa Maria (HSM),
in Lisbon, for the microbiological surveillance of Bcc
respiratory infections (Coutinho et al., 2011, Front Cell
Inf Microbio, 1:1-11; Coutinho et al., 2011, Infect Im-
mun, 79:2950-2960). We have published the first
studies describing the epidemiology of Bcc in CF res-
piratory infections in Portugal and gathered a large
collection of over 700 clinical isolates, which includes
numerous clonal variants retrieved during long-term
infection of several CF patients.
During 2012-13, the collaboration with the
HSM CF Treatment Centre and Clinical Pathology
Service was continued and the epidemiological sur-
vey of Bcc respiratory infection amongst the CF popu-
lation followed in this hospital. Also, we have provided
support to Portuguese hospitals regarding the identifi-
cation of the Bcc species to which clinical isolates
belong, using molecular biology approaches, whenev-
er this was considered critical.
Species identification as well as strain geno-
typing and monitoring of clonal variants during long-
term colonization are critical tasks for the systematic
epidemiological surveillance of Bcc bacteria obtained
from CF patients at Hospital de Sta Maria, during the
hospital routine. We have been using state of the art
techniques for species identification and differentia-
tion of specific lineages based on multilocus se-
quence typing (MLST). However, this technical ap-
proach is still limited to the analysis of a small number
of isolates due to the high cost and time-consuming of
this procedure. During 2012-2013, we contributed to
the optimization of a protocol (the SNaPBcen assay)
for extensive bacterial population studies. The strate-
gy used for the SNaPBcen assay is based on target-
ing single nucleotide polymorphisms (SNPs) located
in MLST genes instead of sequencing full MLST se-
quences.
2. Adaptive evolution and persistence in the CF lung
during chronic infection
Bcc bacteria have remarkable genome plasticity,
providing an invaluable advantage for adaptation to
the highly stressful CF lung environment. Widespread
positive selection across the genome of Bcc bacteria
leads to the emergence of multiple phenotypic vari-
ants of the clonal population exhibiting phenotypic
alterations relevant to bacterial pathogenesis. Under-
standing the mechanisms of microbial genetic adapta-
tion during chronic infection, the corresponding impli-
cations for different Bcc species/strains, and the im-
pact of Bcc infections on clinical outcome and disease
Burkholderia cepacia complex bacteria in cystic fibro-
sis: epidemiology, virulence, and adaptive evolution
during chronic lung infection
Isabel Sá-Correia, Carla C. Coutinho, Sandra C. dos Santos
PhD students: Ana S. Moreira, Rita Maldonado
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progression, is vital for the improvement of infection
control policies and therapeutic approaches by CF
Centres.
We have been involved in the characterization
of relevant features in the pathogenesis of sequential
Bcc clonal variants for which patient history is availa-
ble, together with the exploitation of genome-wide
expression approaches and comparative genomic
analyses. Significantly, this includes Bcc species that
are rarely isolated and therefore are poorly studied, in
spite of their damaging effects on CF patients. In fact,
the chronically colonized Portuguese CF population
followed at Hospital de Sta Maria is remarkably atypi-
cal since it includes a high representation of rare Bcc
species in CF, namely, B. cepacia, B. dolosa and B.
contaminans. In particular, the abnormal prevalence
of B. cepacia, a unique case worldwide, was associ-
ated with intrinsically contaminated non-sterile saline
solutions for nasal application, detected, with our col-
laboration, during routine market surveillance by the
Portuguese competent authority (Cunha et al., 2007,
J Clin Microbiol, 45:1628-33).
We have also contributed to the study of mi-
crobial adaptation and evolution within the host envi-
ronment based on extensive phenotypic, genotypic
and genome-wide expression analysis of selected B.
cenocepacia clonal variants (Coutinho et al., 2011,
Infect Immun, 79:2950-2960); Madeira et al., 2011,
Proteomics, 11:1313-1328; Mira et al., 2011, PLoS
ONE, 6:e28831) [1], recently extended to the pheno-
typic variation of B. dolosa [2] and B. cepacia species
(unpublished results). Previous studies have focused
on 11 sequential B. cenocepacia clonal isolates re-
trieved from a chronically colonized CF patient, from
the onset of infection until his death 3.5 years later
with cepacia syndrome. Three of these 11 isolates
have been particularly scrutinized by genome-wide
expression analyses. The virulence of these 3 iso-
lates was also compared, and it was found that the 2
isolates retrieved during late-stage disease are signif-
icantly more virulent [1]. We also used quantitative
proteomics, at both the intracellular and extracellular
levels, to identify mechanisms that could be associat-
ed with this increased virulence and ability to persist
in the lung. Our findings reinforce the crucial role of B.
cenocepacia adaptation to the host environment in
the development of chronic infections and highlight
the importance of metabolic reprogramming in the
adaptation and increased pathogenic potential of B.
cenocepacia during the course of a chronic infection.
Comparative analysis of the endometabolome of dif-
ferent Bcc clinical isolates can be helpful to gain nov-
el insights into the contribution of metabolites to Bcc
virulence in a CF context. As such, we are currently
comparing the endo-metabolome of four sequential B.
cenocepacia clonal variants using an analytical plat-
form based on nuclear magnetic resonance spectros-
copy.
3. Systems biology approach: integration of the re-
sults and more in-depth analyses
We are currently employing an integrated systems
biology strategy, where metabolomic, proteomic, tran-
scriptomic and genomic data are integrated to
achieve a global perspective of Bcc pathogenesis and
evolution within the CF airways (see Figure). The
most promising research lines are being further ex-
plored by in-depth studies. In particular, genomic al-
terations that are identified by the ongoing compara-
tive analysis can be linked with critical phenotypes in
the respective Bcc clonal variants. Similarly, proteins,
metabolites, transcriptional networks and metabolic
pathways of interest emerging from these studies are
being explored in selected Bcc isolates from our col-
lection, to ascertain their relevance in a clinical con-
text and across different Bcc species.
In this context, during 2012 we were partic-
ularly interested in the impact of adaption to micro-
aerophilic conditions in the persistence and viru-
lence of Bcc bacteria in the CF lung, the variation
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of lipopolysaccharide (LPS) structure among differ-
ent isolates, and the related immune and pro-
inflammatory responses (in the context of interna-
tional collaborations in the framework of a COST
Action). We have already compared the chemical
structure of the LPS of studied B. cenocepacia
clonal variants, and described the chemical struc-
ture of the lipooligosaccharide (LOS) endotoxin
from a B. dolosa isolate [3]. Given the importance
of LPS biosynthesis and structure in virulence and
persistent infections, this is a topic that we are
highly interested in pursuing.
Main Achievements
The virulence potential of 3 thoroughly charac-
terized sequential B. cenocepacia isolates
(IST439, IST4113 and IST4134) was compared in
human epithelial cell lines, and it was found that
isolates IST4113 and IST4134, retrieved during
late-stage disease, are significantly more virulent.
The proteomic profiling of these 3 variants was
performed and 52 proteins were found to be differ-
entially expressed in the two last isolates com-
pared with IST439. These proteins are involved in
metabolic functions, nucleotide synthesis, transla-
tion and protein folding cell envelope biogenesis
and iron homeostasis. The quantitative compari-
son of the different proteomes suggests an im-
portant role played by metabolic reprogramming in
the virulence potential and persistence of B. ceno-
cepacia, in particular regarding bacterial adapta-
tion to microaerophilic conditions [1].
The exoproteome of isolates IST439, IST4113
and IST4134, was quantitatively compared using
an experimental design that mimics growth in the
CF lung under oxygen limitation in biofilm-like lay-
ers. NMR-based metabolomics was also explored
to compare the metabolome of these same clonal
variants (unpublished results).
The chemical structure of the LPSs of the above
referred variants was also compared in the context
of COST BM1003 action and results indicate a
marked variation between early and late isolates
LPSs (unpublished results).
Whole-genome sequences of sequential B.
cenocepacia clonal variants with marked pheno-
typic differences and virulence level were obtained
and are currently being analyzed by comparative
genomics. This analysis is expected to provide
clues into virulence mechanisms and into the ge-
nomic evolution underwent by B. cenocepacia
during chronic colonization of the patient’s lung,
and how it translates to a clinical setting.
A novel chemical structure and the strong pro-
inflammatory activity of the lipooligosaccharide of
an isolate rom the rarely found species B. dolosa
species, obtained in Portugal from a cystic fibrosis
patient, were described [3].
The phenotypic assessment of 14 clonal iso-
lates of B. dolosa obtained over a period of 5.5
years of surveillance from the only CF patient in-
fected with B. dolosa in the major Portuguese CF
center, during the last 2 decades, was carried out.
Results reinforce the concept that clonal expansion
occurs during long-term colonization. The first iso-
late retrieved was found to be more susceptible to
antibiotics and to exhibit a higher swarming motility
compared with most of the isolates obtained during
the later stages of disease progression and antimi-
crobial therapy [2].
32
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Contribution to the development of the SNaPBCen
assay based on targeting SNPs located in B. cenoce-
pacia MLST genes, envisaging the genotyping of B.
cenocepacia strains and clonal variants. This method
allows the differentiation of specific lineages and is
capable to reveal isolates with microvariation, and the
presence of multiple clonal variants in patients chroni-
cally colonized with this species [4].
Our longstanding work in the field of expression
proteomic analyses applied to microbiological research
led to the publication of an invited chapter in the book
“Burkholderia: From Genomes to Function”, describing
the methodologies currently available and providing
researchers with a simple tutorial on the application of
two-dimensional electrophoresis-based expression
proteomics, as well as presenting a broad literature
review on the application of proteomic approaches to
different Burkholderia species in fields such as diag-
nostics, vaccine development, infection and patho-
genicity, and biodegradation [5].
In the context of COST BM1003 action, we have
also contributed to the development of an internation-
al Pseudomonas aeruginosa reference panel [6].
Funded projects
ADHRES - Signature Project: Expression profiling of
adhesive (AHD) and resistance (RES) genes in biofilm
lifestyle in P. aeruginosa, P. putida and B. cenocepacia,
in the frame of the ERA-NET Patho- genomics, ERA-
PTG/SAU/0001/2008, PI: Isabel Sá -Correia.
COST BM1003 action: Microbial cell surface determi-
nants of virulence as targets for new therapeutics in Cyst-
ic Fibrosis, PI at IST: Isabel Sá-Correia.
PhysBioFFM - Biological physics studies based on
Force Feedback Microscopy, EXPL/FIS-NAN/1395/2013,
PI at IST: Isabel Sá-Correia.
Selected Publications
[1] Madeira A, dos Santos AC, Santos PM, Coutinho CP,
Tyrrell J, McClean S, Callaghan M, Sá-Correia I. Proteomic
profiling of Burkholderia cenocepacia clonal isolates with differ-
ent virulence potential retrieved from a cystic fibrosis patient
during chronic lung infection, PLoS One, 8(12): e83065, 2013.
[2] Moreira AS, Coutinho CP, Avezedo P, Lito L, Melo-
Cristino J, Sá-Correia I. Burkholderia dolosa phenotypic varia-
tion during the decline in lung function of a cystic fibrosis patient
along 5.5 years of chronic colonization, Journal of Medical
Microbiology, 63: 594-601, 2014 (Week’s Best Articles: Cystic
Fibrosis February 5-12 2014, according to MDLinx Pulmonolo-
gy).
[3] Di Lorenzo F, Silipo A, Lanzetta R, Fazio LL, Paciello I,
Coutinho CP, Sá-Correia I, Bernardini M, Molinaro A. Struc-
tural elucidation and Biological activity of the lipooligosaccharide
of a Burkholderia dolosa isolate from a cystic fibrosis patient,
ChemBioChem 14(9): 1105-15, 2013.
[4] Eusébio N, Coutinho C, Sá-Correia I, Araújo R. SNaP-
Bcen: a novel and practical tool for genotyping Burkholderia
cenocepacia, Journal of Clinical Microbiology, 51(8): 2646-
53,2013.
[5] Sá-Correia I, dos Santos SC, Madeira A. “Burkholderia
Proteomics: methodologies, challenges, and applications” in
Burkholderia: From Genomes to Function (ed. Tom Coenye &
Eshwar Mahenthiralingam), Horizon Press, 2014.
[6] De Soyza A, Hall A, Mahenthiralingam E, Drevinek P,
Kaca W, Drulis-Kawa Z, Stoitsova S, Toth V, Coenye T, Zlos-
nik J, Burns J, Sá-Correia I, De Vos D, Pirnay JP, Kidd T,
Reid D, Manos J, Lockgether J, Wiehlmann L, Tummler B,
McClean S, Winstanley C. Developing an internation-
al Pseudomonas aeruginosa reference panel, Microbiology
Open, 2(6): 1010-1023, 2013.
Structure of Burkholderia dolosa lipooligosaccharide [3].
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Objectives
Long-term infection of the airways of Cystic Fibrosis (CF)
patients with Burkholderia cepacia complex bacteria has
been associated with emergence of mucoid morphotype
variation. The outcome of disease progression seems to
be dependent on the morphotype of the isolates. Our aim
is to understand the cues and the molecular mechanisms
triggering mucoid morphotype variation by following ex-
perimental approaches covering the areas of genomics,
transcriptomics and phenomics.
Research Topics
1. Mucoid morphotype variation of Burkholderia multi-
vorans during chronic persistence in the airways of cystic
fibrosis patients
We have studied two Burkholderia multivorans clonal iso-
lates displaying different morphotypes from a chronically
infected CF patient to evaluate traits development during
lung infection. Expression profiling of mucoid D2095 and
nonmucoid D2214 isolates, using custom Burkholderia
microarrays, revealed downregulation of genes encoding
products related to virulence-associated traits and metab-
olism in D2214. Furthermore, D2214 showed no exopoly-
saccharide production, lower motility and chemotaxis,
lower virulence in Galleria mellonella, and more biofilm
formation. Overall, adaptation during Bcc chronic lung
infections gives rise to genotypic and phenotypic variation
among isolates, contributing to their fitness while main-
taining their capacity for survival in this opportunistic hu-
man niche.
