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YopD translocator function in Yersinia pseudotuberculosis type III secretion Tiago Rafael Dias Costa Department of Molecular Biology Umeå Center for Microbial Research UCMR Umeå University, Sweden Umeå 2012

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Page 1: YopD translocator function in Yersinia …umu.diva-portal.org/smash/get/diva2:570477/FULLTEXT02.pdfYersinia pestis-plaque agent 1 1.3. Diversion of Y.pseudotuberculosis from Y.pestis

YopD translocator function in Yersinia pseudotuberculosis

type III secretion

Tiago Rafael Dias Costa

Department of Molecular Biology Umeå Center for Microbial Research UCMR Umeå University, Sweden Umeå 2012

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Copyright © Tiago Rafael Dias Costa ISBN: 978-91-7459-483-6 Printed by: Print & Media Umeå, Sweden 2012

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Para o meu Pai To my Father

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Table of contents Table of contents i Abstract iii Abbreviations iv Papers in this thesis vi 1. Introduction 1

1.1. The Yersinia family 1 1.2. Yersinia pestis-plaque agent 1 1.3. Diversion of Y.pseudotuberculosis from Y.pestis 1 1.4. Enteropathogenic Yersinia 3 1.5. Yersinia host diversity and responsiveness 4 1.6. Type III secretion systems (T3SS) in Gram negative bacteria 6

1.6.1. Variability among T3SSs 7 1.6.2. Acquisition of the T3SS 9 1.6.3. Couple heterogenic bacterial T3SSs to its environment 10 1.6.4. Conserved features of type III secretion systems 10

1.6.4.1. OM ring components 11 1.6.4.2. IM ring components 11 1.6.4.3. Periplasmic rod structure 12 1.6.4.4. The needle complex 12 1.6.4.5. T3SS energizer 13 1.6.4.6. T3S export apparatus and subtract recognition 14 1.6.4.7. T3SS chaperones classes 15

1.7. Plasmid encoded Ysc-Yop T3SSs in Yersinia 17 1.8. Environment and regulatory control of Ysc-Yop T3S substrates 19

1.8.1. Temperature and transcriptional control 19 1.8.2. Calcium dependency and post-transcriptional control 21 1.8.3. Secretion of anti-activators and post-transcriptional control 22 1.8.4. Host-cell contact signal transduction and gating of the T3S

machinery 24 1.9. Encountering the host cell – substrate secretion through the

polymerized T3S needle 25 1.9.1. Translocator proteins – facilitators of effector translocation 25

1.9.1.1. LcrV - hydrophilic translocator 26 1.9.1.2. YopD - multifunctional hydrophobic translocator 28

1.9.1.2.1. Domains organization and function 28 1.9.1.2.2. Genetic and functional homology in other T3S

families 31 1.9.1.2.3. YopB – hydrophobic translocator partner 32

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1.9.1.2.4. Role of the hydrophobic translocators chaperone (LcrH) in Yersinia spp. T3SS 33

1.9.2. Yop effector proteins – modulators of host signaling 35 1.9.2.1. Anti-phagocytic modulators 35 1.9.2.2. Immune response modulators 37

1.10. Translocon assembly and translocation dynamics 39 1.10.1. Tip complex assembly platform and activation 39 1.10.2. Translocation of surface localized Yops by an alternative

translocation process 40 1.10.3. The T3SS translocon pore 42 1.10.4. Translocators have effector function? 44 1.10.5. Inhibition of pore formation and feedback regulation 45

2. Objectives of this Thesis 47 3. Results and Discussion 48

3.1. Functional implications of the YopD domain architecture 48 3.1.1. The amphipathic α-helix domain 48

3.1.1.1. Specific residues separate Yops regulation from secretion 49

3.1.1.2. YopD´s molecular interactions facilitate Yersinia T3S 51 3.1.2. Putative N-terminal coiled-coil domain 52

3.1.2.1. A predicted helical structure unique for YopD from Yersinia 53

3.1.2.2. Putative YopD N-terminal coiled-coil contributes for protein self-assembly and Yersinia spp. virulence 54

3.1.3. Putative C-terminal coiled-coil domain 55 3.1.3.1. Predicted α-helical segment that influence translocon

assembly and effectors delivery in vitro 55 3.1.3.2. T3S translocon assembly intermediates attenuate

Yersinia spp. virulence 57 3.2. Influence of the YopD chaperone, LcrH, on Yersinia spp. T3SS

regulation 58 3.2.1. LcrH is not a cofactor of LcrF transcriptional activation 60 3.2.2. LcrH and post-transcriptional regulatory control in Y.

pseudotuberculosis 60 3.2.3. Functional exchangeability of the YscM/LcrQ regulatory

family in Yersinia spp. 62 Main findings in this thesis 63 Future perspectives 64 Acknowledgements 66 References 68

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Abstract

Type III secretion systems (T3SS) are a common feature of Gram-negative bacteria, allowing them to inject anti-host effectors into the interior of infected eukaryotic cells. By this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease.

In pathogenic Yersinia, a crucial protein in this process is YopD. YopD is a T3S substrate that, together with YopB, forms a translocon pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol. The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. How the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD YopB, is still unknown. Portions of YopD are also observed inside HeLa cells. Moreover, YopD functions together with its T3S chaperone, LcrH, to control Yops synthesis in the bacterial cytoplasm. The multifunctional YopD may influence all these processes by compartmentalizing activities into discrete modular domains along the protein length. Therefore, understanding how particular domains and/or residues within these regions coordinate multiple functions of the protein will provide a platform to improve our knowledge of the molecular mechanisms behind translocation through T3SSs.

Comprehensive site-directed mutagenesis of the YopD C-terminal amphipathic α-helix domain, pinpointed hydrophobic residues as important for YopD function. Some YopD variants were defective in self-assembly and in the ability to interact with the needle tip protein, LcrV, which were required to facilitate bacterial T3S activity. A similar mutagenesis approach was used to understand the role of the two predicted coiled-coils located at the N-terminal and C-terminal region of YopD. The predicted N-terminal element that occurs solely in the Yersinia YopD translocator family is essential for optimal T3SS and full disease progression. The predicted YopD C-terminal coiled-coil shapes a functional translocon inserted into host cell membranes. This translocon was seen to be a dynamic structure facilitating at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the other to promote targeted activity of internalized effector toxins.

In Yersinia expression of yop genes and secretion of the corresponding polypeptides is tightly regulated at a transcriptional and post-transcriptional level. If T3S chaperones of the translocator class are known to influence transcriptional output of T3SS genes in other bacteria, we show that in Yersinia the class II T3S chaperone LcrH has no such effect on the LcrF transcriptional activator activity. We also demonstrate that there are possibly additional yop-regulatory roles for the LcrH chaperone besides forming a stable complex with YopD to impose post-transcriptional silencing on Yops synthesis. This mechanism that relies upon an active T3SS, might act independently of both YopD and the regulatory element LcrQ.

In conclusion, this work has sought to delineate the encrypted functions of the YopD translocator that contribute to Yersinia T3SS-dependent pathogenesis. Contributions of the YopD cognate chaperone LcrH in yop regulatory control are also presented.

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Abbreviations 5'-UTR - 5'-untranslated region ATP - Adenosine-5'-triphosphate BCECF - 2′,7′‑bis‑(2‑carboxyethyl)‑5‑(and‑6)‑carboxyfluorescein Bla - Beta-lactamase CBD - Chaperone binding domain CC - Coiled-coil CD - Calcium dependent CI - Calcium independent C-ring - Cytoplasmic ring cryo-EM - Cryo-electron microscopy C-terminus - Carboxy-terminus EHEC - Enterohemorrhagic Escherichia coli EIEC - Enteroinvasive Escherichia coli EPEC - Enteropathogenic Escherichia coli EtBr - Ethidium bromide Fak - Focal adhesion kinase Fyb - Fyn binding protein GAP - GTPase activating protein GDP - Guanosine diphosphate GST - Glutathione S-transferase GTP - Guanosine triphosphate H-NS - Universal nucleoide associated protein IgA - Immunoglobulin A IL - Interleukin IM - Inner membrane Lck - Lymphocyte-specific protein tyrosine kinase LCR - Low calcium response LDC - Lysine decarboxylase LDH - Lactate dehydrogenase LPS - Lipopolysaccharide LRR - Leucin-rich-repeat MAPK - Mitogen activated protein kinase M-cell - Transcytotic epithelial cell MLD - Membrane localization domain MLN - Mesenteric lymph node mRNA - Messenger RNA

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MS - Membrane and supermembranous NF-κB - Nuclear factor kappa-light-chain-enhancer of activated B cells NLR - NOD-like receptor NMR - Nuclear magnetic resonance NOD - Nucleotide oligomerization domain N-terminus - Amino-terminus OM - Outer membrane PAI - Pathogenicity island PAMP - Pathogen-associated molecular patterns PDB - Protein data bank Prk2 - Protein kinase c-like 2 PRR - Pathogen recognition receptor QS - Quorum sensing RBC - Red blood cell RBS - Ribosome binding site Rsk1 - Ribosomal protein S6 kinase 1 SPI - Salmonella pathogenicity island T3SS - Type III secretion system TEM - Transmission electron microscopy TLR - Toll-like receptor TM - Transmembrane TPR - Tetratricopeptide repeat TS - Temperature sensitive Yop - Yersinia outer protein Ysc - Yersinia secretion

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Papers in this thesis This thesis is based on the following publications and manuscripts referred to by their roman numerals (I-IV).

I. Costa, T. R., P. J. Edqvist, J. E. Bröms, M. K. Ahlund, A. Forsberg, and M. S. Francis. 2010. YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis. J Biol Chem 285:25269-25284.

II. Costa, T. R., A. A. Amer, M. Fällman, A. Fahlgren, and M. S. Francis. 2012.

Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis. Infect Genet Evol 12:1729-1742.

III. Tiago R. D. Costa, Ayad A. A. Amer, Salah I. Farag, Hans Wolf-Watz, Maria

Fällman, Anna Fahlgren, Thomas Edgren, and Matthew S. Francis. Active type III translocon assemblies that attenuate Yersinia virulence. (Revised and resubmitted manuscript)

IV. Tiago R. D. Costa, Katrin E. Carlsson, Petra J. Edqvist, and Matthew S.

Francis. Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosis (Manuscript)

Paper I and II are reproduced with the permission from American Society for Biochemistry and Molecular Biology (ASBMB) and Elsevier.

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1. Introduction

1.1. The Yersinia family

The fight for energy is the main driving force in most biological processes.

Living beings strive to do all that is possible in the quest for energy, even if at times they have to sacrifice other life forms. Bacteria are such fascinating microorganisms because of that same reason. Around the time of evolution they created different strategies with the final intention of survival, adaption, and replication, most of the times in harsh environments or in different hosts. Yersiniae are such organisms. The Yersinia genus consists of 11 species, although only Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis are recognized as human pathogens. The remaining species, also referred as Y.enterocolitica-like species, have not been extensively studied, although the role of low-virulence or nonpathogenic in the etiology of human disease should not be overlooked (1). Human pathogenic Yersinia is the causing agent of distinct types of human infections that range from plague to gastrointestinal diseases. Y.pestis causes two different forms of plague in humans. One such form is the ‘bubonic plague’, after the host gets bitten by the contaminated flea carrying the bacilli (obtained from the rodent reservoir), or the highly infectious ‘pneumonic plague’ once the human inhale contaminated respiratory droplets from another infected human (2). The plague life cycle and pathogenesis will be detailed in section 1.2. In comparison, Y. enterocolitica and Y. pseudotuberculosis are found widely in the environment in particular in the soil. Upon consumption of contaminated food or water, it commonly causes gastrointestinal symptoms of moderate intensity (see section 1.4) (3,4).

1.2. Yersinia pestis – plague agent

The plague is an acute zoonotic disease caused by the bacilli Yersinia pestis

that primarily affects wild rodents (reservoir). The transmission between rodents and humans is accomplished with the help of the Xenopsylla cheopis flea (vector) that acquires the bacteria after ingesting a blood meal from the blood stream of an infected animal (2). The bacterium ensures transmission by blocking the foregut of the infected flea (5). This leads to regurgitation of the pathogen in the next feeding attempts on the bite site, ensuring a successful spread to the next host (6). On the site of infection, the bacteria promotes the cleavage of fibrin clots through the activation of the surface protease ‘Pla’, promoting the invasion of mammalian endothelial cells followed by tissue penetration and entrance into lymphatic vessels

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(7,8). The pathogen is also taken up by macrophages that take the pathogens to the nearby lymph nodes, where the bacteria replicates and colonize (9). This creates an acute enlargement of the infected node in result of a massive inflammatory process, giving rise to a ‘bubo’ - which is the hallmark of the bubonic plague. Occasionally, the infection is able to spread into the blood stream becoming systemic, which can lead to lungs colonization where the infection progresses to pneumonic plague or pathogen clearance in the spleen and liver. The form of pneumonic plague is then transmitted from human to human through respiratory droplets, making it highly infectious and usually fatal, unless treated within one day (4). This remarkable disseminative capacity results in three major human pandemics that are associated with three particular Y.pestis subgroups or ‘biovars’. The biovar Antiqua that is present in Africa and Central Asia was responsible for the 6th century Justinian plague pandemics that spread around the Mediterranean Sea; biovar Mediaevalis is currently limited to central Asia and is descendent from the bacteria that caused the Black Death and subsequent pandemics from 1346 to early 19th century. Biovar Orientalis is distributed worldwide and is the cause of the modern pandemic which began in the mid-19th century and spread globally (10). The factors that determine recurrent outbrakes of plague around the world in the last 20 years that result in 100 to 200 deaths each year are still unclear (11). Subtle genetic changes that result in highly virulent strains might be one of many reasons for these epidemics. Remarkably, recent findings support the notion that factors other than microbial genetics - such as climate, social condition, vector dynamics, host susceptibility and synergistic contacts with concomitant diseases - might be critical Y.pestis outbrakes (12).

1.3. Diversion of Y. pestis from Y. pseudotuberculosis

One of the most striking aspects in the Yersinia evolution is the exceptional

genetic relationship between Y. pestis and Y. pseudotuberculosis, despite having entirely different lifestyles. Yersinia pestis causes fatal bubonic plague and is transmitted by a flea vector, whereas Y. pseudotuberculosis utilizes a fecal-oral route to establish a non-fatal infection (they share high identity in the sequences of their 16S rRNAs) (13). Moreover, results in DNA-DNA hybridization indicate that these two species are highly related (14) and have evolved as two distinct species within the last 1,500-20,000 years, just before the first plague pandemics (15). We then ponder, how is it that Y. pestis became an independent pathogen with such successful virulence weaponry? This is a complicated question, more so when we consider that Y. pseudotuberculosis has all the extra genes that Y. pestis needs for

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virulence. Nevertheless, the solution might rely not only on one factor, but in a conjunction of events. In terms of genetic expansion - that seems to be a key element in the evolutionary jump - Y. pestis acquired two plasmids by lateral gene transfer (16). The 9.5-kb plasmid (pPla/pPCP1) that encodes the plasminogen activator Pla degrades fibrin clots and binds to extracellular matrix components promoting bacterial systemic dissemination from intra-dermal site of infection (7). In addition, it harbors a 100-kb plasmid (pFra/pMT1) that encodes the murine toxin gene (ymt) together with the hemin storage locus (hms), which are essential for the bacterial colonization of the flea and transmission to the host (5,17). The pMT1 plasmid also encodes the F1 capsular antigen, which prevents bacterial binding to phagocytic cells evading uptake (18). Host adaptation and niche establishment induced other types of genetic changes that resulted in loss of the insect toxin gene function by which, if active, would prevent the pathogen dissemination utilizing the flea vector (3). In addition, the lack of selective pressure towards genes that encode essential adhesins (YadA and Inv) for the enteropathogen transpose the intestinal barrier resulted as pseudogenes in Y.pestis (19,20).

Y.pestis life cycle requires different hosts and host niches each one with different nutritional availabilities demanding a great plasticity in the bacteria metabolic pathways. This flexibility has been proven important for some pathogens to circulate and establish niches in different hosts (21-23). Therefore, the genetic rearrangements observed in the Y.pestis might be also be trigged by nutrient availability in each of its host niches.

The two pathogens also have remarkable differences in the context of gene regulation which includes the selective inactivation of one type of genes that are common to both organisms, but required for virulence in only one (20,24-26). Remarkably, this is not a host adaptation mechanism specific for Yersinia. It has been described that genome reduction or specific inactivation of some pathways are a common adaptation mechanism contributing to enhance bacterial virulence. For example, complementing Shigella spp. with the E. coli gene coding for lysine decarboxylase (LDC) (absent in the recipient) resulted on attenuation of the bacteria virulence explained by an inhibition of the enterotoxin activity by a product of the LCD (27).

1.4. Enteropathogenic Yersinia

Y. enterocolitica and Y. pseudotuberculosis are the two most frequent

Yersiniae enteropathogens found widely in the environment. Both species can be present in aquatic and soil environments, but also in animals. Indeed,

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Y.enterocolitica has been isolated from different avian and mammalian hosts, swine being the most popular reservoir for the pathogenic strains where they commensally colonize its naso and oropharynx (28). The two subspecies enterocolitica and paleartica comprise 6 biogroups (1A, 1B, 2, 3, 4 and 5) being the biogroup 1B highly pathogenic for humans and mouse-lethal. Y.enterocolitica is responsible for the majority of the yersiniosis outbreak cases as a result of ingestion of contaminated food, water and direct person-to-person or animal-to-human contact (28-32). Y. pseudotuberculosis is a less common human pathogen which derives its name from tuberculosis-like granulomatous abscesses found in liver and spleens of infected animals. Although, only few outbreaks are described in the literature, they are generally severe often requiring patient hospitalization (33). Y. pseudotuberculosis is subgrouped into 21 different serological groups based on variations in the lipopolysaccharide surface antigen (O-antigen) (34).

Both pathogens cause gastrointestinal infections and follow a similar infection route after the consumption of contaminated products. The gastrointestinal tract in the small intestine is the pathogens port of entry. Once in the vicinity of the intestinal lymphoid follicles (Payer’s patches) the intestinal epithelium translocation is granted upon targeting of transcytotic epithelial cell (M-cells) (35). Yersiniae and other enteropathogens such E. Coli, Salmonella and Shigella also take advantage of this transcytosis process that helps in sampling and transport of luminal substances to the underlying immune cells of the follicle to invade and gain access to the submucosa (36). Once in the follicle, the bacteria can replicate extracellularly generating its destruction (37) and promoting dissemination to the mesenteric lymph node. This causes symptoms associated with gastroenteritis, such as self-limiting mesenteric lymphadenitis and terminal ileitis (38). In severe cases, bacteria can spread via the lymphatic system to the liver and spleen causing systemic infection and bacteraemia (38-42).