2. Stress conditions triggering the emergence of mucoid
morphotype variation in Burkholderia and effect on antimi-
crobial compounds resistance and biofilm formation in
vitro
We have shown that in vitro, the mucoid clinical isolate B.
multivorans D2095 gives rise to stable nonmucoid vari-
ants in response to nutrient limitation, presence of antibi-
otics, and osmotic and oxidative stresses. Furthermore,
colony morphotype variation within the Burkholderia ge-
nus occurred in Bcc and non-Bcc strains irrespectively of
their clinical or environmental origin. Infection of Galleria
mellonella larvae with mucoid parental isolates and non-
mucoid variants was isolate specific. Survival under nutri-
ent limitation, exposure to oxidative stress and iron limita-
tion was comparable either for the mucoid isolate or the
respective nonmucoid variant. In addition, most of the
tested nonmucoid variants produced more biofilm bio-
mass. Altogether, these findings suggest that important
virulence traits are increased or not significantly affected
by the nonmucoid morphotype of the Burkholderia isolates
and perhaps their virulence potential is preserved.
Mucoid morphotype variation in Burkholderia
cepacia complex clinical isolates
Leonilde M. Moreira, Inês N. Silva, Ana S. Ferreira
MSc student: Andreia C. Tavares
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3. Comparative genomics of Burkholderia multi-
vorans clinical isolates 3. Comparative genomics
of Burkholderia multivorans clinical isolates
Bcc bacteria are characterized by large size ge-
nomes comprising a high number of protein-
encoding genes that confer bacteria the ability to
rapidly adapt and colonize new environmental
niches. To unveil the global genetic differences
that can explain the mucoid phenotypic variation
and give some insights into the evolution of ge-
nome content during chronic lung infection, a
comparative genomic study of two sequential B.
multivorans clinical isolates – the mucoid D2095
and the nonmucoid D2214 was carried out. Whole
-genome sequence and de novo assembly
showed a genome size of 6.7 and 6.5 Mb for B.
multivorans D2095 and D2214, respectively. Com-
parative genomics identified a large deletion of
175 kb in D2214 genome. The remaining muta-
tions are single base-pair mutations or small inser-
tion/deletions. Some of the mutations are likely to
cause partial or complete loss-of-function in the
protein encoded by the altered gene. Our data
confirms genome reduction during chronic infec-
tion as observed for other CF pathogens and
opens new hypotheses for the mechanisms trig-
gering mucoid-to-nonmucoid variation in Bcc.
Main Achievements
Evidences on the genotypic and phenotypic
traits variation of Burkholderia cepacia complex
bacteria during chronic lung colonization.
Demonstration that nutrient limitation, presence
of antibiotics, and osmotic and oxidative stresses
are triggering Bcc mucoid morphotype variation in
vitro.
Sequence determination, assembly and ge-
nome annotation of the two clinical isolates: mu-
coid B. multivorans D2095 and nonmucoid D2214
in collaboration with Prof. Pedro Santos from Uni-
versity of Minho.
Identification of several mutations putatively
involved in mucoid morphotype variation by com-
parative genomic approaches.
Funded projects
KINASE- Phosphoproteomics in Burkholderia
to assess the role of tyrosine phosphorylation in
virulence determinants expression, PTDC/QUI-
BIQ/118260/2010, PI: Leonilde M. Moreira.
Selected Publications
Silva IN, Tavares AF, Ferreira AS, and Moreira LM.
Stress conditions triggering mucoid morphotype varia-
tion in Burkholderia species and effect on virulence in
Galleria mellonella and biofilm formation in vitro. PLoS
One. 8(12): e825222. doi: 10.1371/
journal.pone.0082522, 2013.
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Objectives
Extracellular polysaccharides (EPS) of bacterial
origin have diverse functions in nature, such as in
bacterium-plant interactions, virulence factors or
protective agents. Due to their unique properties as
rheology modification agents, stabilizers, emulsifiers
and gelling agents, they also represent an important
class of polymeric materials with interest in Biotech-
nology. Research carried out by our group is fo-
cused on the proteins directing EPS biosynthesis,
on EPS-mediated interaction with hosts either in
symbiosis or pathogenesis and on EPS potential
industrial applications. The biological systems under
study are the exopolysaccharide cepacian from
Burkholderia and succinoglycan from Sinorhizobium
meliloti.
Research Topics
1. Proteins required for exopolysaccharide biosyn-
thesis in Burkholderia cepacia complex
The Burkholderia cenocepacia J2315 bceN gene
was shown to encode a protein with GDP-D-
mannose 4,6-dehydratase enzyme activity
(E.C.4.2.1.47). The protein is only active when in
the tetrameric form and catalyzes the conversion of
GDP-D-mannose into GDP-4-keto-6-deoxy-D-
mannose. This sugar nucleotide is the intermediary
necessary for the biosynthesis of GDP-D-rhamnose,
one of the sugar residues of cepacian. The enzyme
kinetics characterization by NMR spectroscopy re-
vealed that the enzyme requires Mg2+ for its activity,
is strongly inhibited by the substrate, and follows a
non-Michaelis-Menten kinetics model. The lack of a
functional bceN gene in a mutant derived from B.
cepacia IST408 slightly reduced cepacian produc-
tion. However, in the B. multivorans ATCC 17616
strain that harbors bceN as the single gene in its
genome with predicted GDP-mannose dehydratase
activity, a bceN mutant did not produce cepacian,
indicating that the bceN gene product is required for
cepacian biosynthesis.
2. Analysis of the Burkholderia cepacia tyrosine
kinase bceF mutant reveals a role in tolerance to
stress, biofilm formation and interaction with host
cells
The bacterial tyrosine (BY) kinase family comprises
the major group of bacterial enzymes endowed with
tyrosine kinase activity. We have previously shown
that BceF protein from Burkholderia cepacia IST408
belongs to this BY-kinase family and is involved in
the biosynthesis of the exopolysaccharide cepacian.
However, little was known about the extent of regu-
lation by this protein kinase activity. In order to ex-
amine this we performed a comparative transcrip-
tome profile between the bceF mutant and the wild-
type B. cepacia IST408. Genes with decreased ex-
pression in the bceF mutant were related to stress
response, motility, cell adhesion, and carbon and
Bacterial exopolysaccharides: biosynthesis
and biological role
Leonilde M. Moreira, Jorge H. Leitão, Ana S. Ferreira, Sílvia A. Sousa, Inês N. Silva
PhD and MSc students: Mário R. Santos, Joana R. Feliciano, Vítor H. Oliveira, Sílvia Matos, Micaela Torrado; Research
Assistant: Andreia Silva
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energy metabolism. Genes with increased expres-
sion were related to intracellular signaling, type VI
secretion and lipid metabolism. Mutation of bceF
gene led to reduced survival under heat-shock
stress and UV-light exposure, reduced swimming
motility and alteration of biofilm architecture when
grown in vitro. The bceF mutant showed decreased
ability to adhere and invade human lung epithelial
cells and to disrupt tight junctions, which enables
bacteria to reach paracellularly the basolateral sur-
face. As homologues of BceF occur in a number of
pathogenic and plant-associated Burkholderia
strains, the modulation of bacterial behavior through
tyrosine kinase activity is most likely a widely occur-
ring phenomenon.
3. Sinorhizobium meliloti proteins with a role in ex-
opolysaccharide biosynthesis and in nitrogen fixation
symbiosis
Members of the TetR family of repressors are known
to control genes whose products are involved in mul-
tidrug resistance, catabolic pathways, biosynthesis
of antibiotics, osmotic stress and pathogenicity. We
have characterized a TetR member of S. meliloti
encoded by gene SMc03169. A deletion mutant ex-
hibited lower tolerance to acidic conditions and in-
creased susceptibility to detergents and oxidative
stress agents. This mutant also showed defects in
symbiosis with Medicago sativa, since the number of
pink nodules decreased by one-half and it was una-
ble to directly compete with the wild-type strain for
nodulation. Expression profiling showed downregula-
tion of genes involved in Nod factor and siderophore
biosynthesis, phosphate uptake systems, and genes
belonging to the heat-shock regulon. Among the
upregulated genes in the mutant we found genes
directing succinoglycan biosynthesis, lipopolysac-
charide modification and genes whose products are
involved in metabolism.
Main Achievements
Functional and kinetics characterization of the
GDP-mannose 4,6-dehydratase from Burkholderia
cepacia complex bacteria [1,2].
Demonstration that the BceF tyrosine kinase from
Burkholderia cepacia is required for exopolysaccha-
ride biosynthesis, stress response, biofilm formation
and virulence [3].
Demonstration that Burkholderia cepacia BceF
protein is required for enhancement of CFBE41o-
cell invasion and paracellular translocation and mod-
ulation of the proinflammatory response.
Demonstration that the Sinorhizobium meliloti
TetR repressor encoded by SMc03169 is involved in
regulatory networks targeting cell envelope and me-
tabolism and is required for the biological nitrogen
fixation symbiosis with Medicago plants [4].
Funded projects
ROOT-INT– Role of a two-component regulatory
system in the early interaction between Sinorhizobi-
um meliloti and plant root hairs, PTDC/BIA-
MIC/113733/2009, PI: Leonilde M. Moreira.
Exploiting the type II phosphomannose isomerise
BceAJ as a new target for the development of new
antimicrobials and for biotechnological applications,
PTDC/EBB-BIO/098352/2008, PI: Jorge H. Leitão.
How do Bacterial Phosphotyrosine Phosphatase
Proteins Modulate Host-Pathogen Interactions? Eu-
ropean Society of Clinical Microbiology and Infec-
tious Diseases (ESCMID) PI: Ana S. Ferreira.
Selected Publications
[1] Sousa SA, Feliciano JR, Pinheiro PF, Leitão JH.
Biochemical and functional studies on the Burkholderia
cepacia complex bceN gene, encoding a GDP-D-mannose
4, 6-dehydratase. PLoS One 8 (2), e56902, 2013.
[2] Sousa SA, Feliciano JR, Grilo AM, Leitão JH.
Bioinformatics: a molecular microbiologist’s perspective.
Current Bioinformatics 9:8-17, 2014.
[3] Ferreira AS, Silva IN, Oliveira VH, Becker JD,
Givskov M, Ryan RP, Fernandes F, Moreira LM. Com-
parative transcriptomic analysis of the Burkholderia cepacia
tyrosine kinase bceF mutant reveals a role in tolerance to
stress, biofilm formation and virulence. Applied and Envi-
ronmental Microbiology, 79, 3009-3020, 2013.
[4] Santos MR, Tomás AT, Becker JD, Moreira LM. Sino-
rhizobium meliloti EmrR regulator is required for efficient
colonization of Medicago sativa root nodules. Molecular
Plant-Microbe Interactions, 27, 388-399, 2014.
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Objectives
Our research focused on studying surface exposed
proteins, namely trimeric autotransporter adhesins
(TAAs), in Burkholderia pathogenicity associated
with infection of the human respiratory tract. The
goal of the project is the molecular and functional
characterization of these TAAs, aiming to gain a
better understanding of how pathogenic Burkholderia
initiate infections. The research specifically focused
on three TAA encoding genes that are clustered
together and belong to a unique genomic region of
the cystic fibrosis epidemic strain Burkholderia
cenocepacia J2315.
Research topics
1. The trimeric autotransporter BCAM0224: a Swiss
army knife of Burkholderia cenocepacia
We show that BCAM0224 occurs on the bacterial
surface and adopts a trimeric conformation. Further,
we demonstrated that BCAM0224 is needed for the
earlier stages of biofilm formation and is required for
swarming motility. In addition, BCAM0224 plays an
important role in evasion of the human innate
immune system, providing resistance against the
bactericidal activity of serum via the complement
classical pathway. Finally, BCAM0224 mediates
bacterial adhesion to and invasion of cultured human
bronchial epithelial cells. Together, our data reveal
the high versatility of the BCAM0224 protein as a
virulence factor in pathogenic bacterium B.
cenocepacia [3].
In collaboration with Prof. Yves Dufrêne,
Institute of Condensed Matter and Nanosciences,
Université Catholique de Louvain, Belgium, Atomic
force microscopy (AFM) has been used to examine
the interactions between B. cenocepacia BCAM0224
-BCAM0224 and between B. cenocepacia
BCAM0224 and living airway epithelial cells [4].
We have shown that the adhesin forms
homophilic trans-interactions engaged in bacterial
aggregation, and that it behaves as a spring capable
to withstand high forces. We also founded that
BCAM0224 binds collagen, a major extracellular
component of host epithelia. Both homophilic and
heterophilic interactions displayed low binding
affinity, which could be important for epithelium
colonization. We also demonstrated that BCAM0224
recognizes receptors on living pneumocytes, and
leads to the formation of membrane tethers that may
play a role in promoting adhesion. Collectively, our
results showed that BCAM0224 is a multifunctional
adhesin endowed with remarkable binding
Unraveling the involvement of trimeric autotransporter
adhesins in Burkholderia cenocepacia host-cells interactions
Arsénio M Fialho, Dalila Mil-Homens
BSR
G
Gene organization of the TAA gene cluster in B. cenocepacia J2315 [1,2 and Mil-Homens et al., 2010, Microbiology, 156, 1084-96; Mil-Homens et al., 2011, Front Cell Infect Microbiol, 1:13].
(A) - BCAM0224 structure prediction. Computer modelling
of the putative 3D structure for the membrane-anchor
region of BCAM0224. (B) – Membrane localization (M-
monomers; T – trimmers) of BCAM0224 in the wild type B.
cenocepacia K56-2. (C) - Fluorescence confocal
microscopy images of B. cenocepacia K56-2 and
BCAM0224::Tp mutant using the anti-BCAM0224 antibody.
38
properties, which may represent a general
mechanism among TAAs for strengthening bacterial
adhesion [4].
2. Identifying host cellular receptors targeted by B.
cenocepacia TAA adhesins
The research specifically focuses on identifying host
cell receptors for B. cenocepacia TAAs. To gain
insights into the nature of such receptors, we are
developing a combination of genetic, cellular,
molecular and imaging techniques. As a first
approach and since lipid rafts are primary interaction
sites for pathogens, we are interested in analyzing
the involvement of such microdomains in
Burkholderia-host cell interaction events. In this way,
we have started examining if a pretreatment with
Methyl-b-cyclodextrin (MβCD) (a lipid raft disrupter)
can impair the adhesion of B. cenocepacia to host
cells. As a second approach, we are focusing our
experiments in various putative TAA host cell
receptors, such as glycolipids (aGM1, aGM2,
globoside, Gb2 and Gb3), mucins (mucus
glycoproteins) and proteins (TNF receptor 1, TLR2,
cytokeratin 13). Then, we have examined whether
pretreatment of host cells with enzymes and
antibodies that degrade/block such receptors,
prevents/impairs B. cenocepacia (wild type and TAA
mutants) adhesion. Our ultimate goal in this research
is to gain an improved understanding of the molecular
basis of host-specificity and virulence of B.
cenocepacia and identify potential protein targets for
more effective disease management.
Funded project
Fundação para a Ciência e a Tecnologia (FCT),
Portugal, Grant PTDC/BIA-MIC/118386/2010.
Selected publications
[1] Mil-Homens D, Bernardes N, Fialho AM. The
antibacterial properties of docosahexaenoic omega-3 fatty
acid against the Cystic Fibrosis multi-resistant pathogen
Burkholderia cenocepacia, FEMS Microbiology Letters
328:61-69, 2012
[2] Mil-Homens D, Fialho AM. A BCAM0223 Mutant of
Burkholderia cenocepacia is Deficient in Hemagglutination,
Serum Resistance, Adhesion to Epithelial Cells and
Virulence, PLoS One, 7(7):e41747, 2012.
[3] Mil-Homens D, Leça MI, Fernandes F, Pinto SN,
Fialho AM. Characterization of BCAM0224, a
multifunctional trimeric autotransporter from the human
pathogen Burkholderia cenocepacia, Journal of
Bacteriology, 2014 (doi:10.1128/JB.00061-14)
[4] El-Kirat-Chatel S, Mil-Homens D, Beaussart A, Fialho
AM, Dufrêne YF. Single-molecule atomic force microscopy
unravels the binding mechanism of a Burkholderia
cenocepacia trimeric autotransporter adhesin, Molecular
Microbiology, 89(4):649-659, 2013.
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BCAM0224 from B. cenocepacia K56-2, a multifaceted protein as
revealed by its different biological properties.
39
20 12-13 Biennial Report
12-13
Objectives
Small non-coding RNAs are increasingly recognized
as powerful tools used by bacterial pathogens to
rapidly adapt their physiology and fitness to the host
environment. Work carried out by our group envisag-
es the identification and functional characterization of
sRNAs and their mRNA targets, particularly of those
related to virulence and resistance to stresses mim-
icking those faced by the bacterium during the infec-
tion process. Our goal is to identify sRNAs and path-
ways which might be exploited as targets for thera-
peutic intervention against infections caused by bac-
teria of the Burkholderia cepacia complex.
Research Topics
1. Identification and validation of sRNAs from bacte-
ria of the Burkholderia cepacia complex.
A total of 24 sRNAs from B. cenocepacia J2315 were
identified experimentally, based on the co-purification
of sRNAs with the bacterium Hfq protein, followed by
conversion into cDNA, cloning, computational analy-
sis of sequences and validation by Northern blot
analysis. The sRNAs identified escaped previous
identification in studies based on transcriptomic or
bioinformatics analyses. Three sRNAs were found
exclusively within genome sequences of bacteria of
the Burkholderia cepacia complex and have no hom-
ologues in other bacteria, while the other 21 share
homology, at a different extent, to sRNAs of other
bacterial species.
2. Functional characterization of h2cR
The novel cis-encoded sRNA h2cR from the human
opportunistic pathogen Burkholderia cenocepacia
J2315 was identified and functionally characterized.
The sRNA was found to negatively regulate the hfq2
mRNA, through binding to part of the 5’-UTR region
of the hfq2 mRNA, resulting in accelerated hfq2
mRNA decay and reduced protein levels in exponen-
tially growing cells. Both the h2cR transcript and the
hfq2 mRNA are stabilized by the other B. cenocepa-
cia RNA chaperone, Hfq. Infection experiments using
the nematode Caenorhabditis elegans revealed that
down-regulation of Hfq2 by h2cR decreases the bac-
terium ability to colonize and persist within the nema-
Small non-coding RNAs in Burkholderia cepacia complex
Jorge H. Leitão, Christian G. Ramos, Sílvia A. Sousa
PhD students: André M. Grilo, Joana R. Feliciano
BSR
G
40
tode, suggesting a role for h2cR on bacterial per-
sistence in the host.
3. Functional characterization of MtvR
The 136 nt-long small non-coding RNA MtvR from
the Bcc member Burkholderia cenocepacia J2315
was identified and characterized. MtvR homo-
logues are restricted to the Burkholderia genus.
The mRNA levels corresponding to 17 out of a total
of 19 selected genes were affected when MtvR was
either overexpressed or silenced. MtvR was found
to interact with its target hfq mRNA by binding ex-
clusively to the 5’-UTR of the hfq mRNA, requiring
nucleotides 8-16 and 125-129 for pairing. This in-
teraction resulted in decreased protein synthesis,
suggesting a negative regulatory effect of MtvR on
the RNA chaperone Hfq. Bacterial strains with
MtvR silenced or overexpressed exhibited plei-
otropic phenotypes related to growth and survival
to several stresses, swimming and swarming motili-
ties, biofilm formation, resistance to antibiotics
(especially to aminoglycosides), and ability to colo-
nize and kill the nematode Caenorhabditis elegans.
MtvR is proposed to be a major global regulatory
sRNA in Bcc bacteria.
Main Achievements
A methodology for the experimental identifica-
tion of sRNAs based on their co-precipitation with
Hfq-like proteins was established. This methodolo-
gy led to the identification of 24 sRNAs from the
epidemic strain B. cenocepacia J2315 [1].
The cis-encoded sRNA h2cR was identified.
h2cR was found to be a negative regulator of the
mRNA levels of hfq2, depending on the RNA chap-
erone Hfq for its stability [2].
The sRNA MtvR was functionally characterized.
Work performed showed that this sRNA affects the
transcripts levels of multiple genes pointing out
MtvR as a global regulatory sRNA in bacteria of the
Burkholderia cepacia complex [3].
Funded project
RNomics-Bcc: RNomics studies to identify
small non-coding RNAs of Burkholderia cepacia
complex involved in host-pathogen interactions,
PTDC/BIA-MIC/119091/2010, PI: Jorge H. Leitão.
Selected Publications
[1] Ramos CG, Grilo AM, da Costa PJP, Leitão JH.
Experimental identification of small non-coding regulatory
RNAs in the opportunistic human pathogen Burkholderia
cenocepacia J2315. Genomics 101(2):139-148, 2013.
[2] Ramos CG, da Costa PJP, Döring G, Leitão JH.
The novel cis-encoded small RNA h2cR is a negative
regulator of hfq2 in Burkholderia cenocepacia. PLOS One
7(10): e47896, 2012.
[3] Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,
Leitão JH. MtvR is a global regulatory sRNA in
Burkholderia cenocepacia. Journal of Bacteriology, 195
(16), 3514–3523, 2013.
[4] Ramos CG, Leitão JH. “Caenorhabditis elegans as a
research tool to unveil bacterial virulence determinants:
lessons from the Burkholderia cepacia complex”, In: Nem-
atodes: Morphology, Functions and Management
Strategies (Fausto Boeri and Jordan A. Chung, Eds.),
Nova Science Publishers, Inc., NY, USA, 135-156, 2012
(ISBN: 978-1-61470-784-4).
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lig
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h2cR
hfq2
70
70
h2cR
hfq2
70
70
X
XcepR
Hfq
Exponential Stationary
RNase RNase
Hfq
Hfq
? ?
h2cR
hfq2
70
70
h2cR
hfq2
70
70
X
XcepR
Hfq
Exponential Stationary
RNaseRNaseRNase RNaseRNaseRNase
Hfq
Hfq
? ?
41
20 12-13 Biennial Report
12-13
Our research, in collaboration with IPATIMUP,
Portugal, focused on studying the bacterial protein
azurin as an anti-cancer agent. Azurin is a low
molecular weight (14kDa), water-solube protein
produced by the bacterium Pseudomonas
aeruginosa. It has cytotoxicity towards cancer cell
lines in vitro and in vivo. Azurin can enter
preferentially into cancer cells, acting as a
multivalent agente [1-3]. On entry into cancer cells,
azurin forms a complex with p53, stabilizing it and
inducing apoptosis. Moreover, azurin inhibits
angiogenesis through inhibition of the
phosphorylation of VEGFR-2, FAK and AKT. p28, a
peptide of azurin, has recently completed a Phase I
clinical trial with promising results [4,5].
Research topics
We aimed to study the molecular and cellular events
occurring when breast cancer cells overexpressing
P-cadherin, were treated with azurin. P-cadherin
overexpression occurs in about 30% of all breast
carcinomas representing one of the most
aggressive sub-type of breast cancer.
1. Azurin impairs invasion and causes a specific
decrease of P-cadherin levels in human breast
cancer cell lines
A sub-lethal single dose of azurin (with cell viability
of at least 80%) produced a decrease in the
invasion of two P-cadherin expressing breast cancer
cell models, the luminal MCF-7/AZ.Pcad and the
triple negative basal-like SUM 149 PT through a
Matrigel artificial matrix. In both cell lines, the
decrease in invasion was associated with a
decrease in the total P-cadherin protein levels and a
concomitant decrease of its membrane staining,
whereas E-cadherin remains not altered with high
expression levels and with normal membrane
localization [6].
2. Azurin decreases soluble forms of P- and E-
cadherin
Metalloproteinases (MMPs) are involved in the
degradation of the extracellular matrix components.
The active form of MMP2 has been detected in half
of all human breast carcinomas. In azurin treated
cells the activity of MMP2 in the extracellular media
of cells was decreased. The proteolytic activity of
MMP2 acts, in part, by shedding P-cadherin
extracellular domain itself, releasing a soluble form
of P-cadherin, sPcad12, which was also reduced in
the extracellular media of azurin-treated cells [6].
3. Signaling pathways and matrix remodeling
triggered by azurin
We investigated signaling pathways triggered by
azurin in cancer cell treatments. FAK and Src are
non-receptor tyrosine kinases that mediate a
significant number of signaling pathways with
relevance to cell-cell and cell-matrix adhesions. We
observed that azurin leads to a decrease in the
phosphorylation levels of both these proteins [6].
Bacterial Proteins in Cancer Therapy: azurin, a therapeutic
protein that interferes with oncogenic signaling and blocks
tumor progression
BSR
G
Arsénio M. Fialho, Nuno Bernardes
MSc student: Sofia Abreu
Azurin multivalent action against two P-cadherin expressing breast cancer cell models.
Azurin, a bacterial protein with anti-cancer properties
42
4. Array-based gene expression profile of azurin
treated breast cancer cells
Microarrays analysis reveals genetic pathways
modulated by azurin that are important contributors
to cell proliferation in breast cancer, in particular
pathways that regulate cell-cell adhesion and cell-
matrix interactions. We have performed a microar-
ray analysis to infer about the signaling pathways
that are altered in a p53 wild-type and P-cadherin
overexpressing breast cancer cell model upon az-
urin treatment. Our results strengthen the hypothe-
sis that azurin has a multivalent action towards
cancer cells, promoting the endocytosis of cell sur-
face receptors and the interruption of signaling
pathways hyper activated in cancer cells. The
blockage of these signaling pathways in P-cadherin
overexpressing breast cancer cell models leads to
the abrogation of tumor cell invasion, providing a
possible new therapeutic approach to this specific
type of breast cancer [7].
Altogether, our data show that azurin exhibits a
specific preference to decrease P-cadherin expres-
sion levels, abrogating its invasive effects and,
therefore, may have a potential use in the treat-
ment of breast carcinomas overexpressing this
protein.
Financed project
Fundação para a Ciência e a Tecnologia (FCT),
Portugal, Grant PTDC/EBB-BIO/100326/2008.
Selected publications
[1] Fialho AM, Salunkhe P, Manna SK, Mahali S,
Chakrabarty AM. Glioblastoma Multiforme: Novel Thera-
peutic Approaches, ISRN Neurology, Article ID 642345,
2012.
[2] Fialho AM, Bernardes N, Chakrabarty AM. Recent
patents on live bacteria and their products as potential
anticancer agents, Recent Patents on Anti-Cancer Drug
Discovery 7: 31-55, 2012.
[3] Bernardes N, Chakrabarty AM, Fialho AM. Engi-
neering of bacterial strains and their products for cancer
therapy, Applied Microbiology and Biotechnology, 97
(12):5189-5199, 2013.
[4] Fialho AM, Chakrabarty AM. Patent controversies
and court cases: Cancer diagnosis, therapy and preven-
tion, Cancer Biology and Therapy, 13: 1229 – 1234,
2012.
[5] Avner BS, Fialho AM, Chakrabarty AM. Overcoming
drug resistance in multi-drug resistant cancers and micro-
organisms: A conceptual framework, Bioengineered,
3:262-270, 2012.