1.5. Yersinia host diversity and responsiveness

The use of the mice animal model to study Yersinia pathogenesis has been a

valuable tool over the years. Due to an efficient colonization of the mesenteric lymph nodes and spreading to spleen and liver, enteropathogenic Yersinia was broadly utilized to appreciate the plague infections dynamics without the need of using the lethal Y. pestis (24,43-46). However, new methods to identify novel virulence factors that take into account the critical role of the host in the disease initiation and progression are needed. Importantly, only within the host or in the environmental reservoir the pathogen coordinate the expression of a subset of

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genes which became important for the pathogen to circumvent the immune system and cause disease. Consequently, the recent use of non-mammalian hosts has already proven valuable in further understanding Yersinia pathogenicity. For example, it has been establish that Yersinia species can kill the C. elegans nematode by either a biofilm-dependent (47) or independent process (48). Formation of biofilm by bacteria harboring the hmsHFRS biofilm synthesis operon induces worm’s starvation and ultimately death (49). This observation proved essential to establish that type III secretion blocks biofilm formation and is reciprocally regulated with motility via quorum-sensing (QS) (50). However, Yersinia strains lacking the hms operon and therefore unable to produce biofilm, are still able to invade the nematode’s intestine and kill it by a unknown mechanism (48). Both types of C. elegans infection resulted in the identification of new genes that prevent or reduce biofilm formation and unveiled new virulence factors that were found also important in the intranasal mouse model of Y. pestis (48). Additional advantages in the use of a C. elegans model are that it is: (i) anatomically simple and transparent making it easy to observe under the microscope, (ii) fully sequenced genome and (iii) genetically tractable. However, the most important trait is that it does not possess adaptative immunity, permitting the study of the innate immunity in isolation.

In the intestinal lumen innate immune responses triggered by pathogens-recognition receptors (PRRs) are meant to identify microbial components - termed pathogen-associated molecular patterns (PAMPs) – which are present in all microbes of a given class but absent from the host (51). Two classes of PRRs include the membrane-associated Toll-like receptors (TLRs) and the cytoplasmic nucleotide-binding oligomerization domain-leucine-rich repeat receptors (NLRs). Toll-like receptors are one of the most studied PRRs expressed by intestinal epithelial cells helping in the preservation of a vigorous intestinal barrier; their activation by microbial molecules such as lipopolysaccharide and flagellin induce epithelial cell proliferation, immunoglobulin A (IgA) production, tight junction’s maintenance and antimicrobial peptide production (52). Additionally, other NLRs, termed NOD1 and NOD2, are involved in providing protection against Gram-positive and Gram-negative bacteria by activation of NF-κB and MAPK, which upregulate transcription and production of inflammatory mediators - including cytokines, chemoattractants, adhesive molecules, and inducible molecules (53,54). Yersinia inhibits NF-κB and MAPK signaling during infection of macrophages, inducing caspase-1-independent apoptosis (55) Y. pseudotuberculosis also exploits the intestinal mucosal inflammatory response by subversion of the Nod2 pathway to promote its dissemination and establish disease (56,57). Strikingly, Y.

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pseudotuberculosis is also able to trigger NF-κB activation by an NOD1/2 and caspase-1 independent mechanism underlining the potentialities of the bacteria in subverting the host immune response (58). An intact Yersinia type III secretion system (T3SS) is critical for host signaling modulation and immune response subversion (59,60).

1.6. Type III secretion systems (T3SS) in Gram-negative bacteria

The T3SS is a virulence mechanism found in Gram-negative bacteria with a variety of lifestyles and niches that range from pathogenic for animal and plants as well as symbiotic bacteria (61-64). Gram-negative bacteria utilize several type III secretion systems to transport a selection of virulence factors across the outer membrane. This acquired complex molecular pump guarantees (energized by ATP hydrolysis) the export of bacterial proteins to the extracellular milieu, host cell membrane and cytosol of an infected host eukaryotic cell (65-67). Through this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease (59,68). The most extensively studied T3SSs are found in the human pathogens Yersinia spp., Shigella spp., Salmonella spp., Pseudomonas aeruginosa and E.coli spp. (69-74) and in the plant pathogens Ralstonia solanacearum, Erwinia spp., Xanthomonas spp. and Pseudomonas syringae (64,75). T3SSs are used for different purposes in different bacteria. For instance, the intracellular bacteria Shigella, Salmonella and Chlamydia (69-71) use T3SSs to evade and multiply within the host. On the other hand, Yersinia spp. T3SS helps the bacteria to be extracellular by evading immune cells phagocytosis (76). The plant pathogens also use T3SS to counter plant defenses and release of plant nutrients essential for multiplication and colonization (64,75). Interestingly, T3SSs are not constrained to pathogenic bacteria. Plant and insect symbiotic bacteria also utilize T3SS to interact with the symbiotic partner underling the non-virulence and more ancestral role that T3SSs potentially contain (62).

Yersinia T3SSs are not the only bacterial virulence strategy required to cause disease. For example, Y.pestis utilizes F1 capsular protein to evade clearance from the infectious site (2). Yersinia LPS is utilized to group Y. pseudotuberculosis and Y. enterocolitica in different serotypes according to O-antigen variation, but more importantly to ensure the proper expression and functionality of some virulence factors located in the bacterial outer membrane (77). Ultimately, the two non-fimbrial adhesins, invasin and YadA, the fimbrial pH6 antigen and the outer

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membrane protein Ail mediate Yersinia precise outer membrane architecture, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms (78-80).

1.6.1. Variability among T3SSs

Whole genome-sequencing programs and phylogenetic analysis revealed that

T3SSs are present in all the four branches of proteobacteria (α, β, ϒ, and δ) (62,66,81,82). In addition, T3SSs were also identified in Chlamydiae phylum, including the environmental species that infect amoeba, suggesting that these bacteria might possess the most ancestral T3SS. This idea is reinforced by the observation that Chlamydiae type III secretion (T3S) genes are dispersed throughout the genome, and not localized in one large locus as is often seen in proteobacteria (62,83). Interestingly, evolutionary trees based on 16S RNA sequence or T3SS gene sequence are remarkably different, indicating that T3SSs have been recently acquired by lateral gene transfer (discussed in section 1.6.2.) (84-87). T3SSs phylogenetic trees also provide evidence that T3SSs have evolved into seven different families (Table 1). Despite sequence similarities between components of translocation associated-T3SSs and those of the flagella assembly machineries, these systems appear to be restricted at the moment to two bacterial phyla - the proteobacteria and the Chlamydia - in contrast with the flagellar-T3SS which has now been found in six different bacterial phyla (62). Hence, widespread bacterial genome samples among the current sequencing genome projects may yield novel translocation associated-T3SSs outside of the over-represented proteobacteria phyla.

Nevertheless, these observations raise several fundamental questions on how T3SSs have evolved. Why are T3SSs also found in pathogens with the ability of targeting different hosts? Could this have to do with the peculiarity of some pathogens, which could harbor more than one T3SS generally from different families? Does possessing different families of T3SSs give an evolutionary advantage to a specific pathogen over others? Could bacteria extend its environmental niche by the use of multiple T3SSs? These are significant questions that underline how far we are from the full understanding of T3SSs origin and acquisition.

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Table 1 - List of the seven families of T3SSs based on sequence homologies and phylogenetic analysis.

Family Species System Description Taxon

Ysc

Pathogenic Yersinia spp. Ysc

Block phagocytosis,

cytokine expression

and apoptosis

ϒ-proteobacteria

Pseudomonas aeruginosa Psc

Aeromonas salmonicida Asc

Photorhabdus luminescens Lsc

Vibrio parahaemolyticus Vsc

Bordetella pertussis Bsc β-proteobacteria

Desulfovibrio vulgaris Dsc δ-proteobacteria

Inv-Mxi-Spa

(SPI-1)

Salmonella spp. SPI-1

Trigger bacterial

uptake in non-

phagocytic cells

ϒ-proteobacteria

Shigella flexneri Inv-Mxi-Spa

Yersinia enterocolitica Ysa

Sodalis glossinidius Inv-Spa

E. coli (EIEC) Eiv-Epa

Yersinia ruckeri Inv-Mxi-Spa

Burkholderia pseudomallei Bsa β-proteobacteria

Chromobacterium violaceum Inv-Spa

Ssa-Esc

(SPI-2)

Y. pestis ? ?

ϒ-proteobacteria

Y. pseudotuberculosis ?

E. coli (EPEC) Esc Attaching and

effacing lesions E. coli (EHEC) Esc

Salmonella enterica SPI-2 Intracellular survival

and dissemination Citrobacter rodentium Ssa

Edwardsiella tarda ? ?

Chromobacterium violaceum ? β-proteobacteria

Hrp1

Pseudomonas syringe Hrp1 Trigger

hypersensitive

response in resistant

plants and disease in

non-resistant plants

ϒ-proteobacteria Erwinia spp. Hrp1

V. parahaemolyticus Hrp1

Hrp2

Xanthomonas campestris Hrp2 ϒ-proteobacteria

Burkholderia pseudomallei ? β-proteobacteria

Ralstonia solanacearum Hrp2

Rhizobium Rhizobium spp. ?

Plant symbiosis α-proteobacteria Mesorhizobium loti ?

Chlamydiales Chlamydia trachomatis ? Intracellular survival

and pathogenicity Chlamydiaceae

Chlamydia pneumoniae ?

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1.6.2. Acquisition of the T3SS

All the seven T3SS families not only share high homology among structural and functional components, but also conserve a similar export and regulatory mechanism. Although they clearly have different functions, translocation associated-T3SSs and flagella-T3SSs systems also overlap in many features and mechanisms such as: export apparatus assembly, substrate recognition and secretion of paralogs proteins (88,89). Therefore, it is tempting to ask which system came first? We know there are three possible answers to this question, yet we do not know which one of the following is the correct: First, flagella system came first; second, T3SSs came first; or third, both have common ancestry. Flagella systems are present in a wider variety of bacteria including Gram-positive where T3SSs are absent; T3SSs function is to exclusively promote pathogenic or symbiotic associations with eukaryotic organisms - eukaryotes appeared latter than bacteria and motility is essential for survival of planktonic marine microorganisms, are all arguments supporting the appearance of flagella systems first (67,90,91). In support of T3SSs coming first, endosymbiont (environmental) and pathogenic Chlamydia are non-motile bacteria that harbor an intact T3SS (92). Finally, the hypothesis that T3SSs and flagella systems have evolved from the same precursor is supported by phylogenetic analysis of the most conserved T3SS and flagella paralogs, which indicate an ancient diversion as far as hundreds of million years ago, and much earlier than the appearance of the first multicellular eukaryotes (93). Researchers supporting each of the three views devoted much of their time and efforts trying to answer and ultimately to convince the scientific community of the validity of their arguments. However, until now, no definitive or consensual answer was achieved.

T3SSs are frequently encoded in chromosomal pathogenicity islands (PAIs) or less often in transferable plasmids, both of which can be mobilized by lateral gene transfer to other Gram-negative bacteria (87). T3SSs encoded on dedicated virulence plasmids, can occasionally be integrated into the bacterial chromosome by a typical bacterial conjugative mechanism. PAIs are large genetic clusters encoding virulence traits which play an important role in the evolution of pathogenicity. These large genetic blocks (up to 200 kb of DNA), posses an uncommon ‘G’ and ‘C’ molecular content compared with the rest of the chromosomal DNA, which are usually flanked by phage integrase genes homologous, direct repeats, insertion sequences or even plasmid origins of replication (85). On other occasions, the DNA is transferred by bacteriophage transduction or natural DNA transformation.

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1.6.3. Couple heterogenic bacterial T3SSs to its environment

Many Gram-negative bacteria during their course of evolution did not acquire any translocation associated-T3SS. Some have however integrated one or multiple T3SSs from the same or even different families providing additional capacity to interact with different hosts or to colonize different niches within the same host (Table 1) (81). These exquisite evolution mechanisms have been insulated from others systems already present in the pathogen, by specificity of substrate recognition and temporal, spatial regulatory control and so that they only function when and where required. One of the most studied examples of T3SS dual carriage are the SPI-1/SPI-2 T3SSs from Salmonella enterica. SPI-1 (Inv-Mxi-Spa family) is important for the bacteria to trigger invasion and penetration of the gastrointestinal epithelium in the initial stage of salmonellosis. SPI-2 (Ssa-Esc family) is required for bacteria survival and replication inside the host phagocytes follow by spreading to neighboring cells in later stages of infection (86,94-96). All the three pathogenic Yersinia species encode the Ysc-Yop T3SS. In addition to this, Y. enterocolitica posses the chromosomally encoded Ysa T3SS (Inv-Mxi-Spa family), implicated in bacterial virulence and colonization of the intestinal epithelium (97,98). In contrast, the Ssa-Esc family T3SSs harbored by Y. pestis and Y. pseudotuberculosis have no determined function. Remarkably, Burkholderia spp. has acquired three T3SSs by lateral gene transfer. It not only harbors the Bsa T3SS (Inv-Mxi-Spa family) essential to invade the host cell, but also two Hrp-2 T3SSs which are exclusive for plant-pathogens. Although they have an unclear function, the presence of this family of T3SSs on a, until now, strict animal pathogen might indicate that this pathogen can establish additional contacts with diverse hosts (99,100)

1.6.4. Conserved features of type III secretion systems

An extensive number of vertebrate and invertebrate-interacting Gram-

negative bacteria harbor macromolecular structures called type III secretion systems (T3SSs). These highly complex machineries consist of more than 25 components that span both bacterial membranes, while a needle-like structure protrudes out from the bacterial surface. This structure anchors onto the host cell membrane, forming a translocon-pore through which anti-host virulence factors pass en route to the host cell cytosol (65,101). These synchronous processes may require a temporal control of secretion by a substrate switch control mechanism in the base of the T3S apparatus. Expectedly, components of the external needle

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(early substrates) must be exported prior the translocator proteins (intermediate substrates) which precede the translocated effectors (late substrates).

Notably, translocation-associated T3SSs core proteins share significant structural similarity with components of the flagellar-T3SS, which exports mainly components of the flagellum - such as hook and filament components (102,103). Although the secretion of non-flagellar substrates by the flagellar-T3SS was reported in vitro (104,105), both systems have different transcriptional regulatory mechanisms (102). Although translocation associated- T3SSs and flagellar-T3SSs are two distinct export apparatus, in the context of host-pathogen interaction a translocated-associated T3SS is often referred to as ‘T3SS’.

1.6.4.1. OM ring components

Outer membrane rings are built by proteins belonging to the secretin protein

family not only found in T3SSs, but also in other secretion systems including type II and type IV secretion systems (106,107). In Yersinia spp. the YscC protein is a sec-dependent translocation protein which possesses a C-terminal membrane spanning domain that is responsible for integration and oligomerization of the protein into the bacterial outer membrane (OM) (Figure 1A) (108,109). With the assistance of the YscW lipoprotein, YscC forms a multimeric ring with a diameter of approximately 11 nm (110,111). The N-terminal domain is thought to form a periplasmic neck which connects the OM ring to the inner membrane (IM). The oligomerization and consequent channel formation is facilitated by small OM lipoproteins called ‘pilotins’ (112,113). In the flagella-T3SS the OM ring is formed by the FlgH lipoprotein - the homologue of YscC - which serves as an insulator for the flagellar rotating rod (114).

1.6.4.2. IM ring components The YscJ and YscD family of lipoproteins are able to form two rings in the

bacterial IM commonly called the MS (membrane and supramembranous) ring. The YscJ multimeric ring anchors into the periplasmic side of the IM through the YscJ amino-terminal sequence, while YscD C-terminal periplasmic domain and N-terminal cytoplasmic domain shape a multimeric ring which interacts - not only with the YscJ ring - but also with the OM YscC ring (Figure 1B) (108,115-119). EscJ and EscD the YscJ and YscD homologues in EPEC are able to interact with the needle subunit protein EscF, indicating that the T3S needle might transverse the bacterial periplasmic space (120).

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1.6.4.3. Periplasmic rod structure

The periplasmic rod structure is needed to connect the IM rings to the OM channel. Additionally, it might also serve as an anchoring and stabilization point for the T3S needle which extends into the periplasmic compartment (121-123). In Yersinia spp. multiple copies of the YscI protein are predictably forming this structure (Figure 1C), in contrast with the flagella-T3SS which consist of four different components (e.g. FlgB, FlgC, FlgF and FlgG) that build-up the structure (124). An open question in the rod assembly mechanism from translocation associated-T3SSs, is how can YscI perforate the peptidoglycan layer in order to assemble these high-molecular structures complexes in the periplasmic space. Although, it has been previously shown that the C-terminal domain of the FlgJ flagellar protein assists in peptidoglycan degradation by its muramidase activity (125,126), no evidences of such activity in FlgJ T3S homologues was observed.

1.6.4.4. The needle complex

Transmission electron microscopy (TEM) has been a powerful tool to

comprehensively visualize needle complex structures derived from S. typhimurium, S. flexneri and EPEC (127-129). This cylindrical structure similar to the flagellum basal body, is composed of two pairs of rings embedded in the inner and outer membranes connected by a rod that transverses the periplasmic compartment (Figure 1D). This hollowed structure is connected to the protruding needle that projects from the bacterial surface forming a complete needle complex (65,66,102). In Yersinia spp. the needle is a hollowed tube with an extension of approximately 60 nm in length and is built by a helical polymerization of about 100-150 units of the monomeric YscF (9kDa) protein (117,130). Although substantially bigger, the flagellar hook protein (flagellin – 45 kDa) displays a similar helical organization as the T3SS needle (131). In Y. enterocolitica T3SS, needles have a length of 58 nm ± 10 nm (132) similar to the 45nm of Y. pestis (132). The Y. pseudotuberculosis needle length was not experimentally determined. The S. typhimurium T3SS needle has a length of 80nm for SPI-1 and 150nm for SPI-2 contrasting with the S. flexneri 45nm (127,129,133). In Yersinia spp. the T3SS needle length is controlled by the so-called ‘molecular ruler YscP protein’ by two different mechanisms. In the ruler model, YscP enters the channel after the basal body is completed, followed by export and sequential polymerization of YscF subunits until the needle assembly is completed. The C-terminal domain of YscP switches the substrate specificity to translocators secretion mode, followed by YscP

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secretion (132) (additional substrate switch mechanisms are discussed in section 1.6.4.6). This rule might not be applicable to all the T3SS harboring pathogens, given that structural analysis of S. flexneri T3SS needles revealed a stoichiometric impossibility for subunits and molecular ruler coexistence in the growing needle channel (134). In the alternative ruler model described for S. flexneri, Spa32/YscP and MxiH/YscF travel alternately through the needle complex. The ruler checks the needle length continuously and when the exact length is reached, YscP engages the C-terminal domain of YscU/Spa40 triggering substrate switch specificity from needle components to translocator and effector proteins (135). Similarly to the ruler model, the flagella assembly cup model is constructed on the bases that FliK acts the as a molecular ruler for flagellin subunits assembly followed by substrate specificity switch (136).