[6] Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos
RG, Arraiano CM, Seruca R, Paredes J, Fialho AM.
The bacterial protein azurin impairs invasion and FAK/Src
signaling in P-cadherin-overexpressing breast cancer cell
models, PLoS One, 8 (7), e69023, 2013.
[7] Bernardes N, Ribeiro AS, Abreu S, Vieira AF, Car-
reto L, Santos M, Seruca R, Paredes J, Fialho AM.
High-throughput molecular profiling of a P-cadherin over-
expressing breast cancer model reveals new targets for
the anti-cancer bacterial protein azurin, The International
Journal of Biochemistry & Cell Biology, 50: 1-9, 2014.
Issued patent
Fialho AM, Bernardes N, Gonçalves J, Santos AC.
Compositions to treat HIV infections and methods there-
for, 1126/MUM/2011, March 2012.
R
ese
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lig
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Array-based gene expression profile of azurin treated
breast cancer cells.
43
20 12-13 Biennial Report
12-13
Articles in International Peer-Reviewed
Journals
[1] Avner BS, Fialho AM, Chakrabarty AM. Overcoming
drug resistance in multi-drug resistant cancers and microor-
ganisms: A conceptual framework, Bioengineered, 3, 262-
270, 2012.
[2] Barbas A, Popescu A, Frazão C, Arraiano CM, Fialho
AM. Rossmann-fold motifs can confer multiple functions to
metabolic enzymes: RNA binding and ribonuclease activity
of a UDP-glucose dehydrogenase, Biochemical & Bio-
physical Research Communications, 430(1): 218-24,
2013.
[3] Bernardes N, Ribeiro AS, Abreu S, Vieira AF, Carreto
L, Santos M, Seruca R, Paredes J, Fialho AM. High-
throughput molecular profiling of a P-cadherin overexpress-
ing breast cancer model reveals new targets for the anti-
cancer bacterial protein azurin, The International Journal
of Biochemistry and Cell Biology, 2014 (doi: 10.1016/
j.biocel.2014.01.023).
[4] Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos
RG, Arraiano CM, Seruca R, Paredes J, Fialho AM. The
bacterial protein azurin impairs invasion and FAK/Src sig-
naling in P-cadherin-overexpressing breast cancer cell
models, PLoS One, 8(7): e69023, 2013.
[5] Bernardes N, Chakrabarty AM, Fialho AM. Engineer-
ing of bacterial strains and their products for cancer thera-
py, Applied Microbiology and Biotechnology, 97(12):
5189-99, 2013.
[6] Chelinho S, Moreira-Santos M, Silva C, Costa C,
Viana P, Viegas AC, Fialho AM, Ribeiro R, Sousa JP.
Semi-field testing of a bioremediation tool for atrazine-
contaminated soils: evaluating the efficacy on soil and
aquatic compartments, Environmental Toxicology and
Chemistry, 31, 1564-1572, 2012.
[7] Costa C, Nunes J, Henriques A, Mira NP, Nakayama
H, Chibana H, Teixeira MC. The Candida glabrata drug:H+
antiporter CgTpo3 (ORF CAGL0I10384g): role in azole drug
resistance and polyamine homeostasis, Journal of Antimi-
crobial Chemotherapy, 2014 (doi: 10.1093/jac/dku044).
[8] Costa C, Pires C, Cabrito TR, Renaudin A, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. Candida glabra-
ta drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) con-
fers imidazole drug resistance, being activated by the CgP-
dr1 transcription factor, Antimicrobial Agents and Chem-
otherapy, 57(7), 3159-67, 2013.
[9] Costa C, Henriques AS, Pires C, Nunes J, Ohno M,
Chibana H, Sá-Correia I, Teixeira MC. The dual role
of Candida glabrata Drug:H+ Antiporter CgAqr1
(ORFCAGL0J09944g) in antifungal drug and acetic acid
resistance, Frontiers in Microbiology, 4, 170, 2013.
[10] de Carvalho J, Rodrigues RMM, Tomé B, Henriques
SF, Mira NP, Sá-Correia I, Ferreira GNM. Conformational
and Mechanical changes of DNA upon transcription factor
binding detected by a QCM and transmission line mod-
el, Analyst, 139(8): 1847-55, 2014.
[11] De Soyza A, Hall A, Mahenthiralingam E, Drevinek
P, Kaca W, Drulis-Kawa Z, Stoitsova S, Toth V, Coenye
T, Zlosnik J, Burns J, Sá-Correia I, De Vos D, Pirnay JP,
Kidd T, Reid D, Manos J, Klockgether J, Wiehlmann L,
Tummler B, McClean S, Winstanley C. Developing an
international Pseudomonas aeruginosa reference panel,
Microbiology Open, 2, 1010-23, 2013.
[12] Dias PJ, Sá-Correia I. The drug:H+ antiporters of fami-
ly 2 (DHA2), siderophore transporters (ARN) and glutathi-
one:H+ antiporters (GEX) have a common evolutionary
origin in hemiascomycete yeasts, BMC Genomics, 14, 901,
2013.
[13] Di Lorenzo F, Silipo A, Lanzetta R, Fazio LL, Paciel-
lo I, Coutinho CP, Sá-Correia I, Bernardini M, Molinaro
A. Structural elucidation and biological activity of the lipooli-
gosaccharide of a Burkholderia dolosa isolate from a cystic
fibrosis patient, ChemBioChem, 14(9), 1105-15, 2013.
[14] dos Santos SC, Teixeira MC, Dias PJ, Sá-Correia I.
MFS transporters required for multidrug/multixenobiotic
(MD/MX) resistance in the model yeast: understanding their
physiological function through post-genomic approaches,
Frontiers in Physiology, 5:180, 2014.
[15] dos Santos SC, Mira NP, Moreira AS, Sá-Correia I.
Quantitative- and phospho-proteomic analysis of the yeast
response to the tyrosine kinase inhibitor imatinib, OMICS: A
Journal of Integrative Biology, 16, 537-51, 2012.
[16] dos Santos SC, Teixeira MC, Cabrito TR, Sá-Correia
I. Yeast Toxicogenomics: genome-wide responses to chem-
ical stresses with impact in Environmental Health, Pharma-
cology and Biotechnology, Frontiers in Genetics, 3, 63,
2012.
[17] El-Kirat-Chatel S, Mil-Homens D, Beaussart A, Fi-
alho AM, Dufrêne YF. Single-molecule atomic force mi-
croscopy unravels the binding mechanism of
a Burkholderia cenocepacia trimeric autotransporter adhe-
sin, Molecular Microbiology, 89(4): 649-59, 2013.
[18] Eusébio N, Coutinho C, Sá-Correia I, Araújo R.
SNaPBcen: a novel and practical tool for genotyp-
ing Burkholderia cenocepacia, Journal of Clinical Microbi-
ology, 51(8), 2646-53, 2013.
[19] Ferreira AS, Silva IN, Oliveira VH, Becker JD,
Givskov M, Ryan RP, Fernandes F, Moreira LM. Compar-
ative transcriptomic analysis of the Burkholderia cepacia
tyrosine kinase bceF mutant reveals a role in tolerance to
stress, biofilm formation and virulence. Applied and Envi-
ronmental Microbiology, 79, 3009-3020, 2013.
Publications
44
[20] Fialho AM, Bernardes N, Chakrabarty AM. Recent
patents on live bacteria and their products as potential anti-
cancer agents, Recent Patents on Anti-Cancer Drug Dis-
covery, 7, 31-55, 2012.
[21] Fialho AM, Chakrabarty AM. Patent controversies and
court cases: Cancer diagnosis, therapy and prevention, Can-
cer Biology and Therapy, 13, 1229 - 1234, 2012.
[22] Fialho AM, Salunkhe P, Manna SK, Mahali S,
Chakrabarty AM. Glioblastoma Multiforme: Novel Therapeu-
tic Approaches, ISRN Neurology, Article ID 642345, 10
pages, 2012.
[23] Guerreiro JF, Mira NP, Sá-Correia I. Adaptive re-
sponse to acetic acid in the highly resistant yeast species
Zygosaccharomyces bailii revealed by quantitative prote-
omics, Proteomics, 12, 2303-18, 2012.
[24] Lourenço AB, Roque FC, Teixeira MC, Ascenso JR,
Sá-Correia I. Quantitative 1H-NMR-metabolomics reveals
extensive metabolic reprogramming and the effect of the
aquaglyceroporin FPS1 in ethanol-stressed yeast
cells, PLoS One, 8(2), e55439, 2013.
[25] Madeira A, dos Santos SC, Santos PM, Coutinho CP,
Tyrrell J, McClean S, Callaghan M, Sá-Correia
I. Proteomic profiling of Burkholderia cenocepacia clonal
isolates with different virulence potential retrieved from a
cystic fibrosis patient during chronic lung infection, PLoS
One 8(12), e83065, 2013.
[26] Madeira A, da Silva CL, Dos Santos F, Camafeita E,
Cabral JMS, Sá-Correia I. Human Mesenchymal Stem Cell
Expression Program upon Extended Ex-Vivo Cultivation, as
Revealed by 2-DE-Based Quantitative Proteomics, PLoS
One, 7(8), e43523, 2012 (highlighted in Mesenchymal Cell
News 4.34).
[27] Matos RG, Fialho AM, Giloh M, Schuster G, Arraiano
CM. The rnb Gene of Synechocystis PCC6803 Encodes a
RNA Hydrolase Displaying RNase II and Not RNase R Enzy-
matic Properties, PLoS One, 7(3), e32690, 2012.
[28] Mil-Homens D, Leça MI, Fernandes F, Pinto SN, Fi-
alho AM. Characterization of BCAM0224, a multifunctional
trimeric autotransporter from the human pathogen Burkhold-
eria cenocepacia, Journal of Bacteriology, 2014
(doi:10.1128/JB.00061-14).
[29] Mil-Homens D, Bernardes N, Fialho AM. The antibac-
terial properties of docosahexaenoic omega-3 fatty acid
against the Cystic Fibrosis multi-resistant pathogen
Burkholderia cenocepacia, FEMS Microbiology Letters,
328, 61-69, 2012.
[30] Mil-Homens D, Fialho AM. A BCAM0223 Mutant of
Burkholderia cenocepacia is Deficient in Hemagglutination,
Serum Resistance, Adhesion to Epithelial Cells and Viru-
lence, PLoS One, 7(7), e41747, 2012.
[31] Mira NP, Münsterkötter M, Valada FD, Santos J, Pal-
ma M, Roque FC, Guerreiro JF, Rodrigues F, Sousa MJ,
Leão C, Güldener U and Sá-Correia I. The genome se-
quence of the highly acetic acid tolerant Zygosaccharomyces
bailii-derived interspecies hybrid strain ISA1307 isolated from
a sparkling wine plant, DNA Research, 1-15, 2014.
[32] Moreira AS, Coutinho CP, Azevedo P, Lito L, Melo-
Cristino J, Sá-Correia I. Burkholderia dolosa phenotypic
variation during the decline in the lung function of a cystic
fibrosis patient along 5.5 years of chronic colonization. Jour-
nal of Medical Microbiology, 63: 594-601, 2014 (Week’s
Best Articles: Cystic Fibrosis February 5-12 2014, according
to MDLinx Pulmonology).
[33] Palma M, Madeira SC, Mendes-Ferreira A, Sá-Correia
I. Impact of assimilable nitrogen availability in glucose uptake
kinetics in Saccharomyces cerevisiae during alcoholic fer-
mentation, Microbial Cell Factories, 11, 99, 2012.
[34] Pereira SG, Rosa AC, Ferreira AS, Moreira LM, Pro-
ença DN, Morais PV, Cardoso O. Virulence factors and
infection ability of Pseudomonas aeruginosa isolates from a
hydropathic facility and respiratory infections, Journal of
Applied Microbiology, 2014 (doi: 10.1111/jam.12463).
[35] Ramos CG, Grilo AM, da Costa PJ, Leitão JH. Experi-
mental identification of small non-coding regulatory RNAs in
the opportunistic human pathogen Burkholderia cenocepacia
J2315, Genomics, 101(2):139-148, 2013.
[36] Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,
Leitão JH. MtvR is a global regulatory sRNA in Burkholderia
cenocepacia, Journal of Bacteriology, 195 (16), 3514–
3523, 2013.
[37] Ramos CG, da Costa PJ, Döring G, Leitão JH. The
novel cis-encoded small RNA h2cR is a negative regulator of
hfq2 in Burkholderia cenocepacia, PLoS One, 7(10):
e47896, 2012.
[38] Remy E, Cabrito TR, Baster P, Batista RA, Teixeira
MC, Friml J, Sá-Correia I, Duque P. A Major Facilitator
Superfamily Transporter Plays a Dual Role in Polar Auxin
Transport and Drought Stress Tolerance in Arabidopsis, The
Plant Cell, 25, 901-26, 2013.
[39] Remy E, Cabrito TR, Baptista R, Teixeira MC, Sá-
Correia I, Duque P. The Pht1;9 transporter mediates inor-
ganic Pi acquisition by the Arabidopsis thaliana root during
phosphorus starvation, The New Phytologist, 195, 356–
371, 2012.
[40] Santos MR, Tomás AT, Becker JD, Moreira LM. Sino-
rhizobium meliloti EmrR regulator is required for efficient
colonization of Medicago sativa root nodules, Molecular
Plant-Microbe Interactions, 27, 388-399, 2014.
[41] Silva IN, Tavares AF, Ferreira AS, Moreira LM. Stress
conditions triggering mucoid morphotype variation in
Burkholderia species and effect on virulence in Galleria
mellonella and biofilm formation in vitro, PLoS One 8(12),
e825222, 2013 (doi: 10.1371/journal.pone.0082522).
[42] Sousa SA, Feliciano JR, Grilo AM, Leitão JH. Bioin-
formatics: a molecular microbiologist’s perspective, Current
Bioinformatics, 9 (1):8-17, 2014.
[43] Sousa SA, Feliciano JR, Pinheiro PF, Leitão JH.
Biochemical and functional studies on the Burkholderia cepa-
cia complex bceN gene, encoding a GDP-D-mannose 4,6-
dehydratase, PLoS One, 8: e56902, 2013.
45
20 12-13 Biennial Report
12-13
[44] Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves
JP, Mira NP, dos Santos SC, Cabrito T, Palma M, Costa
C, Francisco AP, Madeira SC, Oliveira AL, Freitas AT, Sá
-Correia I. The YEASTRACT database: an upgraded infor-
mation system for the analysis of gene and genomic tran-
scription regulation in Saccharomyces cerevisiae, Nucleic
Acids Research, 42, D161-D166, 2014.
[45] Teixeira MC, Godinho CP, Cabrito TR, Mira NP, Sá-
Correia I. Increased expression of the yeast multidrug re-
sistance ABC transporter Pdr18 leads to increased ethanol
tolerance and ethanol production in high gravity alcoholic
fermentation, Microbial Cell Factories, 11, 98, 2012.
[46] Viegas SC, Mil-Homens D, Fialho AM, Arraiano CM.
The virulence of Salmonella enterica serovar typhimurium in
the insect model Galleria mellonella is impaired by mutations
in RNase E and RNase III, Applied and Environmental
Microbiology, 79(19): 6124-33, 2013.
[47] Viegas CA, Costa C, André S, Viana P, Ribeiro R,
Moreira-Santos M. Does S-metolachlor affect the perfor-
mance of Pseudomonas sp. strain ADP as bioaugmentation
bacterium for atrazine-contaminated soils?, PLoS One, 7(5),
e37140, 2012.
Book Chapters
[48] Leitão JH, Sousa SA, Ramos CG, Feliciano JR, Grilo
AM, da Costa PJP. “Experimental and bioinformatics
approaches to identify potential targets and develop novel
strategies to fight infections caused by multiresistant
Burkholderia cepacia complex bacteria”. In: Microbial
pathogens and strategies for combating them: science,
technology and education, vol 1, Formatex Research
Center, Badajoz, Spain, 2013. Pages 372-383 (ISBN 978-84
-939843-9-7).
[49] Leitão JH, Simões N. "Identification of novel
antimicrobials using a live-animal infection model". In: M.C.
Barreto and N. Simões (ed), Determination of Biological
Activities: A Laboratory Manual, Universidade dos Açores,
Ponta Delgada, 2012. Pages 29-39 (ISBN 978-972-8612-82-
5).
[50] Mira NP, Teixeira MC, Sá-Correia I. “Characterization
of complex regulatory networks and identification of promoter
regulatory elements in yeast: in silico and wet-lab”, In:
Methods in Molecular Biology - Transcriptional
Regulation: Methods and Protocols (Vancura A, Ed),
Springer, vol. 809, 27-48, 2012. (ISBN 978-1-61779-375-2).
[51] Ramos CG, Leitão JH. “Caenorhabditis elegans as a
research tool to unveil bacterial virulence determinants:
lessons from the Burkholderia cepacia complex”, In:
Nematodes: Morphology, Functions and Management
Strategies (Fausto Boeri and Jordan A. Chung, Eds.), Nova
Science Publishers, Inc., NY, USA, 135-156, 2012 (ISBN:
978-1-61470-784-4).
[52] Ramos CG, Grilo AM, da Costa PJP, Nadais H,
Leitão JH. “Extraction and Purification of DNA from UASB
Reactors Sludge Samples”, In: DNA Binding and DNA
Extraction: Methods, Applications and Limitations
(Chunxu Zhou and Xia Ling, Eds.) Nova Science Publishers,
Inc., NY, USA, 123-139, 2012 (ISBN: 978-1-61470-958-9).
[53] Sá-Correia I, Guerreiro JF, Loureiro-Dias MC, Leão
C, Côrte-Real M. “Zygosaccharomyces”. In: Encyclopedia
of Food Microbiology, Batt, C.A. & Tortorello, M.L. (Eds.),
vol 3. Elsevier Ltd, Academic Press, pp. 849–855, 2014
(ISBN: 9780123847300)
[54] Sá-Correia I, dos Santos SC, Madeira A.
“Burkholderia Proteomics: methodologies, challenges, and
applications” in Burkholderia: From Genomes to Function
Tom Coenye & Eshwar Mahenthiralingam (Eds.), Horizon
Press, 2014 (ISBN: 978-1-908230-35-5)
[55] Teixeira MC. "Complex regulatory interplay between
multidrug resistance and oxidative stress response in yeast:
the FLR1 regulatory network as a systems biology case-
study", In: Oxidative Stress - Molecular Mechanisms and
Biological Effects, Lushchak V.I. & Semchyshyn H. (Eds),
INTECH, Vienna, Austria, 323-340, 2012 (ISBN: 978-953-51-
0554-1).
[56] Viegas CA, Chelinho S, Moreira-Santos M, Costa C,
Gil FN, Silva C, Lima D, Ribeiro R, Sousa JP, Fialho AM.
“Bioremediation of soils contaminated with atrazine and other
s-triazine herbicides: current state and prospects”, In:
Advances in Environmental Research, Justin A. Daniels
(ed), vol 6, Nova Science Publishers, Inc., NY, USA, 2012,
pp. 1-49 (ISBN 978-1-61728-163-1).
Edition of Special Issues
Fontes CMGA, Sá-Correia I, Romão MJ, Sá-Nogueira I,
Prates JAM, Ferreira LMA. Special issue of Biocatalysis
and Biotransformation, vol. 30, issue 3, 2012, with the
proceedings of the 9th Carbohydrate Bioengineering Meeting
(CBM9)
Mira NP, Teixeira MC. Microbial Mechanisms of Tolerance
to Weak Acids, Special issue published in Frontiers in Mi-
crobiology – Section of Microbial Physiology and Metab-
olism (11 articles included), 2013.
Sá-Correia I, Teixeira MC. “Physiological role and regula-
tion of Multidrug/Multixenobiotic resistance membrane trans-
porters”, Frontiers in Physiology, 2014.
Issued Patents
Fialho AM, Bernardes N, Gonçalves J, Santos AC.
Compositions to treat HIV infections and methods therefor,
Indian patent 1126/MUM/2011. Priority date: March 2012.
Sá-Correia I, Cabrito TR, Teixeira MC, Remy E, Duque P.
Utilização de um gene que confere resistência a xenobióticos
em plantas (Use of a gene that confers resistance to
xenobiotics in plants), Provisional national patent number
PT105727. Priority date: 28th October 2011 (Patente de
invenção nacional n.º 105727; Date: 2013.06.21)
46
Patent applications
Martins AMSD, Leitão JH, Alves LGAR, Pinheiro PBF,
Feliciano JRR. “Síntese e uso de ciclamas com atividade
antibacteriana”. Pedido de Patente Nacional nº 107018.
Priority date: 20th June 2013.
Dissertations
Ph.D. Theses
Andreia Madeira, “Genome wide expression approaches
applied to biomedical research: long-term adaptation
Burkholderia cenocepacia to cystic fibroses patient's air-
ways and extended ex-vivo cultivation of human mesenchy-
mal stem cells”, PhD in Biotechnology, IST/UTL
(supervisors: Isabel Sá-Correia; co-supervisor: Joaquim
Sampaio Cabral), 2012.
Catarina Roma Rodrigues, "Global responses to phenol
and myrcene in Pseudomonas at the level of cell membrane
proteome unveiled by quantitative prote-
omics" (supervisor: Isabel Sá-Correia; co-supervisor: Pedro
Santos), 2012.
Dalila Mil-Homens, “Burkholderia cenocepacia pathogene-
sis: unravelling the virulence functions of trimeric autotrans-
porter adhesins and development of alternative therapies to
target this pathogen”, PhD in Biotechnology, IST/UTL
(supervisor: Arsénio M. Fialho), 2012.
Inês Nunes Silva, “Comparative genomics and tran-
scriptomics to study mucoid morphotype variation in
Burkholderia cepacia complex clinical isolates”, PhD in
Biotechnology, IST/UTL (supervisor: Leonilde M. Moreira;
co-supervisor: Jӧrg D. Becker ), 2012.
Nuno Bernardes “Bacterial protein azurin as a new thera-
peutic tool to treat poor prognosis breast cancer with over-
expression of P-cadherin” PhD in Biotechnology, IST/UTL
(supervisor: Arsénio M. Fialho; co-supervisor: Joana F.
Paredes), 2013.
M.Sc. Theses
Ana Cristina Taborda Gomes de Almeida, “Functional
analysis of the yeast multidrug resistance transporters Tpo1
and Pdr18 under stress induced by the agricultural fungicide
mancozeb”, MSc in Biotechnology (supervisor: Isabel Sá-
Correia; co-supervisor: Tânia R. Cabrito), 2012.
André Marques Sales Henriques, “Role of the multidrug
efflux pumps CgAqr1 and CgTpo2/3 in Candida
glabrata resistance to flucytosine and azole drugs”, MSc in
Biomedical Engineering (supervisor: Miguel C. Teixeira),
2012.
Andreia Filipa Campos Tavares, “Biological relevance of
mucoid vs. nonmucoid morphotype variation by
Burkholderia cepacia complex”, MSc in Cellular Biology and
Biotechnology, Faculdade de Ciências, Universidade de
Lisboa (Supervisor at IST: Leonilde M. Moreira; supervisor
at FC-UL: Carlos Farinha), 2012.
Andreia Ponte, "Genome-wide identification of
mechanisms of resistance to the antifungal drug flucytosine:
from S. cerevisiae to C. glabrata", MSc in Biotechnology,
IST (supervisor: Miguel C. Teixeira), 2013.
Camille Payre, “Les méchanismes moléculaires à l'origine
des différences de tolérance à l'acide acétique et
d'utilization du glucose et de l'acide acétique chez
Saccharomyces cerevisiae et Zygosaccharomyces baillii”,
Genie Biologique, Analyses Biologiques et Biochimiques,
IUT Dijon-Auxerre (supervisor: I. Sá-Correia; co-supervisor:
Margarida Palma), 2012.
Carla Alexandra Nunes Mateus, “Study of bioremediation
for soils contaminated with s-triazine herbicides: the
bioaugmentation bacteira Pseudomonas sp. ADP
and Arthrobacter aurescens TC1 compared”, MSc in
Biotechnology (supervisor: Cristina A. Viegas), 2012.
Cláudia Sofia Pires Godinho, “Studies on the role and
regulation of the multidrug resistance efflux pumps Tpo1
and Pdr18 in yeast tolerance to ethanol and weak acid
stresses, to increase yeast robustness”, MSc in Applied
Microbiology, Faculdade de Ciências, Universidade de
Lisboa (supervisor at IST: Isabel Sá-Correia; supervisor
at FC-UL: Ana Tenreiro), 2012.
Cláudia Rita Ferreira Maldonado, "Adaptive strategies
of Burkholderia cenocepacia to long term residence in
the lungs of cystic fibrosis patients: genome-wide
analyses", Applied Microbiology, FC-UL (supervisor at
IST: Isabel Sá-Correia; supervisor at FC-UL: Ana
Tenreiro).
Eunice Penas “Avaliação da interdependência entre
histidinas cinases híbridas (BCAM0218, BCAM0227) e
adesinas triméricas autotransportadas presentes no
agrupamento genético TAA do microrganismo
patogéneo Burkholderia cenocepacia J2315”, Mestrado
em Doenças Infecciosas Emergentes da Faculdade de
Medicina da Universidade de Lisboa (supervisor at IST:
Arsénio M. Fialho; co-supervisor at FM-UL: José
Augusto Cristino), 2013.
Filipa Gomes de Almeida Dias, “Functional analysis of
the Burkholderia cepacia complex bceOSU genes
encoding putative polysaccharide O-acyltransferases”,
MSc in Biotechnology (supervisor: Leonilde M. Moreira;
co-supervisor: Ana Sofia Ferreira), 2012.
Filipe Bravo da Silva, “Improvement of alcoholic
fermentation performance based on highly acetic acid
tolerant wine isolates of Saccharomyces cerevisiae and
genetically engineered strains”, MSc in Biological
Engineering (supervisor: Isabel Sá-Correia; co-
supervisor: Margarida Palma), 2012.
Gonçalo Emanuel Fialho Mourata da Silva
“Discovering and exploiting bacterial proteins as
anticancer agentes”, Mestrado em Biologia Humana e
Ambiente, Faculdade de Ciências da Universidade de
Lisboa (Supervisor at IST: Arsénio M. Fialho, Supervisor
at FC-UL: Ana M. Viegas), 2013.
47
20 12-13 Biennial Report
12-13
Grégoire Chardon, “La résistance à l’acide acétique
chez Zygosaccharomyces bailii”, Diplôme Universitaire
de Technologie - Genie Biologique, Analyses Biologiques
et Biochimiques, IUT Dijon-Auxerre (supervisor: Isabel Sá
-Correia; co-supervisor: Joana F. Guerreiro), 2013.
Guida Filipa Bartolomeu de Campos Camacho,
“Contribution to the functional analysis of the paralogous
gene-pairs CgTpo1_1 and CgTpo1_2 and CgFlr1_1 and
CgFlr1_2 from the human pathogen Candida glabrata”,
MSc in Biological Engineering (supervisor: Miguel C.
Teixeira), 2012.
Janete Simão Gonçalves, "Estratégias de
biorremediação para solos contaminados com o
herbicida terbutilazina com base na bioadição de
Pseudomonas sp. ADP", MSc in Applied Microbiology,
Faculty of Sciences of the University of Lisbon
(supervisor at IST: Cristina A. Viegas; supervisor at FC-
UL: Manuela Carolino; ), 2013.