High resolution pictures obtained by cryo-EM microscopy of purified S. flexneri needle complexes revealed that the channel that extends from the inner-ring to the tip of the needle (a diameter of about 2-3 nm). These observations support the idea that T3SS substrates have to travel through the needle complex in an unfolded or partially folded manner (137). Jin Q and colleagues have elegantly demonstrated that a Hrp-pilus assembled by the P. syringae T3SS, functions as a conduit for protein delivery across the two bacterial membranes (138).

1.6.4.5. T3SS energizer

Secretion of a substrate through the T3SS to the extracellular space requires prior unfolding of the protein by an energy depended process. In Yersinia spp. a member of the YscN family of ATPases (InvC/Spa47/EscN), aids all the T3SS recognized components to travel along the needle complex (Figure 1E) (139). This conserved T3SS family of proteins, needs to oligomerize in different stoichiometry (homo- or double hexameric rings) and establish contact with the peripheral cytoplasmic side of the IM to increase its ATPase activity, providing energy for the secretion process (140-144). Different states of oligomerization can be induced upon contact with cytoplasmic chaperones triggering simultaneous dissociation of T3S substrates from their cognate chaperone (145,146). Moreover, timely unfolding and sequential ATPase-dependent export of unfolded or semi-folded substrates through the narrow ATPase complex (2-3nm) and subsequently T3SS needle seems fundamental for accurate secretion (137,145). This is an interesting additional T3SS recognition mechanism, which raises the question whether different ATPase oligomeric states might alter the multimeric protein affinity for

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different classes of T3S chaperones, creating a timely controlled hierarchical secretion mechanism.

1.6.4.6. T3S export apparatus and subtract recognition The MS ring of the needle complex accommodates in its interior the T3S

export apparatus essential for recognition and timely export of T3S substrates. This transport channel for secreted proteins is composed of five different transmembrane proteins (YscR, YscS, YscT, YscU and YscV), which create a heterogeneous protein complex that spans the bacterial IM (Figure 1F) (147-149). In flagellar-T3SS, these proteins include the family members of the FliP, FliQ, FliR, FlhB and FlhA respectively. In addition to forming an export conduit, two of its components (YscU/FlhB and YscV/FlhA also described as LcrD) are also involved in controlling T3S substrate switch. An additional mechanism of T3S substrate switch different from the YscP-controlled (see section 1.6.4.4), takes place when T3SS substrates are recognized within the export apparatus by the cytoplasmic domain of YscU/FlhB family of proteins (147,150). Upon autoproteolitic cleavage between the asparagine and proline residues from the conserved C-terminal NPTH motif, secretion of translocator proteins is activated whereas effector proteins secretion is blocked (151-155). Recently, Diepold A. and co-workers confirmed that in Y. enterocolitica YscV (aided by the two cytoplasmic proteins YscX and YscY) contributes for substrate recognition. Upon YscX release from the YscX-YscY-YscV complex, the YscY-YscV complex might recognize the subsequent category of substrates, adjusting the T3S from needle components to translocators secretion mode (156). A cytoplasmic sorting platform important for substrate recognition was also found in S. enterica serovar Typhimurium. This complex of proteins made by the C-ring protein SpaO/YscQ and two YscN associated proteins OrgB/YscL and OrgA/YscK revealed different affinities for different classes of T3SS-associated chaperones - implying that a hierarchical secretion of early (T3S structural components) before intermediate (translocon associated) and ultimately late (effector proteins) T3SS substrates - might be controlled by this platform (157). Considering that protein homologues of SpaO, OrgB, OrgA and YscU can be appreciated as substrates for the Ysc-Yop and SPI-1 families of T3SS might indicate that these subtract recognition mechanisms can also be shared by different pathogens harboring distinct T3SS families. However, no homologues are described

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Figure 1 – Structural building-blocks for the Ysc-Yop T3SS. (A) Outer-membrane ring formed by an YscC multimeric channel. (B) Inner-membrane ring shaped by two multimeric YscD and YscJ rings. (C) Periplasmic rod structure produced by multiple copies of YscL connecting the OM channel and the IM rings. (D) Needle complex assembled from an IM base (ring structures in IM and OM are not shown for clarity) and adjoins with an YscF-polymerized extracellular appendage. (E) T3SS energizer is assembled as an YscN ATPase oligomeric ring(s) serving as energy source for the transport of the proteins through the T3S apparatus. (F) Export apparatus formed by five transmembrane proteins (YscR, YscS, YscT, YscU and YscV) involved in substrate docking and secretion control of T3S substrates.

for YscX or YscY implying that his recognition mechanism might be costume made to Yersinia spp.

In summary, upon all the machinery building-blocks are assembled as a functional T3S (Figure 1), the bacteria will respond to specific environmental signals secreting translocators and effector proteins through the polymerized needle (see section 1.9 and Figure 5). This secretion process is highly controlled by precise regulatory mechanisms (see section 1.8)

1.6.4.7. T3SS chaperones classes

Efficient secretion of T3S substrates is promoted by numerous specialized

chaperones. These small, (less than 20 kDa) acidic molecules have a propensity to engage a specific or several T3S substrates to form transient complexes in the bacterial cytosol. According to the function of their T3SS substrates and chaperone

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structure, chaperones can be grouped into three different classes (Table 2). The class I T3S chaperones bind effector proteins that are translocated to the host cell interior whereas class II T3S chaperones target translocator proteins involved in translocon pore formation. The third class engages distal components of the needle complex assisting in their secretion through the emerging T3SS (158-161). The main functions that have been proposed for T3SS chaperones are: i) ensure stabilization of the substrate and prevent unspecific intra- and intermolecular interactions (162), ii) maintain the substrates in a secretion-competent status piloting them to the T3S substrate recognition site (163,164), iii) uphold a secretion hierarchy given by the three dimensional structure of the chaperone-substrate complex (165), iv) particular class I and class II T3S chaperones have an additional role in regulation of T3S genes expression (166-168) (see section 1.8.1) (Paper IV).

The class I T3S chaperones are a class of small acidic proteins (~10-15 kDa) with low sequence homology but similar structural conformation and requirements for dimer assembly (161,169). They can be further classified according to their affinity to one effector (class Ia) (Table 2) or to several effector substrates (class Ib). Although with low homology in amino acid sequence both classes share similar quaternary structures. The α/β fold topology in a homodimer conformation ensures that the effector substrates wraps around the chaperone surface in a non-globular extended conformation (169-171). Previously, it was thought that the dimerization interface heterogeneity displayed by class Ib chaperones was the reason why they can engage multiple T3S substrates, however recent data shows that class Ib chaperones are not promiscuous but rather recognize a conserved effector sequence (172). The Ysc-Yop system does not utilize class 1b chaperones.

The class II chaperones are slightly larger proteins (15-20 kDa) encompassing in its structure a triad of tetratricopeptide repeats (TPRs) (173). These particular motifs adopt a helix-turn-helix conformation where the canonical small and large residues enable the opposite helices to interlock to form a versatile scaffold that mediate protein-protein interactions (174-176). Unveiled structures from the class II T3S chaperone SycD from Y. enterocolitica (177,178) (LcrH in Y. pseudotuberculosis) and its functional homologue IpgC from S. flexneri (179) revealed also a homodimeric conformation with a potential concave and convex surfaces available for substrate binding. This indicates that these interacting patches may be relevant for simultaneous targets of the two cognate hydrophobic substrates (YopB/YopD in Yersinia spp. or IpaB/IpaC in Shigella spp.) preventing their premature binding (Table 2) (see section 1.9.1.2.4) (180). However, the two

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Figure 2 – Cartoon representations of the homodimeric arrangements of SycD (LcrH) and IpgC T3S chaperones. SycD (LcrH) structure (PDB: 2VGX) displays a head-to-head homodimer orientation versus the asymmetrical orientation viewed in the IpgC homodimer structure (PDB: 3GYZ). The monomers in the homodimers complexes are colored in blue and brown. Structures were rendered using the PyMOL program.

structures display different dimeric structural arrangements. While SycD exist as a head-to-head homodimer, IpgC exists as asymmetrical homodimer (Figure 2) (161).

The class III chaperones consist of a biochemically heterogeneous group made up of the remaining T3S chaperones. In Yersinia spp., the heterodimeric T3S chaperone YscE/YscG lies in this class (Table 2). This complex targets the monomeric needle protein YscF preventing premature polymerization of the protein in the cytosol of the bacteria prior to the assembly of the needle (181). Although with no structure determined, it was proposed that LcrG could be the chaperone of the needle tip protein LcrV (Table 2). This was based on the observation of low levels of LcrV being secreted in the absence of LcrG (182). However, LcrG has associated important regulatory roles in the T3S context even in the absence of LcrV, arguing against a role solely as a T3S chaperone for LcrV (183). Thus, a definitive classification for this putative chaperon is still unknown. In addition, the YscY T3S chaperone that pilots the T3SS component YscX for secretion can also be tentatively grouped in this class although it is predicted to consist of TPRs (tetratricopeptide repeats), analogous to class II T3S chaperones (Table 2) (184,185).

1.7. Plasmid encoded Ysc-Yop T3SS in Yersinia The T3SS of Yersinia spp. is encoded in large extrachromossomal virulence

plasmid (~70 Kbp) denoted as pCD1 for Y. pestis, pIB1 for Y.pseudotuberculosis and pYV227 for Y. enterocolitica (68). Each of these plasmids encodes approximately 50 genes required for a functional Ysc-Yop T3SS. Mutations in most of these genes will have an impact on the survival capacity of the bacteria within the host (68). The

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Table 2 - Yersinia spp. plasmid encoded T3S chaperones and their cognate substrates.

Class Chaperone Crystal structure Suggested function

Secretion substrate References

Ia SycE (YerA)

Yes (alone and in complex with

YopE)

Masking YopE membrane localization

domain

YopE effector (186-195)

Ia SycH Yes

(in complex with YscM2)

Secretion regulation

YopH effector & LcrQ (YscM1

& YscM2) negative regulator

(165,190,195-198)

Ia SycO No

Masking YopO membrane localization

domain

YopO (YpkA) effector (162,199)

Ia SycT Yes (alone) Masking protease activity YopT effector (200-202)

Ia1 SycN and YscB

Yes (in complex with

YopN) System regulation YopN

regulator (203-207)

II SycD (LcrH)

Yes (alone and in

complex with a YopD peptide)

Stabilizer (partitioning

factor) System regulation

YopB & YopD translocators

(168,173,177,180,192,195,

208-212)

III YscE and YscG

Yes (in complex with

YscF)

Anti-polymerization YscF needle (181,213,214)

nd LcrG No System regulation LcrV needle tip protein (182,183)

nd YscY No Secretion hierarchy YscX (184,185,212,

215) nd – not determined 1 The two chaperones form a heterodimer complex resembling class Ia structural classification

genes encoding for structural and translocator proteins are generally organized in large polycistronic operons, in contrast with effector and regulatory proteins which are normally encoded in monocistronic operons. In the Y. pseudotuberculosis pIB1 plasmid there are about 40 genes necessary for the bacteria to efficiently translocate anti-host factors to the target cells. These can be organized in four different functional classes: i) ysc (Yersinia secretion) genes encoding structural proteins which built-up the T3S apparatus, ii) translocator genes encoding for proteins involved in translocon assembly, iii) yop genes (Yersinia outer proteins) encoding anti-host effector proteins, and iv) lcr (low calcium response) regulatory genes whose products control gene expression. In addition, other genes are involved in partition and replication of the pIB1 plasmid and a non-functional

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truncated effector gene (216). Only a strict spatial and temporal regulatory control of the different gene classes in response to environmental cues, will allow a successful infection and colonization of the host.

1.8. Environment and regulatory control of Ysc-Yop T3SS substrates

The temporal and spatial expression of virulence determinants encoded in the

Yersinia spp. virulence plasmid is determined by environmental cues. Specific extracellular signals sensed by the bacteria trigger a massive (but highly controlled) T3SS response. This chapter will elucidate the environmental signals sensed by Yersinia and the corresponding genetic mechanisms used to regulate the expression of Ysc-Yop genes.

1.8.1. Temperature and transcriptional control

At any given moment of the Yersinia spp. life cycle, bacteria can contact with

animal hosts to encounter an upshift of temperature to 37 °C. In Yersinia spp. this thermoregulation mechanism is controlled by the master T3SS transcription activator LcrF (VirF in Y. enterocolitica), an AraC transcriptional activator family member. The LcrF protein includes a poorly conserved N-terminal oligomerization domain connected to a C-terminal helix-turn-helix motif that functions as a DNA-binding domain. This positive activator binds to the lcr, ysc and yop genes promoters, activating its transcription at temperatures above 30 °C (217-219). Although some lcrF transcripts can be generated at 26 °C, its mRNA cannot be translated unless the temperature rises to 37 °C. Recently, Bohme and colleagues unveiled the cause of such post-transcriptional inhibition. Indeed, a protective two-stemloop structure within the intergenic region upstream of the lcrF gene and downstream of the yscW gene melts at 37 °C, alleviating the ribosomal binding site facilitating lcrF mRNA translation (Figure 3) (219,220). An additional thermoregulatory mechanism occurs at the level of transcription. For temperatures below 30 °C, the YmoA (nucleoid-associated protein) binds to T3S promoters stabilizing its intrinsic DNA bended architecture (221,222). However, upon host entry or temperature upshift to 37 °C, the DNA structure changes and the YmoA affinity for the DNA decreases and is rapidly degraded by the ClpP and Lon proteases generating an enhanced transcriptional output from T3S genes (Figure 3) (223). A similar thermo-modulated mechanism has been observed for E. coli and Shigella spp. (224). If in E. coli, the YmoA homologue Hha, represses the transcription of the hlyCABD operon encoding the pore-forming hemolysin only at

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Figure 3 – Thermoregulated expression of the Ysc-Yop T3SS transcriptional activator LcrF. At temperatures below 30 °C, YmoA binds to sequences downstream of the transcription initiation site stabilizing intrinsic curved DNA structure and preventing targeting of RNA polymerase. Additionally, translation of the lcrF transcript is prevented by the formation of a complex-stemloop structure located upstream of the lcrF gene which prevents access of the ribosome to the RBS. Upon temperature upshift to 37 °C, DNA architecture changes impairing YmoA affinity promoting its rapid degradation by the ClpP and Lon proteases leading to an enhanced transcription of the yscW-lcrF operon. Thermally-induced melting of the stemloop frees the RBS promoting translation of the lcrF transcript and further LcrF synthesis. Free LcrF molecules enhance transcriptional output of yop genes possibly with the cooperation of the LcrH T3S chaperone. Schematic model adapted from Bohme and colleagues (220).

low temperatures (225), in Shigella spp., H-NS (universal nucleoide associated protein) represses the expression of T3S genes by binding to the promoter region of the transcription activator virF below 32°C (226,227). Although YmoA was shown to interact and form heterodimers with H-NS, a cooperative binding to ysc or yop DNA promoter regions has never been reported.

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Two additional transcriptional regulatory mechanisms occur upon T3S activation. They involve the interaction of a T3S chaperone that acts either as: i) a coactivator of a transcriptional regulator or ii) an anti-antiactivator sequestering the bound antiactivator from the transcription regulator. As an example of the first mechanism, SicA and IpgC elicit their regulatory effect after its substrate secretion, allowing free chaperone to act as a positive cofactor of their respective AraC-like transcriptional activators InvF (166) and MxiE (167,228). In Yersinia spp. this mechanism has not yet been described. Therefore, we investigated whether LcrH (SicA and IpgC homologue) possesses a similar cofactor function for LcrF (Figure 3) (Paper IV). In P. aeruginosa, effector gene expression is repressed by the activator ExsA bound to the antiactivator ExcD. However, upon T3S activation, the chaperone ExsC is released from its ExsE substrate permitting the interaction with the antiactivator ExcD leading to liberation of ExcA which subsequently activates T3S gene expression (229). 1.8.2. Calcium dependency and post-transcriptional control

It has been known since the mid 1950’s that Yersinia spp. require the presence of calcium in the media for normal growth at 37 °C (230). This behavior was later described as the calcium dependent (CD) phenotype (231). Notably, calcium depletion from the media induces bacterial growth arrest at 37 °C, while T3SS-mediated expression and secretion of Yops is activated (232). This poorly understood phenomenon is named low calcium response (LCR). It is an intriguing in vitro regulatory mechanism which was often used as a toolkit to identify and characterize many T3S-mediated regulatory genes (233). Indeed, mutations made in genes harbored in the virulence plasmid created three different types of LCRs that could be measured by bacterial growth. The first were a set of mutations that did not affect bacterial growth at 37 °C maintained the CD phenotype (72,234,235). A second set of mutations that permitted bacterial growth at 37 °C (independently of presence or absence of calcium) induces a CI (calcium independent) phenotype. These mutations usually induce a deficient T3SS assembly which hamper antiactivators secretion, generating a constitutively repression of Yops production (see section 1.8.3). In addition, CI strains are likely to be defective in a T3S positive activator such as LcrF (236,237). A final set are mutations that induce growth arrest at 37 °C (independently of presence or absence of calcium) generate a TS (temperature sensitive) phenotype. These strains possess a constitutive activation of Yops production even in non inducing conditions (presence of calcium), most likely due to a defect in the negative regulator synthesis or function (PAPER I)

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(212,238-241). Whether calcium depletion from culture media mimics an in vitro condition where bacteria encounter a host cell is unclear. What we do know is that the levels of calcium outside of the eukaryotic cells are higher than in its interior and an activation of Yops production upon host cell contact are more mild than triggered by calcium depletion. Although this artificial signal has proven useful to understand many regulatory aspects of Yersinia spp. regulon, this signal might be one of many that the bacteria senses upon host contact, which in isolation triggers a massive uncontrolled Yops production in vitro (242). Therefore, bacteria and host cell contact is an essential negative feedback control mechanism important for fine-tune secretion and translocation of Yops to the host cell interior (see section 1.8.3).