João Miguel Rodrigues da Silva Brazão, “Functional
analysis of Arabidopsis thaliana major facilitator
superfamily (MFS) transporters through heterologous
expression in yeast”, MSc in Biotechnology, IST
(supervisor: Isabel Sá-Correia), 2013.
Jonathan Ribeiro, "Profiling antifungal drug resistance in
a collection of Candida glabrata clinical isolates:
correlation with the expression of multidrug efflux pumps",
MSc in Biomedical Engineering, IST (supervisor: Miguel
C. Teixeira), 2013.
Kaur Alasoo, “Elucidating the transcriptional regulatory
network controlling the TPO1 response to benzoic acid in
yeast”, euSYSBIO Erasmus Mundus Master´s in Systems
Biology, IST (supervisor: Isabel Sá-Correia; co-
supervisor: Tânia R. Cabrito) , 2012.
Maria Raquel Moita, “Towards the production of levulinic
and itaconic acids in Saccharomyces cerevisae: a
contribution for the understanding of the molecular
mechanisms of toxicity of the acids in producing cells”,
MSc in Biotechnology (supervisor: Nuno P. Mira), 2013.
Rúben Bernardo, “Functional analysis of the Candida
glabrata CgHaa1 (ORF CAGL0L09339g) transcription
factor: role in acetic acid resistance and in biofilm
formation”, MsC thesis in Biotechnology. (supervisor:
Nuno P. Mira) , 2013.
Sofia Patrão Neves Alves, “Contribution to the
functional analysis of genes involved in cyclic GMP
signaling in bacteria of the Burkholderia cepacia
complex”, MSc in Human Biology and Environment,
Faculdade de Ciências da Universidade de Lisboa
(supervisor at IST: Jorge H. Leitão; supervisor at FC-UL:
Deodália Dias), 2013.
Sofia de Almeida Santos de Castro e Abreu, “Bacterial
protein azurin as a new candidate anticancer drug by
decreasing cell adhesion through integrins”, Mestrado em
Biologia Celular e Molecular, Faculdade de Ciência e
Tecnologia, Universidade de Coimbra (supervisor at IST:
Arsénio M. Fialho; supervisor at FCT-UC: Carmen Maria
Martins de Carvalho Alpoim), 2013.
Vítor Hugo Jorge de Oliveira, “Studies on the
involvement of quorum sensing in the regulation of
exopolysaccharide biosynthesis by Burkholderia cepacia
complex isolates”, MSc in Applied Microbiology,
Faculdade de Ciências, Universidade de Lisboa
(supervisor at IST: Leonilde M. Moreira, supervisor at FC-
UL: Mário Santos), 2012.
Viviane Motta Varela, "Studies on s-triazines degrading
bacteria: Influence of three methods of culture formulation
on Arthrobacter aurescens TC1 ability to biodegrade the
herbicides terbuthylazine and atrazine", MSc in Biological
Engineering (supervisor: Cristina A. Viegas), 2013.
Other Publications in International
Journals
Editorials
Fontes CMGA, Sá-Correia I, Romão MJ, Sá-Nogueira I,
Prates JAM, Ferreira LMA. Editorial: Biocatalysis and Bio-
transformation, 30(3): 273, 2012 (DOI:
10.3109/10242422.2012.681858)
Mira NP, Teixeira MC. "Microbial mechanisms of tolerance
to weak acid stress", Frontiers in Microbiology (Editorial),
4, 416, 2013.
Papers in Conference Proceedings
Grilo AM, Ramos CG, Sousa SA, Arroja L, Capela I,
Leitão JH, Nadais H. Optimizing Energy Production from
Wastewater by Intermittent Operation of Anaerobic Reac-
tors, European Biomass Conference and Exhibition Pro-
ceedings, pp. 1721 – 1724, 2011. DOI:
10.5071/19thEUBCE2011-VP2.6.4. ISBN-13:978-88-89407-
55-4
Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA. A
bactéria Arthrobacter aurescens TC1 como ferramenta de
biorremediação para ambientes contaminados com os herbi-
cidas terbutilazina e atrazina, In: Borrego, C., Miranda, A.I.,
Arroja, L., Fidélis, T., Castro, E.A., Gomes, A.P. (eds), Actas
da 10ª Conferência Nacional do Ambiente (ISBN: 978-989
-98673-0-7), Departamento de Ambiente e Ordenamento da
Universidade de Aveiro, Aveiro, Portugal, 2013.
Viegas CA, Silva V, Mateus C, Chelinho S, Moreira-
Santos, Gonçalves J, Varela V, Ribeiro R, Fialho AM, and
Sousa JP. Bioremediation strategies based on Pseudomo-
nas sp. strain ADP for worst-case scenarios of soil contami-
nation with herbicidal formulations containing chlorinated s-
triazines. In proceedings of “BioMicroWorld2013 - V Inter-
national Conference on Environmental, Industrial and
Applied Microbiology”, Madrid, Spain, 2013.
Abstracts in international journals
Costa C, Pires C, Ribeiro J, Teixeira MC. "Antifungal drug
resistance mediated by Candida glabrata Drug:H+ Anti-
porters", Yeast, 30(S1), S175, 2013.
48
Bernardes N, Ribeiro AS, Mota B, Matos RG, Arraiano
CM, Seruca R, Paredes J and Fialho AM. “Azurin impairs P
-cadherin-dependent invasion of breast cancer cells via de-
creased FAk/Src signaling”, EACR22- from Basic Research
to Personalized Cancer Treatment, Barcelona, July 7-10,
European Journal of Cancer, 48: 3133:3318, 2012.
Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan
M, Moreira LM. “Role of BceF BY-kinase in host-pathogen
interaction, biofilm formation and survival to stress in the
opportunist pathogen Burkholderia cepacia IST408: Tran-
scriptomic and phenotypic approaches”, FEBS Journal 279,
502-502, 2012.
Ferreira AS, Oliveira VH, Silva, IN, Becker JD, McClean S,
Callaghan M, Moreira LM. “Role of BceF BY-kinase in host–
pathogen interaction, biofilm formation and survival to stress
by the cystic fibrosis pathogen Burkholderia cepacia IST408:
Transcriptomic and phenotypic approaches”, Journal of
Cystic Fibrosis 12, S77, 2013.
Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Genome-
wide transcriptional response to pesticide exposure in the
model yeast Saccharomyces cerevisiae, FEBS Journal, 279
(S1), 230, 2012.
Grilo AM, Ramos CG, Leitão JH. “Identification of regulato-
ry small non-coding RNAs in Burkholderia cepacia J2315”,
FEBS Journal, 279(S1), 510, 2012.
Pinto-de-Oliveira A, Coutinho CP, Ramos CG, Sousa SA,
de Carvalho CCCR, Leitão JH, Sá-Correia I. “The
Burkholderia cepacia small colony variants (SCV) are a more
pathogenic bacterial form that may facilitate persistent respir-
atory infections in CF patients”, Journal of Cystic Fibrosis,
12(S1), S76, 2013.
Ramos CG, Grilo AM, da Costa PJP, Feliciano JR, Leitão
JH. “Unveiling the roles of small non-coding RNAs and RNA
chaperones on the biology of opportunistic pathogens of the
Burkholderia cepacia complex”, Journal of Cystic Fibro-
sis,12 (S1), S77, 2013.
Silva IN, Tavares AC, Ferreira AS, Moreira, LM. “Mucoid
morphotype variation of Burkholderia multivorans: adaptation
to cystic fibrosis lung environment”, FEBS Journal 279, 237-
237, 2012.
Sousa, SA, P Soares-Castro, JR Feliciano, CG Ramos,
JLC Martinez, PM Santos, JH Leitão. “Unveiling the
Burkholderia cenocepacia J2315 RNA chaperone Hfq2 in-
teractome”, Journal of Cystic Fibrosis, 12(S1), S77, 2013.
Sousa SA, Feliciano JR, Ramos CG, Leitão JH. “The
Burkholderia cenocepacia J2315 small non-coding RNA
MavA is required for fitness and virulence”, Journal of Cyst-
ic Fibrosis, 12(S1), S76, 2013.
Papers in National Journals
Bernardes N, Fialho AM. Biotecnologia e inovação
terapêutica: bactérias e produtos seus derivados como
agentes anticancerígenos” Boletim de Biotecnologia, 4: 42-
45, 2013.
Ramos CG, Grilo AM, Sousa SA, Feliciano JR, Leitão JH.
"Post-transcription regulation of gene expression in the hu-
man opportunistic pathogens of the Burkholderia cepacia
complex". SPM Magazine, 3(1), 2014.
Sousa SA, Ramos CG, Grilo AM, Feliciano JR, da Costa
PJP, Leitão JH. “Estratégias de identificação de novos alvos
para combater as infecções por bactérias do complexo
Burkholderia cepacia”, Boletim de Biotecnologia, 4 (série
2), 41-43, 2013.
Teixeira MC, Cabrito TR, Brazão J, Sá-Correia I. “Yeast as
model eukaryote and expression host system: is it still
useful?”, Microbiologia - SPM Magazine, 2 (2), 2013.
Online publications
The YEASTRACT database (http://www.yeastract.com/) was
very recently updated and upgraded with new bioinformatic
tools and more detailed regulatory information to facilitate the
exploitation of the gathered material when answering specific
biological questions. New options were provided for the
ranking of transcription factors according to their relevance in
the regulation of specific gene sets. Furthermore, information
on the environmental condition and experimental setup
underlying each documented regulatory association was
gathered, thus allowing a fine tuning of the queries offered for
the analysis of gene and genomic regulation.
49
20 12-13 Biennial Report
12-13
Communications in International Confer-
ences
Invited Conferences
Leitão JH, “RNA chaperones and sRNAs from bacteria of the
Burkholderia cepacia complex”. FEBS-ASM Workshop on:
Biology of RNA in host-pathogen interactions. 26-29 January
2014, Tenerife, Spain.
Sá-Correia I, "Toward a systems-based understanding of
yeast response and tolerance to acetic acid" in: Yeast and
Food Microbiology Symposium, FEMS Congress 2013, 21-25
July, Leipzig, Germany.
Sá-Correia I, "Genomics of adaptation to the lung” in:
Burkholderia cepacia complex: still a problem? Symposium,
36th European Cystic Fibrosis Conference, 12-15 June 2013,
Lisbon, Portugal.
Oral Communications
Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Pesticide
toxicity studies using gene expression profiling in the model
yeast Saccharomyces cerevisiae”, 7th European Conference
on Pesticides and Related Organic Micropollutants in the
Environment & 13th Symposium on Chemistry and Fate of
Modern Pesticides, 7-10 October 2012, Porto, Portugal.
Matos RG, Fialho AM, López-Viñas E, Gomez-Puertas P,
Schuster G, Arraiano CM. “The evolution of the RNase II-
family of exoribonucleases: singular features in Archaea and
Cyanobacteria” FASEB Meeting: Post-transcriptional control
of gene expression: mechanisms of mRNA decay, June 24-
29 2012, Steamboat Springs, Colorado.
Mil-Homens D and Fialho AM. “Trimeric autotransporter
adhesins in the human pathogen Burkholderia cenocepacia: a
multifunctional family of proteins implicated in virulence” 3rd
Scientific Meeting of Institute for Biotechnology and Bioengi-
neering, 16-17 March 2012, Instituto Superior Técnico, Lis-
boa.
Nadais MH, Capela I, Arroja L, Leitão JH, Grilo AM, Cour-
as C. “Effects of operational shocks on UASB microbial popu-
lations”, 8th Conference on sustainable development of ener-
gy, water and environment systems. 22-27 September 2013,
Dubrovnik, Croatia.
Ramos CG, Grilo AM, da Costa PJP, Feliciano JR,
Leitão JH. “Towards the unveiling of the biological roles
played by small non-coding RNAs in the opportunistic patho-
gens of the Burkholderia cepacia complex”. FEBS Internation-
al Workshop “New Developments in RNA Biology, 1-4 Sep-
tember 2012, Tavira, Portugal.
Silva IN, Santos PM, Becker JD, Moreira LM. “Within-cystic
fibrosis patient genoptypic variation in Burkholderia multi-
vorans sequential isolates”, International Burkholderia cepa-
cia working group (IBCWG), 9-12 April 2014, Nimes, France.
Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.
“Arthrobacter aurescens TC1 as an efficient bioaugmentation
bacterium for soils contaminated with s-triazine herbicides”, V
International Conference on Environmental, Industrial and
Applied Microbiology, 2-4 October 2013, Madrid, Spain.
Teixeira MC. “Systematic functional analysis of the drug:H+
antiporter family from the pathogenic yeast C. glabrata”,
Yeast Genetics and Molecular Biology Meeting, July 31 -
August 5 2012, Princeton, NJ, USA.
Viegas CA, Chelinho S, Moreira-Santos M, Silva V, Ma-
teus C, Ribeiro R, Fialho AM, Sousa JP. “Pseudomonas sp.
strain ADP as a bioaugmentation bacterium in soil micro-
cosms contaminated with herbicidal formulations containing s
-triazines”, Symposium on Pollutant degradation under envi-
ronmental stress: towards rational bioaugmentation -
BACSIN2012, 29-30 March 2012, Amsterdam, The Nether-
lands.
Viegas CA, Chelinho S, Silva V, Mateus C, André S,
Moreira-Santos M, Viana P, Ribeiro R, Fialho AM, Sousa
JP. “Performance of a bioremediation tool based on Pseudo-
monas sp. strain ADP in soils contaminated with s-triazine
herbicides”, 7th European Conference on Pesticides and Re-
lated Organic Micropollutants in the Environment & 13th Sym-
posium on Chemistry and Fate of Modern Pesticides, 7-10
October 2012, Porto, Portugal.
Viegas CA, Silva V, Mateus C, Chelinho S, Moreira-
Santos M, Gonçalves J, Varela V, Ribeiro R, Fialho
AM, Sousa JP. “Bioremediation strategies based on
Pseudomonas sp. Strain ADP for worst-case scenarios of
soil contamination with herbicidal formulations containing
atrazine or terbuthylazine”, V International Conference on
Environmental, Industrial and Applied Microbiology, 2-4
October 2013, Madrid, Spain.
Poster Communications
Costa C, Pires C, Henriques A, Camacho G, Nunes J,
Teixeira MC. “Systematic functional analysis of the
drug:H+ antiporter family from the pathogenic yeast C.
glabrata”, Yeast Genetics and Molecular Biology Meeting,
July 31 - August 5 2012, Princeton, NJ, USA.
Costa C, Cabrito TR, Pires, C., Sá-Correia I, Teixeira
MC. “Role of the uncharacterized Candida glabrata
drug:H+ antiporter CgQdr2 (ORF CAGL0G08624g) in
antifungal drug resistance: a functional analysis”, 11th
ASM Conference on Candida and Candidiasis, 29 March -
2 April 2012, San Francisco, California, USA.
Costa C, Henriques A, Pires C, Nunes J, Sá-Correia I,
Teixeira MC. “The Candida glabrata drug:H+ antiporter
CgAqr1 plays a role in acetic acid and antifungal drug re-
sistance”, Fifth FEBS Advanced Lecture Course in Human
Communications
50
Fungal Pathogens, 25-31 May 2013, La Colle sur Loup,
France.
Costa C, Pires C, Ribeiro J, Teixeira MC. “Antifungal drug
resistance mediated by Candida glabrata Drug:H+ Anti-
porters”, 26th International Conference on Yeast Genetics and
Molecular Biology, 29 August - 3 September 3 2013, Frank-
furt, Germany.
da Costa PJP, CG Ramos, AM Grilo, JH Leitão.
“Experimental identification of regulatory small non-coding
RNAs from bacteria of the Burkholderia cepacia complex”,
FEBS International Workshop: New Developments in RNA
Biology, 1-4 September 2012, Tavira, Portugal.
da Costa PJP, Ramos CG, Grilo AM, Feliciano JR, Leitão
JH. “The small noncoding RNA MtvR is a global regulator of
gene expression in Burkholderia cenocepacia”, The Non
Coding Genome EMBO-EMBL Symposium, 9-12 October
2013, Heidelberd, Germany.
Dias PJ, Sá-Correia I. “New Insights on the Evolution of the
Hemiascomycete Yeasts Major Facilitator Superfamily Multi-
drug Resistance Transporters, Experimental Approaches to
Evolution and Ecology using Yeast”, EMBL Advance Training
Centre, 17-21 October, 2012, Heildelberg, Germany.
dos Santos DJVA, Leitão JH, Guedes RC. “Development
and validation of a homology model of Type II phosphoman-
nose isomerase”, 6th International Theoretical Biophysics
Symposium (TheoBio 2013), 24-27 June 2013, Gothenburg,
Sweden.
Ferreira AS, McClean S, Callaghan M, Moreira LM.
"Involvement of the bacterial tyrosine kinase BceF and the
phosphotyrosine phosphatase BceD in Burkholderia cepacia
interaction with the cystic fibrosis epithelial cell line
CFBE41o", EMBO/FEBS Host-Microbes Interactions course,
30 August – 7 September 2013, Spetses, Greece.
Gil FN, Gonçalves AC, Becker JD, Viegas CA. “Genome-
wide transcriptional response to pesticide exposure in the
model yeast Saccharomyces cerevisiae”, 37th FEBS and 22nd
IUBMB Congress, 4-9 September 2012, Seville, Spain.
Grilo AM, CG Ramos, JH Leitão. “Identification of regulatory
small non-coding RNAs in Burkholderia cepacia J2315”. 37th
FEBS and 22nd IUBMB Congress, 4-9 September 2012, Se-
ville, Spain.
Grilo AM, Ramos CG, da Costa PJP, Feliciano JR, Leitão
JH. “Experimental identification of small non-coding RNAs
from the human pathogen Burkholderia cenocepacia”, The
Non Coding Genome EMBO-EMBL Symposium, 9-12 Octo-
ber 2013, Heidelberd, Germany.
Guerreiro JF, Mira NP, Sá-Correia I. “Adaptive response to
acetic acid in the highly tolerant yeast species Zygosaccharo-
myces bailii, revealed by quantitative proteomics”, EMBO
Meeting 2012, 22-25 September, Nice, France.
Madeira A, dos Santos SC, Santos PM, Coutinho CP,
Tyrrell J, McClean S, Callaghan M, Sá-Correia I. “Adaptive
mechanisms associated with increased virulence and persis-
tence of Burkholderia cenocepacia during chronic lung infec-
tion: A quantitative proteomic analysis”, 36th European Cystic
Fibrosis Conference, 12-15 June 2013, Lisbon, Portugal.
Maldonado RF, dos Santos SC, and Sá-Correia I.
“Quantitative exoproteomic analysis to better understand the
mechanisms underlying Burkholderia cenocepa-
cia persistence and virulence in CF lung infections”, 36th Eu-
ropean Cystic Fibrosis Conference, 12–15 June 2013, Lisbon,
Portugal.
Mil-Homens D, Fialho AM. “Unraveling the involvement of
trimeric autotransporter adhesins in Burkholderia cenocepa-
cia host-cells interactions”, EMBO|EMBL Symposium: New
Approaches and Concepts in Microbiology, 14 - 16 October
2013, EMBL Heidelberg, Germany.
Mira NP, Henriques SF, Teixeira MC, Palma M, dos San-
tos SC, Sá-Correia I. “Haa1p-dependent regulatory network
of the yeast response to acetic acid stress”, Yeast Genetics
and Molecular Biology Meeting, July 31 - August 5 2012,
Princeton, NJ, USA.
Mira NP, Madeira A, Santos P, Coutinho CP, Moreira AS,
Pinto-de-Oliveira A, Sá-Correia I. “Transcriptomic and pro-
teomic analyses reveal Burkholderia cenocepacia adaptive
strategies to long-term colonization of the lungs of a cystic
fibrosis patient under antimicrobial therapy”, 3rd Seminar on
EraNet Pathogenomics, 23-25 January 2012, Tenerife, Spain.
Moreira AS, Coutinho CP, de Carvalho CCCR, Azevedo P,
Lito L, Melo-Cristino J, Sá-Correia I. “Phenotypic variation
of Burkholderia dolosa along 5.5 years of chronic colonization
with decline of lung function of a cystic fibrosis patient”.
36th European Cystic Fibrosis Conference, 12-15 June 2013,
Lisbon, Portugal.
Pereira M, Coutinho CP, Lopes A, Sá-Correia I, Videira
PA, Cabral MG. “Modulation of dendritic cell functions by
clonal variants of Burkholderia cenocepacia isolated from a
cystic fibrosis patient”, 36th European Cystic Fibrosis Confer-
ence, 12-15 June 2013, Lisbon, Portugal.
Pinto-de-Oliveira A, Coutinho A, Ramos CG, Sousa SA,
de Carvalho CCCR, Leitão JH, Sá-Correia I.
“The Burkholderia cepacia small colony variants (SCV) are a
more pathogenic bacterial form that may facilitate persistent
respiratory infections in CF patients”, 36th European Cystic
Fibrosis Conference, 12-15 June 2013, Lisbon, Portugal.
Ramos CG, Grilo AM, da Costa PJP, Feliciano JR, Leitão
JH. “The MtvR sRNA is a major regulator of Burkholderia
cenocepacia cell physiology”. FEBS-ASM Workshop on: Biol-
ogy of RNA in host-pathogen interactions, 26-29 January
2014, Tenerife, Spain.
Ramos CG, Grilo AM, Feliciano JR, Leitão JH. “Post-
transcriptional regulation in bacterial pathogens: Small non-
coding RNAs and RNA chaperones”, Molecular Biology in
Portugal and EMBL, 18 July 2013, Lisbon, Portugal.
Ramos CG, da Costa PJP, Döring G, Leitão JH. “Hfq and
sRNAs: A new layer of complexity in virulence regulation”,
The 4th EMBO Meeting, 22-25 September 2012, Nice,
France.
Ramos CG, da Costa PJP, Leitão JH. “The Burkholderia
cenocepacia RNA chaperone Hfq2 is negatively regulated by
the novel cis-encoded small RNA h2cR”, FEBS International
Workshop: New Developments in RNA Biology, 1-4 Septem-
ber 2012, Tavira, Portugal.
51
20 12-13 Biennial Report
12-13
Santos MR, Marques AT, Becker JD, and Moreira LM.
“Characterization of the TetR transcriptional regulator
SMc03169 and its contribution to symbiosis between Sinorhi-
zobium meliloti and leguminous plants”, 10th European Nitro-
gen Fixation Conference, 2-5 September 2012, Munich, Ger-
many.
Viegas SC, Mil-Homens D, Fialho A, Arraiano CM.
“Mutations in RNase E and RNase III attenuate S. Typhimuri-
um virulence”, FEBS-ASM Workshop on: Biology of RNA in
host-pathogen interactions, 26-29 January 2014. Tenerife,
Canary Islands, Spain.
Viegas SC, Mil-Homens D, Silva IJ, Saramago M,
Domingues S, Fialho A, Arraiano CM. “Endoribonucleases
mutations impair Salmonella typhimurium virulence in the
model host Galleria mellonella”, FEBS International Work-
shop on “New Developments in RNA Biology" 1 – 4 Septem-
ber 2012, Tavira, Portugal.
Viegas SC, Mil-Homens D, Silva IJ, Saramago M,
Domingues S, Fialho A and Arraiano CM. “The virulence of
Salmonella typhimurium is attenuated by mutations in endori-
bonucleases E and III”, in FASEB Meeting: Post-
transcriptional control of gene expression: mechanisms of
mRNA decay, from 24-29 June 2012, Steamboat Springs,
Colorado, USA.
Communications in National Conferences
Invited Conferences and Seminars
Fialho AM. “Bacterial proteins, a new class of multifunctional
anticancer drugs” 3rd PhD Meeting ITQB, 10-12 October
2012, Oeiras.
Leitão JH. “Post-transcription regulation in bacterial patho-
genesis: roles played by RNA chaperones and sRNAs”, Mi-
crobiotec´13 Portuguese Congress of Microbiology and Bio-
technology, 6-8 December 2013, Aveiro.
Leitão JH, “Small non-coding regulatory RNAs from
Burkholderia cepacia complex bacteria:
potential targets for therapeutic intervention?” DBE Seminars,
Instituto Superior Técnico, 28 May 2012, Lisboa.
Mira NP. “Involvement of the Haa1p-dependent regulatory
network in S. cerevisiae response to acetic acid stress: ge-
nome-wide approaches guided by bioinformatic analysis”, 1st
symposium in Bioinformatics, Universidade de Trás-os-
Montes e Alto Douro, 15 February 2012, Vila Real.
Sá-Correia I. "Systems-based understanding of chemical
stress defense mechanisms in yeast: challenges and opportu-
nities", António V. Xavier Seminars, ITQB, 29 November
2012, Oeiras.
Teixeira MC. "Unveiling antifungal drug resistance mecha-
nisms in the human pathogen Candida glabrata: from gene to
genome-wide approaches", CBAA seminars, Instituto Superi-
or de Agronomia, 30 January 2013, Lisboa.
Teixeira MC. "Antifungal drug resistance in yeasts: elucida-
tion of the biomolecular mechanisms", DBE Seminars, Insti-
tuto Superior Técnico, 16 April 2012, Lisboa.
Oral Communications
Costa C, Ponte A, Teixeira MC. “Unveiling 5-flucytosine
resistance mechanisms in yeast, using chemogenomics and
membrane proteomics approaches”, Microbiotec'13 Portu-
guese Congress of Microbiology and Biotechnology, 6-8 De-
cember 2013, Aveiro.
Costa C, Pires C, Henriques A, Camacho G, Cabrito T, Sá-
Correia I, Teixeira MC. “CgQdr2 drug:H+ antiporter from the
pathogenic yeast Candida glabrata: role and regulation in
azole drug resistance”, XIX Jornadas de Leveduras Prof.
Nicolau van Uden, 15-16 June 2012, Caparica.
Dias PJ, Sá-Correia I. “Evolution of the 12-spanner drug:H+
antiporters of family 1 (DHA1) in the pathogenic Candida
species: phylogenetic analysis of Mdr1 and Flu1 proteins”,
XIX Jornadas de Biologia de Leveduras “Nicolau van Uden”,
15-16 June 2012, Caparica.
Dias PJ, Sá-Correia I. “The major facilitator superfamily mul-
tidrug resistance transporters (mfs-mdr) in hemiascomy-
cetous yeasts: phylogenetic and syntenic analyses”, Microbi-
otec’13 Portuguese Congress of Microbiology and Biotechnol-
ogy , 6-8 December 2013, Aveiro.
dos Santos SC, Palma M, Tenreiro S, Mira NP, Moreira
AS, Becker J and Sá-Correia I. "Yeast as a model in phar-
macogenomics: genome-wide studies applied to quinine and
imatinib", XIX Jornadas de Biologia de Leveduras Professor
Nicolau van Uden, 15-16 June 2012, Caparica.
Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan M,
and Moreira LM. “Involvement of the BceF tyrosine kinase in
stress response, biofilm formation and virulence in Burkhold-
eria cepacia IST408 clinical isolate”, XXXVII Jornadas de
Genética, 28-30 May 2012, Lisboa.
Henriques SF, Mira NP, Matos R, Arraiano C and Sá-
Correia I. “Gene expression regulation in yeast mediated by
the transcription factor Haa1p: identification of residues re-
quired for functional Haa1-DNA associations and role of cop-
per in DNA-binding”, XIX Jornadas de Biologia de Leveduras
Professor NIcolau van Uden, 15-16 June 2012, Caparica.