1.8.3. Secretion of antiactivators and post-transcriptional control In Yersinia spp. the expression of yop genes is activated by bacteria-host cells

contact coupled to the secretion of the antiactivator YopD from the bacterial cell in a T3S-dependent manner (168,212,243). In complex with its chaperone LcrH, YopD acts as a negative regulator of yop genes expression inside the bacterial cell by binding to the 5’-untranslated region (5’-UTR) of yops mRNAs at short AU-rich sequences (212,244). This repression mechanism presumably hampers the access of the ribosome to the Shine-Delgano sequence facilitating mRNA degradation by ribonucleases which accelerate the degradation of the untranslatable mRNA leading to impaired production of Yops by the bacteria (Figure 4) (245). Although these sequences are required for a YopD-dependent binding, they might not be an exclusive mechanism necessary for regulation. In addition to this regulatory mechanism, LcrH together with YscY might also have a regulatory role in suppression of yops expression by an alternative mechanism independent of YopD.

Post-transcriptional control of yops expression in Yersinia spp. requires an additional antiactivator LcrQ, which in Y. enterocolitica is the dimer composed by YscM1 and YscM2 (246,247). The negative regulatory element LcrQ, has to be maintained inside the bacteria to exert its negative regulatory effect (197,248). This effect is dependent on the presence of YopD and LcrH since a ΔyopD or ΔlcrH mutant display a derepression of Yops production even when LcrQ is overexpressed (PAPER IV) (212,243,249). Thus, yops mRNA translation might be modulated by a tripartite complex formed by YopD and its cognate chaperone LcrH in addition to the negative regulatory element LcrQ (Figure 4) (250). Although the idea of a tripartite complex has been put forward because the regulatory function of YopD and LcrQ are similar, that concept has never been proved experimentally.

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Figure 4 – YopD-dependent T3SS post-transcriptional regulation. In a repressed state, the complex formed by YopD-LcrH binds to short AU-rich sequences within the 5’-UTR of the yops mRNA hampering their translation. It is possible that another negative regulatory element LcrQ could function in this regulatory complex in Y. pseudotuberculosis. Upon host cell contact, disruption of the putative YopD-LcrH-LcrQ tripartite complex might be triggered by SycH, which guides LcrQ for secretion and thus promoting the release of the YopD-LcrH complex. LcrH can then direct YopD to the secretion apparatus promoting its secretion allowing the ribosome to initiate translation of yops mRNA.

When calcium is depleted or upon host cell contact, the destabilization of this complex is triggered by the binding of the SycH anti-activators (that is also a chaperone of the YopH effector) to LcrQ (165,198). This leads to a secretion of LcrQ (248) and presumably to a release of the YopD-LcrH complex from the 5’UTR of the mRNA, allowing the ribosome to start the translation process. YopD can finally be piloted by the LcrH chaperone to the T3SS apparatus in order to be recognized and secreted (Figure 4). In paper IV, we also describe efforts to clarify the mechanisms underlying the functional interplay between the action of LcrH and YopD together with LcrQ or its homologues YscM1 and YscM2 in the context of yops genes repression.

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1.8.4. Host-cell contact signal transduction and gating of the T3S machinery

Physical contact between the bacterial and the host cells triggers an activation of Yersinia spp. T3SS resulting on translocation of Yop effectors into the interior of the target cell (248). However, how bacteria senses host cells and transduces the signal to the bacterial cell cytoplasm is still not properly understood. In Shigella spp. is it has been suggested that the host cell is sensed by the needle tip complex composed of four molecules of IpaD (LcrV in Yersinia spp.) and one molecule of IpaB (YopB in Yersinia spp.) (251-254). Upon host cell contact, IpaB gets inserted into the host cell membrane, triggering a conformational change in the T3S needle resulting in secretion of the second translocator IpaC (YopD in Yersinia spp.) (252). IpaB in cooperation with IpaC form the pore allowing secretion and translocation of Shigella spp. effector proteins (255,256). To obtain a better understanding of how the signal is propagated from the host to the bacterial cytoplasm, Fujii and coworkers tried to structurally visualize needles from mutants unable to transduce such signals. Unfortunately, these predicted conformational changes were not preserved when needles were isolated from their base and tip complex (134). Consistently, signal transducing-deficient mutants in the Yersinia spp. YscF needle protein were also reported. These deregulated bacteria, were able to secrete virulence proteins in the presence or absence of calcium and prior to contact with eukaryotic cells (257,258).

Nevertheless, a conserved α-helical coiled-coil in both needle subunits and tip proteins suggests a similar mechanism of oligomeric assembly (251,257,259). Biophysical analysis of those structures demonstrate a potential for conformational amplification involved in mechanotransduction of a possible nanoswitch signal (260). Another model proposes that an ‘extracellular plug’ made by the needle complex proteins could act as a physical block of the T3SS and only cell contact could trigger a conformational change that could open the T3S channel (251,252). Lastly, the ‘intracellular plug’ model suggests that in Yersinia spp. a complex made by YopN-TyeA is partially inserted into the secretion channel blocking Yop effectors secretion before host cell contact (205,261,262). Upon host cell contact a conformational change in the T3S needle is transduced to the basal body dislodging TyeA from the complex, relieving the secretion of YopN followed by the Yop effectors (263). Although Yersinia spp. putative gate-keeping proteins are highly conserved in Salmonella spp., Shigella spp., and E.coli, it remains unclear whether this regulatory mechanism behind bacterial-host cell contact is functionally conserved among those pathogens. Remarkably, in the plant-pathogen R. solanacearum T3S genes expression are controlled via a three component system

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that transduces a contact-dependent signal to the bacterial cell interior (264). Therefore, the existence of a specific receptor-ligand interaction between a bacterial adhesin and a host cell receptor that could instigate a bacterial T3S-dependent response cannot be discarded.

In summary, all the above mentioned bacterial secretion control mechanisms, aim to spatially and timely control secretion and translocation of translocators and effector proteins via the functional polymerized needle.

1.9. Encountering the host cell – substrate secretion through the polymerized T3S needle Yersinia spp. T3SS can secrete a variety of substrates that essentially can be

divided into two different classes according to function and timing of secretion. The first group of proteins being secreted upon host cell contact are the translocator proteins. They are involved in the formation of the needle tip platform essential for translocon pore assembly in addition to integration in the host cell membrane (255,265-267). What follows is the secretion of the effector protein group, which interferes with signal transduction pathways that regulate the host actin cytoskeleton, phagocytosis and pro-inflammatory response (59,268). The secretion of these proteins groups via the polymerized T3S needle is assisted by small bacterial proteins termed T3SS chaperones essential for presecretory stability, secretion pilot (see section 1.6.4.7), regulation of T3S gene expression (see section 1.8.1 and 1.8.3) and substrate secretion recognition by the T3SS (see section 1.6.4.6) (Figure 5).

1.9.1. Translocator proteins – facilitators of effector translocation Activation of T3S initiates the assembly of a translocation complex, called

translocon, in the host cell membrane by translocator proteins. The Yersinia spp. translocator proteins LcrV, YopB and YopD (IpaD, IpaB and IpaC in Shigella spp.) are critical for translocon assembly and effectors passage to the host cell cytosol (261,269-271). All three proteins are encoded in the large lcrGVHyopBD polycistronic operon together with their cognate chaperones that pilot the proteins to the T3S apparatus (272,273). It is assumed that the hydrophobic translocators YopB and YopD form a translocon pore in the host cell membrane and the

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Figure 5 – Schematic representation of the T3SS from Yersinia. Upon bacteria-target cell contact, hydrophobic translocators are presumed to be secreted first after being directed to the T3S export apparatus by their cognate class II T3S chaperone. Following their integration in the host cell membrane to shape a functional translocon, effectors are secreted and they gain access to the host cell cytosol probably via the translocon pore. Effectors secretion through the T3S apparatus is facilitated by dedicated class I T3S chaperones.

hydrophilic translocator LcrV serves as a scaffold or assembly platform for translocon pore assembly (274,275). Although pore formation requires the presence of all three translocators, purified membranes from infected erythrocytes show the presence of only the two hydrophobic translocators (269,276,277). When the functional translocon pore is fully assembled, effector proteins are delivered to the host cell cytosol most probably through the translocon pore to elicite a host immune reaction (59,278).

1.9.1.1. LcrV – hydrophilic translocator LcrV (also known as V-antigen) is a soluble protein important for virulence and

is a protective antigen against plague (279). A tip complex built-up by the

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hydrophilic translocator LcrV makes the physical connection between the polymerized T3S needle and the translocon pore inserted in the host cell membrane. The LcrV tip complex was first identified by scanning transmission EM at the tip of purified needles from Y. enterocolitica grown on either non inducing (+Ca2+) or inducing (-Ca2+) secretion conditions, supporting the notion that the tip complex might have multiple proposes in Yersinia spp. pathogenesis (265). The observation of LcrV atop of T3S needles in non T3S inducing conditions (265) or before host cell contact (270), indicates that the complex can serve as a sensor for host contact. Indeed, the N-terminal domain of LcrV has been reported to engage both TLR2 and CD14 receptors from immune host cells, which could be a mechanism of host contact (280). In addition, tip complexes might also work as an assembly platform for the translocon since antibodies against LcrV prevent the formation of the translocation pore and block the delivery of Yop effectors (281,282). Tip-complexes can also serve to control secretion of the hydrophobic translocators prior to host cell contact (255). Although these ideas are supported by observations in Shigella spp., a ΔlcrV mutant in Yersinia spp. does not constitutively induce T3S, contrary to a deletion in the tip protein IpaD from Shigella spp. (253,283-286). This emphasizes that the tip complex from the two pathogens might have distinct roles in the control of T3S, although they share a conserved role in translocon assembly.

This disparity is also viewed in the tip complex morphology and stoichiometry from the two pathogens. In Yersinia spp. the tip complex is assumed to consist of five molecules of the LcrV protein that oligomerizes in vitro forming a ring-like shape with an internal diameter of about 3 to 4 nm that resembles the central size of the needle channel (274,287). This contrasts with biochemical observations from the Shigella spp. tip complex which contains one molecule of the hydrophobic translocator IpaB in addition to four molecules of IpaD (251,252,254,255,286). Although the tip complex shows variability among the two pathogens, the monomeric proteins share a common topology consisting on long central rigid coiled-coil with one globular domain on each end (265,288). Moreover, the presence of a coiled-coil region in the hydrophilic proteins (IpaD and LcrV) and in the respective needle subunits (MxiH and YscF) suggest a shared mechanism of assembly (289). Thus, a conserved scaffold function for the translocon assembly is possible.

The fact that in Yersinia spp. the hydrophobic translocator proteins are not detected in the tip of the needle before host cell contact and that the tip complex formation process proceed even in the absence of YopB or YopD in vitro, implies that they may only be secreted once host cell contact is achieved. Since interaction

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between LcrV and the two translocator partners YopB and YopD has been established, we can therefore envisage that integration and stabilization of YopB and YopD in host eukaryotic membranes might be facilitated by this complex in a dynamic assembly process (Paper I) (see section 1.10.1) (290).

1.9.1.2. YopD – multifunctional hydrophobic translocator The translocator YopD is a crucial protein involved in governing many steps of

Yersinia spp. type III secretion. It negatively regulates the synthesis of other T3S substrates (Yops) in conjunction with other regulatory proteins (LcrH and LcrQ) (165,243,244). The mechanism behind this negative regulatory loop is post-transcriptional, whereby the translation of mRNAs of specific Yop proteins is inhibited. Additionally, secreted YopD also forms a translocator pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol (291). The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. It is still not known how the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD and the second hydrophobic translocator YopB. In addition, YopD may associate with pores only transiently, since a portion of the molecule is localized in the cytoplasm of infected cell monolayers (see section 1.10.4) (249). Although it is difficult to conceptualize how YopD actually integrates and coordinates all these multiple activities, it does appear to possess a mosaic domain organization that may provide important insights how all these functions are coordinated to promote Yersinia spp. pathogenicity (292).

1.9.1.2.1. Domains organization and function Several studies have demonstrated the role of the 306-residue YopD protein

in the context of T3SS by pathogenic Yersinia spp. (243,249). Until now, structural determination of the full length protein has been hampered by difficulties in obtaining large quantities of soluble protein due to its high hydrophobic character (and perhaps because of a dynamic conformation). Consequently, the only limited structural data that are available at the moment are: i) an internal polypeptide of YopD that exists in a stable unfolded state (293) consistent with the requirement of extended conformation for secretion through the T3S needle; ii) the C-terminal amphipathic α-helix domain whose structure was solved by NMR (Figure 8) (209). Therefore, most of the genetic and biochemical approaches implemented to

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functionally characterize specific amino acids sequences of the protein, are a result of in silico predictions (271,291,294,295).

The YopD protein incorporates a likely chaperone-independent secretion signal in its extreme N-terminal domain. Although the secretion signal might not strictly be dependent on the protein sequence, its full secretion requires the presence of isoleucines at position 3 and 5. These two codons were a part of an absolute minimal secretion sequence incorporated in the first five residues of YopD (Figure 6) (292,296).

As initially revealed by in silico predictions and later confirmed experimentally, YopD possess an amphipathic α-helix domain in its C-terminal sequence (Figure 6) (249). This secondary structure is important for the full function of YopD. Notably, when the amphipathic α-helix coding sequence was deleted, the mutant was phenotypically indistinguishable from a ΔyopD mutant. The bacteria was unable to either translocate Yop effectors into the HeLa cell monolayers nor to translocate itself to the host cell interior, presenting an intriguing scenario where the YopD protein has to be translocated in order to polarize translocation of Yop effectors. This domain was also important in the pore forming ability of Y. pseudotuberculosis, as measured by hemoglobin release upon red blood cells (RBCs) infections. In addition to this, the negative regulatory function in the bacterial cytoplasm was also lost, probably due to impaired interaction with is dedicated chaperone (LcrH) yielding no capacity to engage yop mRNA transcripts. In summary, the YopD amphipathic α-helix domain is involved in multiple functions critical for Y. pseudotuberculosis Ysc-Yop T3S (292). Its role in such a diversity of T3S events created the need to scrutinize individual residues within this domain. Thus in paper I, we endeavored to identify particular residues within this domain that could specifically be linked to individual functions of YopD.

Two more α-helical domains that may be putative coiled-coil domains were predicted in YopD sequence; one downstream of the N-terminal secretion signal motif and the second immediately upstream of the amphipathic α-helix domain (Figure 6). Although with different degrees of probability (<70% for the first and 99% for the second), likely heptad-repeat sequences, a hallmark of coiled-coil motifs, were appreciated in the amino acids sequences of both segments (294,295). Initial insights in the function of these domains based on their deletion, revealed that the putative N-terminal coiled-coil (CC1) had an exclusively role in the translocation of Yop effectors to the cell cytosol, while the putative C-terminal coiled-coil (CC2) was critical for Yops translocation and pore assembly in RBC membranes (292). In paper II and paper III we therefore aimed to take advantage of these regulatory intact domains to introduce secondary structure disruptive

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Figure 6 – Schematic diagram of the 306-amino-acid YopD protein. The structural features include a minimal secretion sequence (SS) located within the first 5 residues of the N-terminus and two putative coiled-coil domains located at the N-terminus between residues 31 and 44 (CC1) and at the C-terminus between residues 251 and 274 (CC2). The predicted coiled coil regions were identified using the Coiled-coil webserver (http://npsa-pbil.ibcp.fr/). A putative transmembrane domain (TM) identified by the TMPRED webserver (http://www.ch.embnet.org/software/TMPRED_form.html) is located between residues 128 and 149. At the C-terminus lies an amphipathic α-helix that was initially identified between residues 278 and 292 by the helical wheel projections (Antheprot v3.2) and later confirmed in the NMR structure of a synthetic peptide incorporating this domain (209). Two chaperone binding domains, one located internally between residues 44 and 149 (CBD1) and the other between residues 278 and 292 mediate the interaction with the YopD (and YopB) cognate T3S chaperone LcrH (211).

point mutations to study in isolation the involvement of these predicted structures in the process of translocation and virulence.

A putative transmembrane (TM) domain is predicted in the central region of the protein (Figure 6) (271). Surprisingly, a deletion mutant encompassing this hydrophobic region maintains some capacity for pore formation without mediating effector translocation, indicating that YopD’s role extends beyond pore formation. This mutant also reveals a defective regulatory control phenotype in which Yops synthesis is constitutive even in repressive conditions (+Ca2+), which is corroborated by a deficient interaction with is dedicated chaperone. YopD seems to possess two chaperone binding domains (CBDs), which when disturbed causes Yersinia spp. mutants to uncontrol Yops production in suppressive conditions. The CBD1 spans from the predicted CC1 until the end of the TM region, while the CBD2 encompasses the C-terminal amphipathic α-helix (Figure 6) (292).

In summary, YopD is a multifunctional protein built-up by a mosaic of domains that have encrypted in its sequence information on how the protein controls regulation and secretion of Yops, pore formation and effectors translocation. Thus an in-depth structural-functional analysis of these particular domains in the future will help us to decipher the complex YopD molecular puzzle.

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1.9.1.2.2. Genetic and functional homologues in other T3S families The YopD translocator from Y. pseudotuberculosis is exceptionally conserved

among the other pathogenic Yersinia spp. species, exhibiting sequence identities of 99.7% to YopD from Y. pestis and >97.1% to YopD from Y. enterocolitica isolates. Notably, only one amino acid is changed in the 306 residues of Y. pestis YopD, consistent with the close evolutionary relationship between the two species that have diverged more recently than Y. enterocolitica. Consequently, a higher degree of heterogeneity is appreciated in the YopD sequence from Y.enterocolitica isolates in comparison to the Y. pseudotuberculosis homologue (Paper II). As a result, all the three YopD variants from Yersinia spp., retained indistinguishable functions both as a regulatory factor inside the bacteria or as a translocator that self-oligomerizes to form pores in host cell membrane (276).