Matos RG, Fialho AM, Schuster G, Arraiano CM. ”The
evolution of the RNase II-family of exoribonucleases selected
enzymes with peculiar features: examples from Archaea and
Cyanobacteria”, XXXVII Jornadas Portuguesas de Genética,
28-30 May 2012, Lisboa.
Mira NP, Münsterkötter M, Dias-Valada F, Santos J, Palma
M, Roque FC, Guerreiro JF, Rodrigues F, Sousa MJ, Leão
C, Guldener U, Sá-Correia I. “Genome sequence and anno-
tation of the highly acetic acid-tolerant Zygosaccharomyces
bailii derived interspecies hybrid strain
ISA1307”, Microbiotec’13 Portuguese Congress of Microbiolo-
gy and Biotechnology, 6-8 December 2013, Aveiro.
Palma M, Madeira S C, Mendes-Ferreira A and Sá-Correia
I. “Impact of assimilable nitrogen availability in glucose up-
take kinetics in Saccharomyces cerevisiae during alcoholic
fermentation”, XIX Jornadas de Biologia de Leveduras Pro-
fessor NIcolau van Uden, 15-16 June 2012, Caparica.
52
Palma M, Roque F, Guerreiro JF, Mira N and Sá-Correia I.
“Screening of a genomic library of the Zygosaccharomyces
bailii derived interspecies hybrid strain ISA1307 to search for
genes responsible for acetic acid resistance”, Microbiotec’13
Portuguese Congress of Microbiology and Biotechnology, 6-8
December 2013, Aveiro.
Ramos CG, Grilo AM, da Costa PJP, Feliciano JR, Leitão
JH. “The MtvR sRNA is involved in the regulation of Hfq”,
Microbiotec'13 Portuguese Congress of Microbiology and
Biotechnology, 6-8 December 2013, Aveiro.
Santos MR, Marques AT, Becker JD, and Moreira LM.
“Involvement of the TetR transcriptional regulator SMc03169
in pH sensitivity and contribution to nodulation competitive-
ness in Sinorhizobium meliloti”, 3rd Meeting of the Institute for
Biotechnology and Bioengineering, 16-17 March 2012, Lis-
boa.
Silva IN, Ferreira AS, Becker JD, Tavares AC, and Moreira
LM. “Genotype and phenotype variation of Burkholderia multi-
vorans sequential isolates during long-term colonization of the
airways of cystic fibrosis patients”, XXXVII Jornadas de Gen-
ética, 28-30 May 2012, Lisboa.
Silva V, Mateus C, Gonçalves J, Varela V, Viegas CA.
“Arthrobacter aurescens TC1 as a bioaugmentation tool for
bioremediation of environments contaminated with the herbi-
cides atrazine and terbuthylazine”, 10ª Conferência Nacional
do Ambiente, 6-8 November 2013, Aveiro.
Poster Communications
Bernardes N, Ribeiro AS, Matos R, Arraiano CM, Seruca
R, Paredes J, Fialho AM. “Azurin impairs P-cadherin-
dependent invasion of breast cancer cells via decreased FAK/
Src signalling” I3S Scientific retreat, 10-11 May 2012, Póvoa
do Varzim.
Ferreira AS, Oliveira VH, Moreira LM. “Quorum-Sensing
inhibitors as antimicrobial agents against bacteria belonging
to Burkholderia cepacia complex”, XXXVII Jornadas de Gen-
ética, 28-30 May 2012, Lisboa.
Ferreira AS, Silva IN, Becker JD, McClean S, Callaghan M,
Pilkington R, Givskov M, Ryan RP, Fernandes F, Moreira
LM. “Involvement of the BceF tyrosine kinase in stress re-
sponse, biofilm formation and virulence in Burkholderia cepa-
cia IST408 clinical isolate” 3rd Meeting of the Institute for Bio-
technology and Bioengineering, 23-24 November 2012, Lis-
boa.
Grilo AM, Ramos CG, da Costa PJP, Feliciano JR, Leitão
JH. “Identification of small non-coding RNAs and their roles
on the biology of opportunistic pathogens of the Burkholderia
cepacian complex”, Microbiotec’13 Portuguese Congress of
Microbiology and Biotechnology, 6-8 December 2013, Aveiro.
Isidoro D, Santos MR, Ferreira AS, and Moreira LM.
“Studies on gellan gum modification aiming new biotechno-
logical applications”, XXXVII Jornadas de Genética, 28-30
May 2012, Lisboa.
Ramos CG, Grilo AM, da Costa PJP, Feliciano JR, Leitão
JH. “The MtvR sRNA is involved in the regulation of Hfq”,
Microbiotec’13 Portuguese Congress of Microbiology and
Biotechnology, 6-8 December 2013, Aveiro.
Ramos CG, Grilo AM, Silva IN, Ferreira AS, Becker JD, da
Costa PJP, Feliciano JR, Sousa SA, Moreira LM, Leitão
JH. “Building an Atlas of Hfq-dependent samll non-coding
RNAs from the opportunistic human pathogen Burkholderia
cenocepacia J2315”, Microbiotec´ 13 Portuguese Congress of
Microbiology and Biotechnology, 6-8 December 2013, Aveiro.
Santos MR, Marques AT, Becker JD, Moreira LM.
“Characterization of the TetR transcriptional regulator
SMc03169 and its contribution to symbiosis between Sino-
rhizobium meliloti and leguminous plants”, XXXVII Jornadas
de Genética, 28-30 May 2012, Lisboa.
Tavares AC, Silva IN, Becker JD, Ferreira AS, Moreira LM.
“Mucoid morphotype variation of Burkholderia isolates under
stress conditions: Effects on gene expression, phenotypic
properties and virulence”, IV Symposium on Bioengineering,
23-24 November 2012, Porto.
Tavares AC, Silva IN, Becker JD, Ferreira AS, Moreira LM.
“Effect of mucoid morphotype variation of Burkholderia iso-
lates on gene expression, resistance to antimicrobial environ-
ments and virulence”, XXXVII Jornadas de Genética, 28-30
May 2012, Lisboa.
Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves JP,
Mira NP, dos Santos SC, Cabrito TR, Palma M, Costa C,
Francisco AP, Madeira SC, Oliveira AL, Freitas AT, Sá-
Correia I. YEASTRACT: an upgraded information system for
the analysis of gene and genomic transcription regulation in
Saccharomyces cerevisiae, Microbiotec'13 Portuguese Con-
gress of Microbiology and Biotechnology, 6-8 December
2013, Aveiro.
53
20 12-13 Biennial Report
12-13
Guest Editors of International Journals
Fontes CMGA, Sá-Correia I, Romão MJ, Sá-Nogueira I,
Prates JAM, Ferreira LMA - Guest editors of the special
issue “Biocatalysis and Biotransformation”, Proceedings of
the 9th Carbohydrate Bioengineering Meeting (CBM9), 2012.
Mira NP, Teixeira MC. Guest editors of the special issue
“Microbial Mechanisms of Tolerance to Weak Acids”, Fron-
tiers in Microbiology – Section of Microbial Physiology
and Metabolism (11 articles included), 2013.
Sá-Correia I, Teixeira MC. Guest editors of the special issue
“Physiological role and regulation of Multidrug/Multixenobiotic
resistance membrane transporters”, Frontiers in Physiolo-
gy, 2014.
Editorial boards of International Journals
“OMICS: A Journal of Integrative Biology” (2012-2013),
“FEMS Yeast Research” (2012-2013), “Journal of Biomedi-
cine and Biotechnolology/BioMed Research Internation-
al” (2012-2013), “International Journal of Microbiology” (2012-
2013) , “Microbial Cell” (2013) (I. Sá-Correia)
“International Journal of Microbiology”, “The Open Microbiolo-
gy Journal”, “Bioengineered Bugs” (A.M. Fialho)
“Dataset papers in Science”, Hindawi Publishing Corporation
(J.H. Leitão)
Editorial boards of National Journals
“Magazine de MICROBIOLOGIA” (http://
www.spmicrobiologia.pt/ ), the journal of the Portuguese Soci-
ety of Microbiology (I. Sá-Correia and C.G. Ramos)
“Boletim de Biotecnologia” (J.H. Leitão)
Organization of Scientific Events
5th PYFF- Physiology of Yeast and Filamentous Fungi, 4-7
June 2013, Montpellier, France (I. Sá-Correia, scientific com-
mittee and chairperson of session “Response and tolerance
to stress”)
36th European Cystic Fibrosis Conference, 12-15 June 2013,
Lisboa, Portugal (I. Sá-Correia, scientific & organizing com-
mittees and chairperson of the Symposium "Translating what
we have learned from bacterial genomics to CF care")
FEMS Congress 2013, July 21-25, Leipzig, Germany (I. Sá-
Correia, co-chair of “Yeast and Food Microbiology” Symposi-
um)
2012 and 2013 editions of the Winter School in Systems Biol-
ogy held within the framework of the Erasmus Mundus
euSYSBIO Masters´ in Systems Biology, Instituto Superior
Técnico-Alameda (Organizers: I. Sá-Correia and M.C.
Teixeira)
Coordination of post-graduation degrees
Master’s program in Microbiology, University of Lisbon (IST
and the Faculties of Sciences, Medicine and Veterinary Medi-
cine) (I. Sá-Correia, coordinator)
Master’s Program in Biotechnology, IST (I. Sá-Correia, coor-
dinator)
Ph.D. Program in Biotechnology, IST (I. Sá-Correia, coordi-
nator)
Erasmus Mundus euSYSBIO Master’s Programme in Sys-
tems Biology (Instituto Superior Técnico, KTH (Sweden) and
Aalto University (Finland)) (I. Sá-Correia, coordinator at IST)
Other Scientific Activities
2012 2013
54
General and Committee Chairs or Committee
Memberships
Portuguese Society of Microbiology (I. Sá-Correia, Presi-
dent; A.M. Fialho, 2nd secretary)
Council of the Federation of European Microbiological Soci-
eties (FEMS) (I. Sá-Correia, member)
Study group on fungal infections of the European Society of
Clinical Microbiology and Infectious Diseases (ESCMID)
(N.P. Mira)
Biological Sciences Research Group of IBB at Instituto Su-
perior Técnico (I. Sá-Correia, coordinator)
Department of Bioengineering of IST (I. Sá-Correia, vice-
president)
Scientific Board of IST, Universidade de Lisboa (I. Sá-
Correia, member)
General Council of Universidade de Lisboa (I. Sá-Correia,
member)
Evaluation panels
Member of the Evaluation Panel "Genetics, Genomics,
Bioinformatics and Systems Biology" of Advanced
grants, European Research Council (ERC)(2012
applications) (I. Sá-Correia)
Coordinator/Member of the evaluation panel "Biology, Bio-
chemistry and Biotechnology" of University study cycles for
the Portuguese Agency for the Evaluation and Accreditation
of Higher Education (A3ES) (I. Sá-Correia)
Awards
Miguel C. Teixeira - Honorable Mention within the scope of
the UTL/Santander scientific awards 2012, in the area of
Biochemical Engineering, Biochemistry and Biotechnology,
as a recognition of the impact of his scientific work during
the past five years.
Sandra C. dos Santos - Young Researcher Award (ex-
equeo) SPM - Isabel Spencer-Martins, awarded by the Por-
tuguese Society of Microbiology, 2012.
Sandra C. dos Santos - Best Poster Award, granted by the
European Cystic Fibrosis Society at the 36th European Cyst-
ic Fibrosis Conference, with the communication “Adaptive
mechanisms associated with increased virulence and per-
sistence of Burkholderia cenocepacia during chronic lung
infection: A quantitative proteomic analysis”, 2013.
Nuno P. Mira - Best oral communication Award (ex-aqueo),
awarded by Portuguese Society of Microbiology and
Alfagene at the Microbiotec13 meeting with the oral commu-
nication “Genome sequence and annotation of the highly
acetic acid-tolerant Zygosaccharomyces bailii-derived inter-
species hybrid strain ISA1307”, 2013.
55
20 12-13 Biennial Report
12-13
Faculty Staff Isabel Sá-Correia Arsénio M. Fialho Cristina A. Viegas Jorge H. Leitão Leonilde M. Moreira Miguel C. Teixeira Nuno P. Mira
Post-doctoral Fellows Sílvia A. Sousa Ana S. Ferreira Sandra C. dos Santos Margarida Palma Paulo J. Dias Carla P. Coutinho Tânia R. Cabrito Christian G. Ramos Dalila Mil-Homens Inês N. Silva
PhD Students Catarina Rodrigues Andreia Madeira Mário R. Santos Nuno Bernardes Fátima Gil Catarina Costa André M. Grilo Ana S. Moreira Joana F. Guerreiro Joana R. Feliciano Sílvia F. Henriques Filipa C. Roque Rita Maldonado
Research Assistants Vera P. Silva Filipa Valada Paulo JP da Costa Andreia Silva
Master Students Ana Almeida André Henriques Andreia Tavares Andreia Ponte Carla Alexandra Mateus Carla Pires Cláudia P. Godinho Eunice Penas Filipa G. Dias Filipe Silva Gonçalo Silva Guida Camacho Janete Gonçalves João Brazão Jonathan Ribeiro Kaur Alasoo Maria Moita Rúben Bernardo Sofia Abreu Sofia Alves Vítor Oliveira Viviane Varela
Technical Assistants Mónica Rato Sílvia Rana
BSRG Members (2012-2013)
56
BSRG
Biological Sciences Research Group
Institute for Biotechnology and Bioengineering
Instituto Superior Técnico
Av. Rovisco Pais
1049-001 Lisbon
Portugal
http://ibb.pt/bsrg/