Outside of the Yersinia genus, but in different bacterial genus also harboring an Ysc-Yop T3SS, the YopD-like proteins share some sequence and functional homologies. These sequences identities can vary from 49.3 % (A. veronii) to 27.8% (Pb. damselae) (Paper II). The YopD translocator and its P. aeruginosa homologue PopD, share a sequence identity of 41.3 % and one central putative transmembrane domain in addition to a putative C-terminal amphipathic α-helix (297). This level of identity makes possible to a Y. pseudotuberculosis strain lacking YopD to synthesized and secreted PopD even in the absence of its native chaperone (PcrH); however this did not restore the YopD-dependent yop-regulatory control or effector translocation. This suggests that key residues in YopD sequence, which are not conserved in PopD, are crucial for efficient Yersinia spp. type III secretion (298). However, the aptitude to oligomerize (299,300) and to form translocons in host cell membranes (277,301) and in liposomes (300) is fully conserved in PopD and in the majority of all the YopD homologues present in other members of the Ysc-Yop T3SS family (302,303). Indeed, the ability for translocon formation is the central characteristic of the all the YopD-like proteins being its regulatory functions accessory, deriving exclusively from specificities of the pathogen lifestyle.

More distant related are SipC and IpaC, two functional YopD homologues in Salmonella spp. and Shigella spp. that are part of the Inv-Mxi-Spa T3S family (291). Despite low amino acids identity with YopD (<15%), these large two proteins also share a conserved ability to interact with membranes and to form pores in eukaryotic membranes (267,304). Likewise YopD, IpaC also possesses a distinct functional domains organization that are important for S. flexneri to deploy its T3SS

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(305). Although IpaC N-terminus and the central immunogenic domain are not involved in IpaC-dependent invasiveness, its N-terminus is critical from IpaC secretion from the bacteria, as observed for YopD (292,305). The central hydrophobic region of the protein exhibit avidity for membrane phospholipids while its C-terminus that encompasses the putative coiled-coil is important for the protein to establish contacts with itself, resembling what is observed also for YopD (305,306). Although SipC and IpaC share biochemical properties that enhance bacterial entry into culture cells (304,307), interaction with phospholipid membranes (256,305,308) and oligomerization in vitro (305,306,309) - they differ in either the ability to be translocated into host cells or in their function of effectors of host cell invasion (310). Although, YopD can also self-assemble and is translocated to the cell interior, given the lifestyle of Yersinia spp., it is unlikely that YopD also promotes bacterial uptake. Thus, distantly related translocators maintain central functions, but have diverge sufficiently to evolve new functions tailored to the individual bacterial needs, such as invasiveness and intracellular survival versus extracellular survival.

1.9.1.2.3. YopB – hydrophobic translocator partner The 401-amino acid protein YopB is the hydrophobic translocator partner of

YopD. In Yersinia spp. insertion of YopB and YopD into liposomes or eukaryotic cell membranes was associated with channel formation (269,311). YopB is predicted to possess two central transmembrane helices that presumably insert into the host cell membrane in addition to two putative coiled-coils (297,312). Insertion of helix-disruptive mutations in the first putative TM domain impairs translocation and pore formation (313). This domain organization is also shared by genetic and/or functional homologues from P. aeruginosa, EPEC, Salmonella spp. and Shigella spp. (i.e. PopB, EspD, SipB and IpaB) (297). The SipB and IpaB insert into synthetic membranes where each TM helix similarly spans the bilayer creating a hairpin structure with the amino and carboxyl-terminus exposed (314,315). Like YopB, intimate association of PopB and EspD with eukaryotic membranes was also observed upon activation of T3SS (277,316). Insertion of YopB into host membranes activates a YopD-independent pro-inflammatory signaling response from the host cell (317). The response of epithelial cells to YopB is counteracted by the effectors YopE and YopT (317). The mechanism by which YopB stimulates this response is unknown. However, one possibility is that YopB engages a membrane receptor triggering a specific host signaling cascade. This is not unprecedented, since functionally equivalent IpaB interacts with the CD44 receptor host as a

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requisite for Shigella spp. invasion in early steps of the host entry process (318). Moreover, IpaB and SipC can also bind cholesterol with high affinity, suggesting that targeting of cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells (308).

1.9.1.2.4. Role of the hydrophobic translocators chaperone (LcrH) in Yersinia spp. T3S As already mentioned briefly in section 1.6.4.7, LcrH (SycD in Y. enterocolitica)

is the chaperone of both translocators YopB and YopD. The interacting domains between the class II LcrH/SycD chaperone and its two cognate translocators substrates YopB and YopD have been difficult to map. While deletion mutants within the YopB translocator have failed to unveil discrete CBDs, the same strategy uncovered two specific CBDs in the YopD sequence that lie in a long region of its N-terminus in addition to the C-terminal amphipathic α-helix domain (see section 1.9.1.2.1 and Figure 6) (210,211). In relation, yeast-two-hybrid assays mapped the IpaB (YopB in Yersinia spp.) CBD to residues 58-72 and IpaC (YopD in Yersinia spp.) CBD to residues 73-122 (319). However, it was also proposed that IpaC possess an additional CBD located at residues 50-80 (320). Despite the hydrophobic and intrinsic aggregative nature of the two translocators, structural and biochemical efforts have also been devoted to detail the interacting patches of LcrH homologues, with the two cognate hydrophobic translocators.

Structural characterization attempts were made to understand whether IpgC (LcrH homologue in Shigella spp.) binds IpaB as a homodimer or monomer. Some research groups have proposed that IpaB N-terminus mediates an interaction with an IpgC homodimer implying a 1:2 stoichiometry (179,321). Others have shown that both IpaB N-terminal and IpgC exist as homodimers in solution and when the two are mixed, these two homodimers dissociate and form heterodimers implying a 1:1 stoichiometry (322). Therefore, only co-crystallization of full length IpaB with soluble IpgC in solution might unveil the IpaB-IpgC structural intricacies.

In all class II chaperones known to date, LcrH/SycD adopts and superhelical structure of tandem TPRs resulting in a curved structure with a convex outer surface and a hydrophobic concave inner grove (see section 1.6.4.7) (161). The crystal structure of SycD and their homologues from Shigella spp. (IpgC) and P. aeruginosa (PcrH) revealed that the hydrophobic concave groove provides the binding site for small N-terminal translocators peptides (177-179,323). This data confirmed the prediction made previously, that a hexapeptide with consensus

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hydrophobic residues in three positions mediate the binding between the translocators and class II chaperones (179). Importantly, these peptides are bound in an extended conformation agreeing with the unstructured profile of the N-terminus region and a putative timely control mechanism for translocators secretion.

Recently, Schreiner and colleagues have presented the structure of SycD chaperone in complex with a synthetic N-terminal YopD peptide. Although SycD is presented as a monomer in this study, the hydrophobic pocket accommodates the synthetic peptide incorporating the sequence motif 58PELIKP63 in an extended manner (178). In addition, the highly similar motif 49VELNAP54 from the N-terminal PopD (YopD analogous) was also demonstrated to bind to the concave groove of dimeric PcrH (323). Likewise, in Shigella spp., an N-terminal IpaB synthetic peptide binds to the monomeric IpgC concave groove via the incorporated short binding sequence motif 65PELKAP70 (179). Remarkably, a soluble IpgC homodimer unveil an additional CBD which in complex with IpgC is essential for maintenance of the IpaB-IpgC complex stoichiometry. This additional interacting CBD might explain how the molecule blocks the putative binding site of the IpgC homodimer for additional interactions (321). Chaperone homodimerization seems to be of physiological relevance also for SycD. When a SycD variant harboring two point mutations in the dimerization interface is expressed as a stable SycD monomer in Y. enterocolitica, it does not rescue the phenotype of a sycD null mutant, suggesting functional relevance for the dimerization interface (177).

With all structural data taken into consideration, it is unlikely that both hydrophobic translocators can be engaged by the same chaperone simultaneously since both substrates occupy the same concave binding pocket. However, experimental data have not clearly shown both hydrophobic translocators belonging to the same bacterial specie compete for the same binding groove. Our group proposed that some LcrH residues are more important for the interaction with one translocator, but not with another (180). Hence, residues outside the binding groove can be important for translocator interaction specificity. This is particular important in the YopD context because it exists in a partially unfolded molten globule state (293). Therefore, one can envisage that the partly unfolded region of the protein could wrap around the convex outer surface of the LcrH/SycD homodimer so that the YopD CBDs are engaged by the concave hydrophobic pocket.

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1.9.2. Yop effector proteins – modulators of host signaling All the six Yop effectors found in pathogenic species of Yersinia have a

tendency to mimic or capture a eukaryotic activity in order to modulate specific molecular host signaling pathways. Although inert inside the bacterial cell, they become highly potent upon translocation to the host cell interior. They then either inhibit a particular pathway or over-activate it causing deregulation of normal cell functions. Upon translocation, it is presumed that a set of Yop effectors (i.e. YopH, YopE, YpkA and YopT) are translocated early to target the actin cytoskeleton hampering the host phagocytic activity (Figure 7). A second set of effectors (i.e. YopM and YopJ) might only be translocated after since they diminish the host innate immune response by targeting critical signaling pathways (Figure 7). Modulating these host functions, guarantee bacterial survival, replication and dissemination within the infected host.

1.9.2.1. Anti-phagocytic modulators YopH is one of the most powerful tyrosine phosphatases described. It was

previously assumed that bacteria lacked machinery for this type of post-translational modification, however recent bacterial genome sequencing projects reveal the presence of tyrosine kinases and phosphatases in bacteria (324). Since YopH N-terminus is both the host substrates interacting domain and the CBD, a probable mechanism to maintain YopH inactive in the bacterial cytoplasm is through the association with its chaperone SycH (325,326). YopH catalytic site mimics what is observed in eukaryotic tyrosine phosphatases supporting the idea that YopH targets host cell substrates and arguably have a eukaryotic origin (327,328). The effect on YopH in the host is remarkably rapid. Immediately after bacteria-cell contact, translocated YopH causes a global decrease of host tyrosine phosphorylated proteins (329) and abrogates calcium signaling in neutrophils within seconds (330). YopH mainly targets proteins from focal adhesion complexes essential to link actin cytoskeleton and the extracellular matrix (331,332). One of those is the p130cas protein which functions in concentrating and transmitting signals from the integrin binding and clustering sites (333). Since it was reported that Yersinia spp. invasin engages β1-integrin receptors (334), is not surprising that YopH targets proteins in focal adhesion sites (Figure 7). Additionally, YopH was also reported to interact with focal adhesion kinase (Fak), paxilin, Fyn-binding protein (Fyb) and Lck (101). Inactivation of these targets lead to disruption of integrin

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signaling, focal complexes and ultimately disruption of actin structures and subsequent inhibition of phagocytosis (Figure 7).

YopE is involved in cytotoxicity and actin filaments disruption upon its translocation to eukaryotic cells (261,335). This non-canonical GTPase activating protein (GAP) targets small Rho-like G-proteins such as RhoA, Rac1 and Cdc42 promoting its conversion from an active GTP-bound state to an inactive GDP-bound state (Figure 7) (336-338). The GAP domain is located in the carboxyl-terminal region of YopE and enclose a conserved ‘arginine finger’ that is introduced into the GTPase catalytic site accelerating the hydrolysis of GTP into GDP (339). This has implications in critical cellular function in the host cell, since inactivation of these proteins block activation of downstream effectors resulting in unregulated depolimerization of actin fibers and ultimately inhibition of phagocytosis (59). Moreover, a yopE mutant lacking the critical arginine at position 144 is avirulent in a mouse infection model, demonstrating that the GAP activity of YopE is essential for Yersinia spp. virulence (338). While the arginine finger is conserved, its tertiary structure does not reveal homologies to eukaryotic GAPs, although the structure is similar to ExoS from P. aeruginosa and SptP from S. enterica (340). Crucial for YopE stabilization and translocation to the host cell interior is an interaction with the T3S chaperone homodimer YerA (SycE in Y. enterocolitica) (186,190,195,341,342). This CBD region overlaps with the membrane localization domain (MLD) that has been attributed a role in localizing and autoregulating the protein when inside the host cell (Figure 7) (343,344). Indeed, YopE may also play a role in regulating levels of effectors transport into the host cell considering since a ΔyopE mutant displays a ‘hyper translocation’ phenotype characterized by failed down-regulation of Yop synthesis in the presence of eukaryotic cells (345).

YpkA (YopO in Y. enterocolitica) is a multifunctional serine/threonine kinase. Inside the bacteria, YpkA solubility is granted when its dedicated chaperone SycO binds the CBD which overlaps with the MLD of the protein (162). Upon translocation to the host cell in an inactive status, the MLD localizes the protein in the inner side of the plasma membrane (346). Here its C-terminus engages the actin fibers activating YpkA kinase activity that trigger subsequent actin phosphorylation and inactivation (Figure 7) (347). Both the N-terminal kinase domain and the C-terminal Rho-GTPase binding domain act synergistically engaging RhoA and Rac1 inhibiting the turnover exchange of GTP for GDP (347-349). Interestingly, YpkA specifically phosphorylates Ser47 of the Gαq GTPase. As a result, Gαq-dependent cellular processes including RhoA activation are impaired (Figure 7) (350). These mechanisms of actin cytoskeleton disruption promote Yersinia spp. resistance to phagocytic activity by macrophages.

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YopT is a cysteine protease, a functional version of which is found exclusively in Y. enterocolitica (101). Inside the bacteria, the YscT chaperone binds to YopT domain responsible for the protease activity of the protein presumably maintaining the protease inactive prior to secretion (200,202). Upon translocation, it cleaves a site near the C-terminus of the small G-proteins Rho, Rac1 and Cdc42 (351). Cleavage of this group of proteins prevents their correct localization to the host cell membrane, which inhibits their function (Figure 7). This obstructs actin fiber assembly and ultimately host cell phagocytosis (351,352). Y. enterocolitica ΔyopE mutant exhibit cytotoxicity towards HeLa cells, whereas the same Y. pseudotuberculosis mutant no longer is able to induce cytotoxicity. This has been attributed to the role of YopT function in Y. enterocolitica that is lacking in Y. pseudotuberculosis (353). This questions the specific role of YopT in Yersinia spp. virulence.

1.9.2.2. Immune response modulators YopJ (YopP in Y. enterocolitica) is an acetyl-transferase that targets and

acetylates serine and threonine residues from several kinases (354,355). Those molecular targets include MAPK kinases from the MAPK signaling pathway and IKK-β kinase from the NFκB signaling pathway (Figure 7) (356,357). Therefore, YopJ inhibits their phosphorylation and consequently their activation resulting in reduced production of inflammatory cytokines and promotion of cell death (354). Inhibition of these pathways by preventing downstream signaling is required to limit the host immune response and induce apoptosis of macrophages during infection. However, the precise role of YopJ in vivo is less well defined. For example, YopJ is not critical for Y.pestis followed by intravenous infection (358), while oral infections with a Y. pseudotuberculosis ΔyopJ mutant display a 64-fold reduction in virulence due to a reduced ability to establish a systemic infection (359,360). Moreover, a YopP-defective Y. enterocolitica was also attenuated in an oral mice infection assay. Although, these bacteria were able to spread systemically and to colonize organs, the mice were able to control the infection (361). Together these observations indicate that YopJ might be important for enteropathogenic Yersinia spp. to establish critical infections within the host. Curiously, a YopJ-specific T3S-chaperone has not yet been identified. This raise interesting questions concerning its spatial and temporal secretion control.

YopM is one of the less understood Yersinia spp. Yop effectors. The tertiary structure of the protein revealed two antiparallel N-terminal alpha-helices serving as nucleation points for the folding of the leucine-rich-repeat (LRR) domain, and

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Figure 7 – Modulation of host signaling cascades by Yersinia T3SS Yop effectors. Schematic representation of how translocated Yop effectors subvert the host cell signaling pathways. Two major immune cell processes are disrupted (X); phagocytosis/bacterial uptake (via YopE, YopH, YopT and YpkA) and pro-inflammatory immune cell signaling (YopJ and perhaps YopM).

the monomers can stack together as tetramers forming a hollowed cylinder (362). YopM effectors are composed of a variable number of LRRs that can vary from 13 to 21 (363). However, the functional implications of such heterogeneity are unclear. YopM has a C-terminal nucleus localization signature responsible for its accurate trafficking to the nucleus compartment via a vesicle-associated pathway (364,365). Since YopM localizes within the host nucleus, it was assumed to play a role in host gene transcriptional regulation (Figure 7). This function, however, is still controversial (366,367). At least in in vitro conditions, YopM can target two kinases: ribosomal S6 protein kinase 1 (Rsk1) and protein kinase c-like 2 (Prk2). YopM assumingly prevents dephosphorylation of Rsk1 promoting its interaction with, and activation of Prk2 (368-370). However, additional evidence that the YopM-Rsk1-Prk2 complex phosphorylates other cytosolic or nuclei substrates is still lacking. Recently, it was demonstrated that YopM stimulation of the anti inflammatory cytokine IL-10 contributes to Y. pseudotuberculosis virulence (371). Despite lacking any measurably enzymatic activity, YopM is an important virulence determinant for

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all pathogenic Yersinia spp. In enteropathogenic Yersinia spp. species, YopM is fundamental for bacterial propagation from the intestinal tract to the spleen and virulence (361,370,372). YopM is also important for Y. pestis virulence injected intradermally or intravenously a mice model (373-375). Like YopJ/P, YopM may also lack the requirement for a dedicated T3S chaperone.

1.10. Translocon assembly and translocation dynamics Defining the structure and composition of a native T3S translocon pore

inserted into host cells membranes by Gram-negative bacteria is one of the major challenges in the T3S field. The events that precede and follow translocon assembly into the host cell membranes also need to be elucidated. In particular, the spatial and timely positioning of the hydrophobic translocators in the needle tip assembly platform before and during host cell contact has been difficult to fully unveil in the Yersinia spp. pathogens. In relation to translocation dynamics, it is still not obvious how we can integrate the proposed two models (one step vs. two step) for Yops translocation to the host cell interior. In addition, it is not known whether the translocon have a gating mechanism which controls effectors translocation and ultimately its function. It is however interesting that some hydrophobic translocators have a dual role as translocator and effector. How translocated effectors control pore formation and relay a feedback regulatory signal presumably through the translocon is also unclear. Despite this, elegant experimental evidence originating from the Shigella spp. field might help us to better comprehend the intricacies of T3S translocation in general, including by Yersinia spp. In this section, an overview is presented on how diverse T3SS-harbouring pathogens control chronologically and spatially the events that occur just before the translocon is fully assembled, until the signaling for pore formation inhibition is triggered from translocated effectors.

1.10.1. Tip complex assembly platform and activation The observation that LcrV forms a structure at the tip of the T3S needle,

before contact with host cells, suggests a model by which an LcrV homopentamer acts as a scaffold for translocon assembly (265,274,289). Although, there is no evidences to support the presence of the hydrophobic translocators associated with this assembly platform prior host cell contact, the N-terminal globular domain of LcrV that forms the base of the platform can mediate the interaction with YopB (274). The fact that there is a good correlation between an LcrV-YopB interaction

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and insertion of YopB into the target cell membrane leading to formation of a functional translocon (274,281), suggests a mechanism by which the Yersinia spp. LcrV assembly platform aids to properly fold secreted unfolded YopB, and in its insertion into the host cell membrane (274). Since binding studies suggest that LcrV can also engage monomeric or self-assembled YopD, one can predict that the assembly platform might have a similar function with the other hydrophobic partner (Paper I). This mechanism is supported by similar observations in Shigella spp. where the heteropolymeric assembly platform is formed by four molecules of IpaD and one molecule of IpaB (252,285). Upon host cell contact, IpaB is inserted into the host cell membrane which in turn triggers a needle conformational change relayed by an IpaD-dependent mechanism to the base of the T3S apparatus to permit secretion of IpaC. Secretion activation of IpaC leads to the protein assembly into the host cell membrane shaping a functional pore with its partner IpaB (255,256). Therefore one can predict that in Shigella spp. IpaB acts as a sensor for host cell membranes as the pentameric LcrV complex does for Yersinia spp. Despite IpaD and LcrV having no clear sequence homology they share similar overall architecture, suggesting that the assembly mechanism of the translocators into a functional pore might be analogous in both bacteria (252).

This mechanism thus supports a single step translocation model where the T3S needle serves as a continuous conduit to connect the bacterial cytoplasm to the translocon granting efficient polarized delivery of unfolded or semi-folded effector proteins into the host cell interior.

1.10.2. Translocation of surface localized Yops by an alternative translocation process A long-standing dogma in the T3SS field is the apparent one-step polarized

delivery of effector proteins into the host cell interior. In reality however, several reports in the literature are not entirely compatible with this accepted model. For example, Watarai and colleagues have reported that in Shigella spp. IpaC, IpaB and IpaD are bacterial-surface localized prior to host cell contact and its release occurs once host cell contact is established. This indicates a bacterial sensing mechanism that triggers the release of the proteins (376). Moreover, Ménard and colleagues later showed that latex beads coated with secreted IpaB and IpaC are internalized into epithelial cells, inducing cellular events associated with the Shigella spp. host entry (377).

Recently, Akopyan and colleagues have shown by electron microscopy that YopE, YopH and YopD are localized on the surface of Y. pseudotuberculosis before

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target cell contact (378). Moreover, these and other Yops were accessible to proteinase K degradation also suggesting that a large proportion of the Yops were already present on the surface of the bacteria before target cell contact. Furthermore, Y. pseudotuberculosis coated with recombinant purified YopH, induced an YopH-mediated inhibition of early calcium response in neutrophils. The translocation of the YopH-bla reporter was dependent on the presence of a discrete translocation domain located downstream of the secretion domain, indicating that effector secretion and translocation are two independent processes. The translocation of surface localized YopH was dependent on a functional T3SS, including YopB and YopD translocators. Translocation of YopH was also achieved when S. typhimurium was coated with the recombinant protein, suggesting that translocation of surface-localized effectors might be a conserved feature among different T3SS-harbouring pathogens (378). This is not entirely surprising, since other studies have ably demonstrated that S. typhimurium is capable of secreting the Yersinia spp. YopE effector in a T3SS-dependent fashion (379).

The two-step translocation mechanism also proposes a sensory role for T3S needle tip complex, rather than an injection device as in the one-step model. Upon engaging an unknown host factor, alterations to the needle triggers the release of complexes shaped by translocators and effectors from the bacterial surface. The Yops complex would insert into the host cell membrane by a mechanism that could resemble the delivery of tripartite AB toxins. The T3S translocators could resemble the role of the B-moiety that binds to host cell membranes eliciting the transport of Yop effectors which reflecting the role of the enzymatic active A-moiety (378,380). The presence of comparable soluble complexes has been reported in Y.pestis. These were shaped by YopB-YopD-YopE and elicit antibodies, protective against Y. pestis infection. However, with that particular experimental setup they could only be obtained under in vitro T3SS-inducing conditions (381). Crystal structures of a protease-stable fragment from N-terminal regions of IpaB from S. flexneri and SipB from S. enterica serovar Typhimurium reveal considerable relationship to the coiled-coil regions of the pore-forming proteins from other Gram-negative pathogens such as colicin Ia (382). Although the long N-terminal coiled coil domain of the toxin is important for recognition of bacterial outer membrane receptors which mediate the protein translocation and formation of an ion channel inducing bacterial cell lyses, IpaB N-terminus binding to host membrane CD44 receptor is also essential for Shigella spp. invasiveness (318). It may therefore be possible that the B-moiety (translocators) of surface localized complexes engage the host membrane CD44 receptor to promote the translocation of the A-moiety (effector) into the host cell cytoplasm.

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The data mentioned support the idea of a two-step translocation model where T3SS substrates are secreted prior to host cell contact, followed by their translocation across the host cell membrane upon host sensing. However, the one-step and the two-step models are not mutually exclusive and can possibly operate in parallel. One can envisage that the Yersinia spp. surface-localized T3SS complexes might have an initial protective role against accidental immune cell recognition during bacterial transit through target tissues. When the host target cell membrane is sensed by the pentameric LcrV platform these ready-made extracellular T3SS complexes might be assembled into the host cell membrane rapidity translocating their effector cargo presumably to arrest the very initial host responsive events. Newly synthesized hydrophobic translocators would be consistently recruited to the needle tip to assemble a functional translocon pore for one-step polarized translocation of Yop effectors.

1.10.3. The T3SS translocon pore Different mammalian pathogens harboring a T3SS have been shown to form

translocon pores in different eukaryotic nucleated cell types, erythrocytes or even liposomes. The ability of Yersinia spp. to form translocons into host cell membranes is recurrently monitored by indirect methods. Pore formation aptitude is often measured by i) capacity to induce RBC hemolysis; ii) uptake or release of impermeable dyes such as BCECF or EtBr from eukaryotic cells iii) quantification of the cytoplasmic enzyme LDH released into supernatants of culture cells. (269,275,383,384). However, pore formation in vitro can only be observed in multi-effectors mutant or ΔyopK background, indicating that these assays might not represent what occurs in in vivo (269,385). Håkansson and colleagues suggested if Yop effectors are not present to fill the translocon channel, the formed pore leads to leakage of measurable cytoplasmic contents (383). Similarly, YopK (YopQ in Y. enterocolitica) is thought to influence the pore size by an unknown mechanism. When YopK is absent from Yersinia spp. infections, the bacteria forms larger pores into the host membrane as observed by osmoprotection experiments (385).

Translocon assembly process was first performed by measuring a YopB and YopD-dependent lytic activity in sheep erythrocytes and electrophysiological experiments on artificial liposomes (311,383). These were the first evidences that directly involved the hydrophobic translocators in the process of translocon assembly. In addition, these studies also show that mutants devoid of YopD did not affect YopB-mediated lysis of sheep erythrocytes which in the liposome model also led to current fluctuations even though different from the wild-type (311,383). This

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confirmed the idea that translocon assembly in either natural or artificial lipid membranes is mostly dependent on the YopB translocator presence. Additionally, the role of the hydrophilic translocator LcrV in the pore formation process is controversial. If the needle tip protein is strictly required for translocon insertion in vivo, it is dispensable to assemble YopB/YopD-dependent pores in liposomes (265,274,299-301). This suggests that native pores inserted into host membranes might be structurally different from the ones resulted from purified hydrophobic translocators inserted into artificial membranes. Although artificial lipid by-layers are a fruitful tool to study the interplay between the two hydrophobic translocators in the translocon assembly context, only a truly and translocative pore assembled in vivo can yield the native assembly mechanism and the innate structure of the translocon.

The internal diameter of translocons assembled in eukaryotic cells determined by osmoprotection experiments lead to estimated pore dimensions that can range from 1.2 nm to 5.0 nm depending on the pathogen (266). Infected RBC with Y. pseudotuberculosis, for example, generated pores with an estimated average size of 2.3 nm (383), fitting with the 2.5 nm inner-diameter of the T3S needle (131). Montagner and colleagues have recently purified native translocon from RBC membranes upon infections with wild-type Y. enterocolitica (276). In this study, YopB and YopD are integral membrane proteins and form heteropolymeric complexes with an estimated mass between 500 and 700 kDa (276). However, no evidence of a defined size was observed, suggesting the dynamic character that the translocon might adopts when inserted into eukaryotic membranes.

The two P. aeruginosa translocators PopB and PopD, bind and disrupt artificial liposomes revealing a ring-like structure with an internal diameter of 4 nm when visualized by electron-microscopy (EM) (299). When both proteins were mixed in an equimolar ratio in vitro, they were able to lyse liposomes in a cholesterol-dependent manner, similarly to what was observed in S. flexneri and Salmonella spp. infections (299,308,386). Although no stoichiometric ratio between the two hydrophobic translocators was established to date, the same inner diameter of pores formed by PopB/PopD into liposomes was also confirmed by osmoprotection experiments. Using fluorescein-labled dextrans entrapped in liposome vesicles Faudry and coworkers determined that purified PopB/PopD proteins induce pores with an estimated size ranging from 3.4 nm to 6.1 nm fitting with the heteromeric ring like-structures visualized by EM (301). Attempts were also made to visualize wild-type EPEC translocons assembled in RBC membranes (387). The funnel-like structures analyzed by atomic force microscopy (AFM) had an outer-diameter of 55 nm to 65 nm and an estimated inner diameter of 8 nm, contrasting with the 3 nm

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to 5 nm determined by osmoprotection experiments. This could explained by the morphology of the observed funnel-like structure that displayed a large opening to the outside of the membrane narrowing towards the inside (387). However, no direct evidences were shown to prove that these structures were indeed an EPEC T3S translocon.

Efforts to obtain insights on the adopted stoichiometry by the hydrophobic translocators upon host membrane insertion, point towards the presence of 6 to 8 translocators in EPEC infections, six translocators (one IpaB and five IpaC) for Shigella spp. and a ratio of ~2.4 YopD molecules per YopB in Yersinia spp. (252,387,388). This level of stoichiometric heterogeneity may account for the observed pore sizes differences induced by different pathogens measured in osmoprotection experiments.

Regardless of large amount of data concerned the biochemical characterization of T3SS translocon components from many bacterial species, structural data that could elucidate the shape that the translocon adopts into the membrane is still elusive. This might be explained by the intrinsic dynamic property of the translocon that impedes a clear snapshot of a stable structure.

1.10.4. Translocators have effector function? The observation of a pool of YopD located intracellularly upon eukaryotic cell

infections with Y. pseudotuberculosis raised a possible additional function for the protein besides regulating Yops production in the bacterial cytosol and formatting the T3SS translocon on target cell membranes (249). Although YopD translocation to the host cell interior is dependent on its C-terminal amphipathic α-helix, the exact functions of these translocated fractions are unknown since no obvious phenotypically changes in morphology of YopD-laden eukaryotic cells was observed. Thus, no obvious effector function has been assigned for the protein. Nevertheless, one can assume a possible role of the molecule as a chaperone maintaining each effector in a translocation competent status or even promoting suitable effectors localization in the host cytoplasm. This idea may be supported from the fact that YopD can interact with the YopE effector (389). Interestingly, a cooperative role has already been assigned for SipC (YopD analogous) and SipA (effector of Salmonella spp.) as SipA potentiate SipC effector activity for actin nucleating and bundling. Additionally, some translocators from other T3SSs that are translocated to the host interior have been assigned effector functions. IpaC from Shigella spp., for example, has been described to possess multiple effector-related functions such as: i) triggering cytoskeletal changes in host cells (390-392);

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ii) induce uptake of S. flexneri lacking a functional T3SS (307); iii) promoting S. flexneri invasiveness (306,307,391) and iv) interaction with phospholipids membranes (390-392). Like IpaC, YopD C-terminus mediates its self-assembly (305,390,393). Indeed, the C-terminal domain of IpaC is required specifically for Shigella spp. invasiveness but not for pore formation, as some parts of this domain might be involved in actin nucleation (394). Similarly (to IpaC), Salmonella spp. SipC is also translocated into host cells in a T3S-dependent manner as it can induce actin nucleation in vitro as mentioned above (304,393,395,396). This actin nucleation activity is promoted by SipC multimerization and contributes to Salmonella spp. invasiveness (304,309,395).

Effector function is not restricted to translocators belonging to the YopD-like family such as IpaC and SipC. Although (until recently) there has been no evidence suggesting that YopB could possibly have an effector function, that possibility cannot disregarded. In fact, IpaB from S. flexneri has been recently suggested to possess an additional function to facilitating translocation of other effectors molecules. Indeed, purified IpaB inserts into host cytoplasmic membranes after spontaneous oligomerization forming monovalent selective cationic channels which disturb ion homeostasis within the endolysosomal compartment. It follows membrane disintegration with endolysosomal leakage and Caspase-1 activation that ultimately leads to macrophages cell death (397). This supports the idea that YopB might possess additional functions in Yersinia spp. pathogenesis.

1.10.5. Inhibition of pore formation and feedback regulation The observation reported by Viboud and colleagues that the effectors YopE

and YopT counteract pore formation activity once they have been translocated to the host cell interior, may unveil one possible mechanism by which Yersinia spp. tightly controls effectors translocation and uncontrolled host cell lysis (398). Infection of eukaryotic cells with multi-Yop effectors mutant Y. pseudotuberculosis strain (ΔyopEHJT) induced the activation of a host signaling pathway leading to pore formation and production of pro-inflammatory cytokine IL-8 in a YopB-dependent fashion (317). However, when YopE and YopT where expressed in the same background, pore formation was inhibited by the two effectors whereas YopB-induced IL-8 production was inhibited mostly by YopE (399). Additionally, catalytic inactive YopE and YopT failed to inhibit pore formation (398). These observations suggest that Rho GTPase activation and actin depolimerization induced by translocated YopE and YopT are required for closing of the translocon and prevention of subsequent osmotic lysis. By this mechanism, Yersinia spp.

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prevents release of the host intracellular content and consequently a massive host immune response. Insertion of Yersinia spp. T3S translocons triggers maturation and release of pro-inflammatory IL-1β independently of cell lysis, suggesting that pores are intrinsically pro-inflammatory inducers. Consistently, this effect is also counteracted by the action of anti-inflammatory effectors upon their translocation into the HeLa cell cytoplasm (400).

Releases of pro-inflammatory cytokines from activated macrophages are an important innate immune response to bacterial infections. Therefore, control of spontaneous or uncontrolled release of pro-inflammatory cytokines as a result of inserted translocons in the host cytoplasmic membrane, has to be tightly controlled by the bacteria. Yersinia spp. utilizes a particular effectors repertoire that negates pore formation upon effectors translocation. The bacteria have developed different strategies to down regulate host inflammatory response by ensuring bacterial proliferation during early stages of the infection.

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2. Objectives of this thesis Critical aspects of Yersinia T3S translocation are scantily understood. The main

objective of this thesis is to therefore elucidate aspects that govern the Yersinia T3S translocon assembly dynamics and regulatory control of Yops secretion.

Specific aims:

• Identify residues within the YopD amphipathic α-helix domain specifically linked to discrete functions of the protein.

• Investigate the biological relevance of the N-terminal predicted coiled-coil that occurs solely in Yersinia YopD sequences.

• Examine the influence of the YopD C-terminal putative coiled-coil in Yersinia Yop effectors, translocation and virulence.

• Explore the complex regulatory mechanisms displayed by the YopD chaperone LcrH.

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3. Results and discussion 3.1. Functional implications of the YopD domain architecture The 306-residue YopD is a multifunctional protein built-up by a mosaic of

domains that presumably permit involvement of the protein in multiple steps of Yersinia T3S (see section 1.9.1.2.1 and Figure 6) (292). Indeed, discrete domains of the protein are necessary to facilitate Yops secretion, LcrH chaperone binding, maintenance of yops regulation, translocon assembly into host cell membranes and Yop effector translocation into host cells. Pore formation and the injection of effectors into the host cell interior are separate events controlled by particular domains of YopD, which may indicate that complex functions can be encrypted in specific domains and/or residues of the protein (292). To understand how YopD may integrate this multitude of functions by attributing them to specific structured domains and/or residues within those domains, will bridge gaps in our understanding of the molecular mechanisms behind the delivery of effectors toxins to the host cell interior.

3.1.1. The amphipathic α-helix domain An in silico prediction of the YopD C-terminal domain indicated a region that

had a high propensity to form an amphipathic α-helix structure (271). This initial assumption was later confirmed when the structure of a synthetic peptide encompassing the secondary structure was solved by NMR confirming the predicted hydrophobic and hydrophilic sidedness of the domain (Figure 8) (209).

Amphipathic domains are known to mediate molecular interactions with proteins and lipid rafts (401). A possible reason why this versatile secondary structure is present in multifunctional YopD is the intrinsic need to mediate multiple molecular interactions in different environments. The involvement of the YopD amphipathic α-helix domain in establishing molecular contacts with lipid membranes or formation of homo- or hetero-oligomeric complexes has been little investigated prior to Paper I. A previous phenotypic analysis of a YopD variant completely lacking the amphipathic domain (YopDΔ278-292), indicated it to possess a central role in YopD translocator function, perhaps via the hydrophobic side of the helix integrating the protein into host cell membranes (Figure 8) (292). It was also implied that the hydrophobic residues may mediate interactions with the cognate chaperone LcrH in the bacterial cytosol (Figure 8) (211). As a consequence of

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Figure 8 – Structure of the YopD amphipathic α-helix domain. Spatial modeling of YopD amphipathic α-helix (PDB: 1KDL) illustrating the hydrophobic (red) and hydrophilic (blue) sidedness of the peptide. Modified from Tengel et al. (209). Structure was rendered using the PyMOL program.

mediating complexes with LcrH, this domain is therefore implicated in Yops regulatory control. It is however unclear whether this extends to engaging yops mRNA transcripts to mediate post-transcriptional control. The YopD C-terminal amphipathic α-helix is a central player in Yersinia T3S influencing Yops regulation, LcrH binding, pore formation and effector translocation. It is therefore likely that these YopD activities require at least transient connections to several additional T3S components; contacts presumably established by specific residues within the amphipathic α-helix domain.

In paper I, the aim was to uncouple the various YopD functions by identifying individual contributing residues within the amphipathic α-helix.

3.1.1.1 Specific residues separate Yops regulation from secretion

To understand the critical role of the 15 residues placed in the YopD amphipathic α-helix domain we performed a widespread site-directed mutagenesis generating 38 individual in cis point mutants in total. These mutations were introduced in hydrophobic amino acids (18 mutations) or hydrophilic amino acids (13 mutations) in addition to 7 double mutations. Each original amino acid was substituted by either alanine or a residue with opposite chemical properties (Paper I, Fig. 1A). These 38 bacterial strains each expressing a uniquely modified YopD variant were

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then subjected to a series of phenotypic tests to characterize the T3S function. These tests included maintenance of yops regulatory control, assembly of translocon pores measured by RBC lysis and delivery of effector toxins into eukaryotic cell. Interestingly, substitutions in the hydrophilic residues did not reduce any facet of YopD function (Paper I, Table 2). However, some substitutions of hydrophobic residues generated YopD variants with a profound phenotypic effect on the protein function and T3S activity (Paper I, Table 2). Substitutions in the hydrophobic residues Val284 and Ile288 induced defects in yops regulatory control, similar to that observed in the full-length yopD deletion mutant (Paper I, Fig. 2A). These same substitutions also compromised effector delivery (Paper I, Fig. 4A). These two residues must have dual function in both maintaining yops regulatory control in the bacteria cytoplasm and establishing important molecular interactions at the bacterial surface to permit Yop effector translocation (Figure 9C).

Critically, alterations in Phe280 and Met281 generated derivatives solely defective in yops regulatory control (Figure 9B). This was evidenced by creating a series of progressively more severe substitutions in the Phe280 residue (e.g. YopDF280Y, YopDF280A and YopDF280R) that in turn created a proportional gradient in extend of deregulation of Yops production in non-inducing conditions. It should be noted that the YopDF280R mutation was severe enough also to affect Yop effector delivery. Additionally, GST fused to these YopD variants could still engage the cognate LcrH chaperone (Paper I, Fig. S2A). This finding was not unanticipated, given that the YopD N-terminus also contains a CBD for LcrH chaperone binding (see Figure 6). Therefore, the regulatory defects presented by these YopD mutants must result from impairment in other molecular interactions. It is assumed that YopD cooperates with LcrH, and possibly with LcrQ, to maintain yops post-transcriptional regulatory silencing in non-inducing conditions (see section 1.8.3 and Figure 4). It is thus conceivable that these YopD variants compromise a possible YopD-LcrQ or YopD-mRNA interaction.

Being solely defective in yops regulatory control, these derivatives will permit the future study of the YopD’s role in yops regulatory maintenance in isolation from other YopD-dependent T3S events. The utilization in Paper IV of such YopD derivatives in conjunction with the LcrH and LcrQ regulatory elements will benefit our understanding of the molecular interactions underlining the tripartite regulatory complex involved in post-transcriptional silencing on Yops synthesis.

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3.1.1.2. YopD’s molecular interactions facilitate Yersinia spp. T3S Successful pore formation and Yop effector delivery is presumed to require

multiple molecular interactions between the pore constituents as well as host cell membranes. In our mutagenesis scanning of the YopD amphipathic α-helix domain, we identified the residues Val284 and Ile288 as critical for the bacteria to assemble translocon pores into eukaryotic membranes and to delivery effector toxins into the host cell cytosol (Paper I, Fig. 4A and 5A) (Figure 9C). To mechanistically understand why T3S translocon assembly was deficient in YopD derivatives where those residues were substituted (i.e. YopDV284R and YopDI288K), we determined the ability of these YopD variants to engage either the hydrophilic needle tip LcrV or the second hydrophobic translocator YopB utilizing a protein interaction assay based on GST pull-downs. We found that all the tested YopD variants could engage YopB (Paper I, Fig. S2B). This indicates that a YopD-YopB interaction may not be the sole requirement for translocon assembly and Yop effectors delivery. However, reduced binding to LcrV was evident which suggests that the YopD amphipathic α-helix domain may be critical in order to establish molecular interactions between YopD and LcrV (Paper I, Fig. 8). Therefore, it infers that only an intact YopD amphipathic α-helix can act as interacting interface to engage LcrV ensuring assembly of a functional pore capable to translocate effector toxins.

Formation of translocon pore into host cells membranes probably needs the hydrophobic translocator constituents to oligomerize (299). Multimerization can be mediated by amphipathic α-helix domains (401,402). Therefore, using in vitro cross-linking studies we explored the propensity for YopD to self-assemble. Although native YopD was able to assemble in high order oligomers, the deletion mutant lacking the amphipathic α-helix domain failed to form some of these oligomers, which suggests that this domain does contribute to YopD self-assembly (Paper I, Fig. 9). Reduced tendency to self-assemble was also observed for YopDI288K, YopDV284R and YopDF280R consistent with their reduced ability to form translocon pores into RBC. Significantly, these observations demonstrate the biological importance of the amphipathic α-helix domain of YopD in its oligomerization which in turn impacts on T3S activity.

In combination, these observations might suggest a potential scenario where upon bacterial-host cell contact, YopD secretion is triggered via the T3S conduit in an unfolded or semi-folded status. The nascent YopD protein interacts with LcrV via its amphipathic α-helix domain at the tip of the needle. YopD oligomerization would then proceed either induced by LcrV the needle tip or through contact and/or insertion into the host membrane (Figure 10).

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Figure 9 – Spatial localization of distinctive site-directed mutants mapping to the YopD amphipatic α-helix. (A) Molecular surface positioning of phenotypically relevant hydrophobic residues within the amphipathic α-helix structure (PDB: 1KDL). (B) Highlighted in red are the Phe280 and Met281 residues which are primarily important for yop regulatory control. (C) Highlighted in orange are the Val284 and Ile288 residues which have functions in both maintaining yops regulatory control and establishing relevant molecular interactions that elicit Yop effector translocation. Structure was rendered using the PyMOL program.

3.1.2. Putative N-terminal coiled-coil domain Based on several in silico predictions, T3SS substrates (including the

translocators class) have a propensity to form α-helical coiled-coils (294,403). Although limited structural data supporting such predictions for hydrophobic translocators are present in the literature, initial reports have experimentally confirmed their presence. Barta and colleagues, for example, have demonstrated the presence of N-terminal coiled-coils in the IpaB and SipB translocators from S. flexneri and S. enterica Typhimurium, suggesting that this motif might be important for protein integration and assembly of the translocon into host cell membranes (382). In addition, other reports connecting this structural motif to critical protein functions exerted in a T3SS context are also available. Coiled-coils likely help in needle polymerization, assembly of the needle tip complex, insertion of a functional translocon pore, T3S control and stabilization of secreted substrates or

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even in the proper localization of translocated T3S effectors in the host cell (253,289,404-410).

In paper II we analysed the predisposition of YopD derived from the three pathogenic Yersinia spp. to form putative coiled-coil domains along the sequence using the web server Coiled-coil prediction (http://npsa-pbil.ibcp.fr/) that runs an implementation of the algorithm first described by Lupas and colleagues (411). In addition, we strived to understand whether the N-terminal coiled-coil predicted only in YopD of pathogenic Yersinia spp. is important for full T3SS activity.

3.1.2.1. A predicted helical structure unique for YopD from Yersinia The YopD sequences are broadly conserved among the pathogenic Yersinia

spp. species. Utilizing the BLASTP webserver and the YopD sequence from Y. pseudotuberculosis (serotype 0:3) as a bait, sequences identities of 99.7% for all known YopD sequences from Y. pestis and identities of at least >97.1% to all known YopD sequences from Y. enterocolitica isolates available were retrieved from the NCBI database (Paper II, Table 2). These observations are consistent with the close evolutionary relationship between Y. pseudotuberculosis and Y. pestis contrasting with the earlier divergence between Y. pseudotuberculosis and Y. enterocolitica. Among those bacteria that also harbour T3SSs belonging to the Ysc-Yop family, the level of YopD-like proteins identities drops to between 49,3% (AopD protein from A. Veronii) and 27.8% (VDA_000190 in Pb. damselae) (Paper II, Table 2).

Using the method described by Lupas and colleagues, we further scrutinized the YopD and YopD-like sequences for the tendency to form putative coiled-coil segments using the coiled-coil prediction web server (http://npsa-pbil.ibcp.fr/). We focused on this secondary structure motif because it has been reported to possess critical functions in a variety of T3SS substrates (405,408,409,412). Coiled-domains were predicted with a high probability in the C-terminus region of the YopD protein from pathogenic Yersinia spp. corroborating earlier reports indicative of functional role of this region in the bacteria T3SS activity (Paper II, Fig. S1 and Table 2) (292). Moreover, a similar segment was also predicted in the C-terminal region of YopD-like sequences from other bacterial genera, suggesting a functional conservation of this motif in other pathogens. However, two exceptions were detected in the LopD protein from Ph. luminescens and PopD from P. aeruginosa, indicating that some structural deviations might be necessary to support individual bacterial T3SS specificities (Paper II, Fig. S1 and Table 2).

The same algorithm also predicted (with a much lower probability) a second modestly sized coiled-coil structure (propensity <70%) between residues 31-44 of

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the YopD N-terminus from pathogenic Yersinia spp. species (Paper II, Fig. S1 and Table 2). Analysis of all the YopD-like sequences from the other unrelated bacteria consistently failed to predict a putative N-terminal coiled-coil, which may suggest an exclusive contribution to YopD function during Yersinia spp. pathogenesis.

3.1.2.2. Putative YopD N-terminal coiled-coil contributes for protein self-assembly and Yersinia spp. virulence In the absence of any tertiary structure of YopD it is difficult to know if this α-

helical segment constitutes a coiled-coil. However, if our in silico predictions are correct, any disruptive alteration of this functional secondary element might lead to differences in YopD structure which may in turn impact in T3SS activity. Knowing this, we used site-directed mutagenesis to generate the variants YopDI32K or YopDI32P. These substitutions were predicted to disrupt the coiled-coil from 80% to 60% and 0% respectively (Paper II, Fig. S1). In addition, we also took advantage of the poorly secreted YopDΔ23-47 that lacks the putative N-terminal coiled-coil (292).

The introduction of a proline helix-breaker in Ile32 induces a subtle structural alteration shown by an altered chymotrypsin digestion profile (Paper II, Fig. 2) and a modest loss of homoligomerization capacity (Paper II, Fig. 3). This was most likely due to the secondary structural alteration of the protein. Curiously however, disruption of this putative element did not have any impact on T3S activity in vitro. This includes the ability to shape a translocon pore in RBC membranes (Paper II, Fig. 5) and to translocate YopE and YopH into eukaryotic cells to promote anti-phagocytosis and extracellular survival (Paper II, Fig. 6 and 7). Thus, elementary YopD interactions with recognized T3S substrates or even unknown of the host cell appear to remain intact in these mutants.

All these YopD mutant bacteria maintained yops post-transcriptional control and normal growth (Paper II, Fig. 4) consequently their biological significance could be investigated in an in vivo murine infection model. Oral infections of female BALB/c mice with bacteria producing YopDI32K and YopDI32P clearly displayed milder signs of infection at lower infection doses (107 CFU/ml) including a reduction in body weight (Paper II, Fig. 8B). Furthermore, 50% of the mice infected with the mutants were able to survive the entire 14 day experiment (Paper II, Fig. 8A).

It therefore appears that when confronted with a massive immune response systemic progression of the disease caused by bacteria producing a sub-optimal T3SS (YopDI32K and YopDI32P) was hampered, particularly at low inoculated doses of 107 CFU/ml. However, when the inoculums rose to 108 or 109 CFUs/ml the balance favored the bacteria, suggesting that even a massive infection of bacterial with a

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sub-optimal T3SS can hamper the animal immune response. This data highlights the limitations of current in vitro assays that intrinsically cannot reproduce all the biological complexities associated with the in vivo animal environment.

Analyzed collectively, the data suggests that YopD requires an intact putative N-terminal coiled-coil for the full disease progression in orally infected mice. Although we cannot indefinitely prove that this YopD N-terminal region is a true coiled-coil, our overall data suggest that this region confers some structural constraint to YopD that is necessary for optimal T3SS activity in defense against in vivo killing by naturally activated immune cells.

3.1.3. Putative C-terminal coiled-coil domain In Paper II, we appreciated that YopD and most YopD-like proteins belonging

to the Ysc-Yop T3SS family seemed to possess a high propensity (~99%) to form coiled-coils in their C-terminal regions. However, experimental evidence that definitely confirms the presence of this secondary structure or demonstrates its influence on the protein function is lacking. In silico predictions indicate that YopD might possess a long α-helical coiled-coil between residues 250 and 275 (294). This secondary structure could be relevant for the protein to establish intra or/and intermolecular interactions with other coiled-coil helices. We thus attempted in paper III to understand the contribution of the predicted YopD C-terminal coiled-coil to overall translocator function in Ysc-Yop T3S.

3.1.3.1. Predicted α-helical segment that influences translocon assembly and effector delivery in vitro To gain an understanding of how this secondary structure might benefit

protein function, we specifically introduced the helix breaking residue Proline at selected positions within the helix. The four generated YopD variants YopDI262P, YopDA263P, YopDK267P and YopDA270P reduced the predicted capacity to form a coiled-coil from 99% for YopDwild-type to values ranging from 40% to 0% (Paper III, Fig. 1 and S1). In addition, we also took advantage of the previously constructed YopDΔ256-275 derivative that essentially lacks the entire α-helix motif (292). A thorough in vitro phenotypic analysis was preformed to comprehend whether these derivatives were dysfunctional in any aspect of Ysc-Yop T3S. Two mutant categories were unveiled on the basis of various in vitro tests. The first class composed of YopDΔ256-275, YopDI262P and YopDK267P producing bacteria, assembled low levels of translocon pores in eukaryotic cell membranes and the T3SS was dysfunctional when in the

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presence of target cells. The other class, composed of mutants producing YopDA263P and YopDA270P, maintained a fully functional T3SS even though smaller pores were found in target cell membranes. Table 3 summarizes all the results obtain in our rigorous in vitro phenotypic analysis for both classes of the generated YopD variants.

Table 3 - Phenotypic in vitro characterization of putative coiled-coil YopD variants.

YopD variants

C-terminal coiled coil propensity (Fig. 1 and

S1)

Post-transcriptional

regulatory control

(Fig. 3, S3 and S4)

Contact-dependent hemolysis and pore formation

(Fig. 6)

Translocators insertion into

RBC membranes

(Fig. 7 and S8)

YopD complexes formation (Fig. 8, S9 and S10)

Yop effectors translocation (Fig.5, S5 and

S6)

Resistance to

macrophageuptake (Fig. 4)

Wild type 99% Yes Yes Yes Yes Yes Yes

YopDΔ256-275 0% ↔ ↓ ↓ ↔ ↓ ↓

YopDI262P 40% ↔ ↓ ↓ ↔ ↓ ↓

YopDK267P 0% ↔ ↓ ↓ ↔ ↓ ↓

YopDA263P 10% ↔ ↔1 ↔ ↔ ↔ ↔

YopDA270P 30% ↔ ↔1 ↔ ↔ ↔ ↔

↔ Mutant is functional equivalent to wild-type ↓ Mutant has partially lost function compared to wild type 1 YopD variants that produce smaller size pores compared to wild type

The observation that putative YopD coiled-coil mutants generated two

distinct phenotypes may be suggestive that this domain is likely to be a long α-helix rather being a coiled-coil per se. More importantly, this is the first report presenting single point mutants (YopDI262P and YopDK267P) defective in host contact-dependent T3SS, while maintaining regulatory control and growth at 37°C. Furthermore, these mutants are affected in insertion of both hydrophobic translocators into host cell membranes despite maintaining YopD-YopB and YopD-YopD complexes. These will therefore serve as valuable tools to study in isolation the dynamic process of translocon assembly in artificial or natural lipid membranes. One can assume that these particular mutations may have impaired the capacity of these proteins to target a specific domain/receptor of the target cell membrane. It is also possible that other bona fide protein-protein interactions with additional Yops have also been compromised, such as the YopD-LcrV interaction shown to be critical for the full function of the Yersinia spp. T3SS (Paper I).

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3.1.3.2. T3S translocon assembly intermediates attenuate Yersinia spp. virulence Given that all mutants maintained yops regulatory control and grew normally,

oral infections of BALB/c mice were preformed (Paper III, Fig. 9). Independent of the amount of ingested bacteria, the mutants YopDΔ256-275, YopDI262P and YopDK267P displayed a 100-fold ID50 attenuation in comparison to the parent. Unexpectedly, mutants producing YopDA263P and YopDA270P also displayed a degree of attenuation despite exhibiting no defect associated to T3S effectors delivery in several in vitro assays. Clearly, the whole animal creates a multifactorial environment that is far more stringent in simultaneously testing of multiple aspects of T3SS activity then our in vitro assays that examine only one aspect in isolation. However, mice infected with higher doses (108 and 109 CFUs/ml) of these mutants did experience some disease symptoms such as diarrhea and weight loss. Since this could indicate a different pattern of organs colonization, the bacterial load was measured in mesenteric lymph nodes (MLNs) and spleens of orally infected mice (Paper III, Fig. 10). All the five tested mutants were less efficient at colonizing the MLNs (2 to 40 fold reduction) and completely failed to colonize the spleen after 6 days of infection. Thus, even the mutants producing YopDA263P and YopDA270P that displayed no defect in T3S effectors delivery in vitro and some initial symptoms of infection in mice were critically affected in organ tissue spreading. This suggests that the symptoms observed in the infected mice with those strains must have been related to a massive immune response triggered by a hampered T3SS activity in vivo. Nevertheless, in these two mutants a critical property specifically required for the full in vivo translocon function must be absent from these pore, even though they function normally in vitro. To identify the reason for this defect might be experimentally difficult. One possible explanation for the virulence attenuation is that the assembled translocon has an incorrect stoichiometric form, which reduces optimal localization and function of injected effectors (Figure 10). In this way, the pore would be far more than just a mere conduit for effector passage to the host cell cytosol; the pore could ‘talk’ with effectors and direct their spatial delivery (Figure 10). YopD can also localize intracellularly. Hence, one other cause could be that YopDA263P and YopDA270P cannot gain access to the host cell interior to exert a possible effector function (Figure 10).

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Figure 10 – Molecular model for translocon biogenesis and control of effectors localization. YopD can establish interactions with itself, YopB and LcrV. Shaping a functional pore in the host cell membrane might require the contact of self-assembled YopD with LcrV at the tip of the needle or pre-integrated YopB in the host cell plasma membrane. It is also possible that YopD oligomerization can be triggered upon YopB-dependent host membrane integration. This type III translocon conduit may not be sufficient ensure correct localization of effectors in the host cytosol. Instead, translocated YopD (249) might have a role in ensuring the correct localization of effectors. YopD may even possess an effector function in its own right.

In summary, Paper III describes data suggesting that a C-terminal region of YopD facilitates at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the second to promote targeted activity of internalized effector toxins (Figure 10).

3.2. Influence of the YopD chaperone, LcrH, on Yersinia spp. T3SS regulation A mystery of Yersinia physiology is their requirement for calcium to grow at

37 °C. In contrast, depletion of Ca2+ ions restricts bacterial growth and triggers Yops production and secretion (232). While these conditions might be an in vitro artifact, they do appear to mirror a stimulus associated with intimate bacterial-target cell contact. Whatever the relevance, Yersinia avidity for calcium at 37 °C has been a

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powerful selection for the isolation and characterization of several Ysc-Yop T3SS regulatory components. However, integration of all these regulators into the Yersinia T3SS regulon has proven to be difficult given the complexity of environmental signals to which Yersinia spp. reacts.

Several class II T3S chaperones - those that target the hydrophobic translocators - including the SicA from S. enterica Typhimurium and IpgC from S. flexneri, augment the transcriptional output driven by the cognate AraC-like transcriptional activators InvF and MxiE, respectively (161). Upon secretion of their cognate translocator substrates, intracellular free chaperones then function as cofactors for the transcriptional activator promoting transcription of T3SS genes. It is of interest to known whether T3S chaperone LcrH - that target YopD and YopB - has a similar function towards the Y. pseudotuberculosis AraC-like transcription activator LcrF.

It is known that LcrH is capable of exerting a negative regulatory role at the post-transcriptional level when in complex with the cognate substrate YopD (212). LcrH-dependent negative regulation could be indirect through the stabilization and efficient secretion of its cognate partner YopD, a prominent negative regulator. Alternatively, as has been demonstrated in Y. enterocolitica, this YopD-LcrH complex may cooperate with the YscM1 and YscM2 regulatory elements to specifically bind yop mRNA and impose post-transcriptional silencing on Yops synthesis in the presence of calcium (in vitro), or prior to host cell contact (250). Strangely, neither YopD nor LcrH display any obvious nucleic acid binding motif, so how they then engage the yop mRNA template is unclear. Furthermore, it is not yet known how this binding is influenced by the presence of YscM1 and YscM2. This is a critical point considering that Y. enterocolitica SycD chaperone (LcrH homologue) can directly interact with YscM2 essentially a unique molecule in Y. enterocolitica (192,208). Hence, as both Y. pestis and Y. pseudotuberculosis lack YscM2, harbouring only the equivalent to YscM1 (and termed LcrQ) it is not known how relevant this SycD-YscM2 interaction actually is. It does, however, suggest that possible differences in regulatory control may exist within each of these three species.

In paper IV we further examined Yersinia LcrH (SycD) as a Ysc-Yop regulator In addition, we investigated how reciprocal the LcrQ/YscM proteins are in terms of establishing post-transcriptional regulatory control in Y. pseudotuberculosis and Y. enterocolitica.

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3.2.1. LcrH is not a cofactor of LcrF transcriptional activation Upon secretion of their cognate substrates, several LcrH-like T3S chaperones

act as cofactors of transcriptional activators to enhance transcriptional output of T3S genes. In Yersinia the possibility that free LcrH cooperates with the transcriptional activator LcrF to increase yop promoter activity has never been examined. To test this hypothesis, five different promoters from Y. pseudotuberculosis T3S genes representative of secreted proteins (YopE and YopK), chaperone proteins (SycE/YerA chaperone of YopE), components of the secretion apparatus (YscN) and a cytoplasmic regulator (LcrG), were cloned in a plasmid harboring a lacZYA promoter-less reporter and transferred into a lac deficient E. coli strain (Paper IV, Fig. 6). An additional expression plasmid encoding the lcrF transcriptional activator allele under an inducible promoter was co-transferred into the same background strain. As expected, the promoters were activated between ~5 to ~60 fold in the presence of the transcriptional activator LcrF (Paper IV, Table 3). However, when lcrH allele was also co-expressed in an additional expression vector, its presence did not significantly amplified the activity of the target promoters, indicating that free LcrH does not influence transcriptional output of LcrF on target yop promoters (Paper IV, Tab. 4).

The observation that LcrH does not function as a cofactor for the transcriptional activator LcrF, suggests that T3S class II chaperones might have evolved different regulatory networks to respond to environmental specificities of individual pathogens. Indeed, the intracellular lifestyle of S. enterica Typhimurium and S. flexneri versus the extracellular preference of Yersinia might account for these regulatory differences. Moreover, it is plausible that additional regulatory molecules are needed for LcrH to exert its transcription activation cofactor function. Considering that an intimate interaction between LcrH and YscY is essential for Yops regulatory control maintenance (215), the involvement of an LcrH-YscY complex as a cofactor for LcrF transcriptional activator might have to be analyzed.

3.2.2. LcrH and post-transcriptional regulatory control in Y. pseudotuberculosis From previous work, it was evident that LcrH N-terminus may exert a T3S

regulatory function that is independent of YopD stabilization and secretion (212,215). Indeed, when the LcrHE30G variant is produced in trans in a ΔlcrH mutant, it fails to complement its Ysc-Yop regulatory deficiencies, despite restoring stable

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intracellular YopD and its efficient secretion. Therefore, analysis of this mutant could uncover direct LcrH regulatory effects. When this mutation was introduced in single copy in cis onto the Y. pseudotuberculosis virulence plasmid, the bacteria generated stable YopD and YopB proteins capable of being secreted to the extracellular media, confirming the ability of LcrHE30G to fulfill the chaperone role of the two cognate substrates (Paper IV, Fig. 1). Significantly, this mutant represses Yop and Lcr protein synthesis under non-inducing conditions and produces more protein under inducing conditions, while also display altered low-calcium dependent growth profiles that altogether suggested subtle changes to Ysc-Yop regulatory control (Paper IV, Fig. 1 and Table 2). An obvious deviation from the parental Yops control was observed for LcrV secretion, which from the LcrHE30G mutant was more akin to the levels derived from a ΔlcrH or ΔyopD mutant, suggesting that an LcrH-dependent post-transcriptional regulatory complex might not control translation of all genes to the same extent (Paper IV, Fig. 1). This observation supports data from Chen and colleagues who found that purified LcrH-YopD complexes have greater affinity for yop transcripts rather lcr transcripts possibly ensuring a expression hierarchy during the low calcium response (244).

The behavior of the LcrHE30G mutant was additionally examined when the established negative regulatory factors YopD and LcrQ, were trapped inside the bacterial cytoplasm. This was achieved either by inducing a defect in T3S (via a ΔyscU mutation) (Paper IV, Fig. 1) and over-expressing LcrQ in trans (Paper IV, Fig. 2). The former resulted in a repression of Yop synthesis, although not to the extent of the ΔyscU mutant alone (Paper IV, Fig. 1), while in the second assay LcrHE30G was blind to high LcrQ levels (Paper IV, Fig. 2). The data indicate that LcrHE30G can respond to YopD accumulation, but not to LcrQ. This suggests that the over-riding negative repressive element involves LcrH and YopD, while LcrQ might be important but plays some other role.

It is not clear what is the role of LcrQ. YscM2 of Y. enterocolitica interacts with SycD (LcrH) and YscM2 is thought to function with YscM1 (LcrQ) in the tripartite regulator complex with LcrH-YopD. However, YscM2 is unique in Y. enterocolitica. Therefore, it was necessary to test if LcrH could interact with LcrQ. Using GST fusions, a LcrH-LcrQ interaction was not verified; only the established LcrH-YscM2 interaction (Paper IV, Fig. 4). Hence, data in Paper IV suggest that Y. pseudotuberculosis, LcrH-YopD complex are repressive for Yop synthesis, and this may not necessarily involve LcrQ. However, LcrQ must still be important since in its absence Yop synthesis control is lost.

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3.2.3. Functional exchangeability of the YscM/LcrQ regulatory family in Yersinia spp. Although YscM1 and YscM2 from Y. enterocolitica and LcrQ from Y.

pseudotuberculosis are considered functionally equivalent, experimental evidence of functional exchangeability between the bacterial proteins has not been confirmed. This is particularly relevant given that the YscM1 and YscM2 share a divergent protein sequence homology to LcrQ of 99.1% and 59.1% respectively (Paper IV, Fig. 3). To test for functional exchangeability, a complementation assay was preformed where yscM1 or yscM2 alleles were expressed in a Y. pseudotuberculosis ΔlcrQ mutant. Surprisingly, YscM1 failed to complement the ΔlcrQ mutant despite the high level of homology to LcrQ (one residue difference) (Paper IV, Fig. 5). This inability might suggest that either YscM1 needs the presence of YscM2 to exert its function, supporting observations that suggest a possible YscM1-YscM2 heterodimer (208), or with less likelihood, the residue at position 48 that differs between LcrQ and YscM1 sequence is critical for the protein to establish critical molecular interactions. Even more intriguing was the expression of YscM2 in the same background caused a potent impairment of Yops production to a far greater extent than when LcrQ was produced ectopically in parental bacteria (Paper IV, Fig. 5). It is possible that the interaction between LcrH and YscM2 reported here and by others has in someway prevented Yop synthesis (192,208) (Paper IV, Fig. 4). Whatever the reason, it is clear from these results that LcrQ, YscM1 or YscM2 are not functionally interchangeable.

In conclusion, in Paper IV we rule out a possible role for free LcrH as a cofactor for the transcriptional activator LcrF. However, a comparison between ΔlcrH, ΔyopD and LcrHE30G mutants revealed alterations in Yops regulatory control that suggests other regulatory functions of LcrH. Finally, the functional differences observed between the YscM and LcrQ regulatory proteins might indicate that the elegant post-transcriptional model proposed for Yops synthesis inhibition presented for Y. enterocolitica might require a refinement for Y. pseudotuberculosis. Therefore, a definitive mechanism for post-transcriptional control on yops mRNA translation imposed by a putative YopD-LcrH-LcrQ (YscM1 and YscM2 in Y. enterocolitica) complex in Y. pseudotuberculosis is still unknown.

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Main findings in this thesis

• Hydrophobic residues within the amphipathic α-helix domain are essential for the multiple functions of YopD in Ysc-Yop T3S.

• YopD mutants solely affected in yops regulatory control have been isolated.

• YopD and YopB strongly interact, and this must be a requirement for translocon assembly and Yops effectors delivery.

• YopD multimerization is a requirement for aspects of YopD function.

• YopD binding to LcrV facilitates T3S.

• A putative N-terminal helical structure predicted only in YopD from pathogenic Yersinia is essential for full disease progression in a murine infection model.

• A predicted YopD C-terminal helical segment is integral for dynamic translocon assembly in eukaryotic cell membranes and Yop effector delivery.

• The function of this region is essential for Yersinia virulence.

• Multiple YopD mutants create a versatile toolbox for dissecting individual functions of YopD in isolation.

• LcrH does not augment LcrF transcription of target genes.

• LcrH possibly possess additional yop-regulatory roles.

• Function of the YscM/LcrQ regulatory family may not be fully conserved within human pathogenic Yersinia spp.

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Future perspectives The work presented in this thesis thoroughly dissected the functional domains

of YopD that enable a significantly better understanding of the underlying structural and regulatory mechanisms of Ysc-Yop-dependent effector delivery by Y. pseudotuberculosis. However, within this field, there are many unsolved questions regarding the mechanism of translocon assembly and how this contributes to translocation control of effectors in route to the host cell interior.

Studying the contribution of particular YopD residues located in structural/functional domains has generated important genetic tools that can be used for the study of translocon assembly and translocation dynamics. We now have mutants that can be used to better comprehend any molecular interplays between the YopD translocator and particular lipid or proteinaceous components from host membranes. Using artificial or natural lipid membranes it might be possible to clarify whether a specific lipid moiety or host receptor is critical to trigger a translocon assembly mechanism. Yet, other YopD derivatives will permit an investigation into the dynamic nature of an assembled translocon, with particular emphasis on defining its multiple roles that appear to extend well beyond just pore formation by ensuring appropriate and efficient effector intracellular localization to hamper host phagocytic activity.

The initiation of the translocon assembly process that precedes translocon assembly in target cell membranes is not fully understood. It would be important to understand how LcrV from the distal end of the needle-tip assists the assembly of the two hydrophobic translocators into a functional translocon. Therefore, to comprehend whether translocons are pre-assembled on the tip complex or prior to contact and/or insertion of YopD and YopB into the host membrane, will raise our understanding of how Yersinia spp. orchestrate translocon assembly. To spatially demonstrate where the translocon is formed will also provide additional experimental indications that can be used to gain insights on the adopted architecture of a functional translocon. This may not be a trivial task given the apparent dynamic nature of the translocon which might create additional difficulties to isolate a defined structure. It is even more difficult to obtain indisputable proof that effectors actually do pass through the translocon in route to the host intracellular environment; no doubt this is one of the ultimate challenges in the T3SS field.

A recurrent mystery of pathogenic Yersinia physiology is the post-transcriptional regulatory mechanism by which the bacteria silence Yops synthesis in the presence of calcium (in vitro) or in the absence of host cells (in vivo). Timely

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repression of yops mRNA translation before host cell contact has been attributed to a tripartite complex formed by YopD-LcrH-LcrQ. However, no direct experimental evidence has demonstrated the involvement of this complex in preventing yops mRNA translation or promoting its degradation. Moreover, the arrangement and the contribution of each individual protein to shape this regulatory complex are not understood. Therefore, some of our isolated LcrH and YopD mutants that are exclusively affected in regulatory control maintenance will be critical to elucidate the molecular mechanisms of this regulatory complex. We will therefore develop assays to determine the cross-talk between our mutants their counterparts as well as to target mRNA. Such studies should provide valuable insights into the validity of this tripartite-dependent regulatory function.

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Acknowledgments During these years at Umeå I was lucky to have the opportunity to know many

persons that have contributed, in one way or the other, to make my life easier in the cold and dark North of Sweden. I am very grateful to all of you.

I would like express my gratitude to my PhD supervisor Matthew Francis for giving me the opportunity of starting my research career within his group. Thank you for everything that you have taught me, contagious enthusiasm for science and for always finding time to discuss science with me. Thank you for your guidance during the writing of this thesis.

I would like also to thank Åke Forsberg, Hans Wolf-Watz and Maria Fällman for various thoughtful suggestions during the course of my PhD studies. I would like to extend my gratitude to all the members of our Yersinia + Vibrio + Pseudomonas journal club for the great scientific discussions along these years. I have learned a lot from you all.

Now to the past and present members of the Francis lab. Junfa – for being so supportive in my initial steps in the lab, it is always a pleasure to discuss whatever topic with you. Ayad – we have started this long marathon together, I wish you all the best for your thesis writing and for your future career. Edvin – for always being friendly and helpful, good luck with your studies. Ikenna – I admire your serenity and efficiency, good luck for you and your family. Katrin, Jyoti, Damien, Salah, Simone….. it has been a pleasure the time we spent together in the lab.

To all the people with whom I collaborated during these years, in particular to Tomas (keep your never-ending ideas) and Anna (thanks for all your help with the mice experiments). To Kemal and Ummehan for your friendship, fishing trips and the nice Turkish coffee. Helen for the nice parties at your place, Frederic for all the laughter, Connie for the nice get-together and spontaneous comments, Buphender for all our nice conversations (and some of your jokes), Nico for your transparency. Shen – for your friendship and for always being available to help me (especially when my car got broken!). Konstantin – I am very grateful for the proofreading that you made in this thesis.

Teresa y Martin, me siento muy afortunado de haberos conocido y sentido tan cercanos durante estos años en Umeå. Gracias por vuestra amistad y apoyo. Katia y Toni, aunque el tiempo que hemos coincidimos fue breve, ha sido suficiente para entablar una bonita y sincera amistad. Gracias por vuestra cercanía y apoyo. Obrigado Lisandro por estares sempre tão proximo, é um prazer enorme ser teu amigo. Os meus melhores desejos para ti, Maria, Alva e Thea.

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Para aquelas pessoas que marcaram a minha infancia e juventude em Valezim: Bruno, Antonio, Alberto, Carlos Jorge, Carlos Martins, Isabel Martins, entre muitos outros, obrigado pela vossa amizade, apoio e interesse na minha vida academica. Obrigado Tiago, Tanea e Afonso pela vossa amizade que perdura e se solidifica apesar da distância geografica.

Para Rosa, Antonio y Javier, muchas gracias por vuesto apoyo, cariño y presencia constante. Ángel, Ascen, Daniel, Nieves, Julio, Mariano, Amparo, Jose, Adrián, Iñaki, Cristina, Javier, Esther.... a cada uno de vosotros os doy las gracias porque desde el primer momento me hicisteis sentir uno más de la familia.

Para a minha familia, pelo seu apoio incondicional em todas as decisões que tomei durante o decurso da minha vida. Obrigado pela educação, principios e valores que incutiram durante o processo de construção da pessoa que sou hoje. O mais profundo agradecimento à minha Mãe pelo legado que deixou em mim para toda a minha vida. Aos meus quatro avós João, Maria, José e Maria por serem os pilares de toda a familia e um exemplo para mim. Obrigado pelo vosso carinho. Aos meus tios Francisco, Margarida, Filomena, Gastão, Alice, Adelino e Manuela assim como a todos os meus primos por me proporcionarem um ambiente familiar ideal. Eduarda, pelo seu apoio e carinho que sempre me demonstra no dia-a-dia.

Pai, esta tese não é só o resultado do meu trabalho, é também o reflexo de toda a educação, exemplo e amor que sempre me transmitiste a cada momento. Dedico-te com todo o meu carinho esta tese. É um orgulho para mim ser teu filho.

Patricia, o teu apoio, carinho e encorajamento constante foram fundamentais para superar os momentos mais dificeis que atravessei durante esta etapa da minha vida. O teu amor, conselhos e sábias opiniões iluminaram inumeras vezes o caminho que iniciamos e percorreremos juntos.

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