Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for host cell invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. The aims of this study are twofold. The first is to expand the repertoire of known effector proteins using modern proteomics techniques. The second is to explore the function of a subset of effector proteins to better understand their interaction with host cells. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain as well as a mutant incapable of effector protein secretion. This study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. To determine the possible function of two effector proteins, BipC and BapA, a yeast two-hybrid system was used to identify host cell proteins the effectors interact with. The proteins were screened against a library of human proteins for interactions. BapA interacted with 2 proteins while BipC interacted with 14. Both BapA and BipC were shown to interact with human C1QBP, a mitochondrial protein involved in inflammation, immunity and autophagy. Finally, the Bsa T3SS protein BipC was characterised in its ability to interact with actin. This study is the first evidence that BipC has the ability to bind to filamentous actin, but not monomeric actin. This binding is direct and no intermediate proteins are required for the interaction. Ectopic expression of BipC in eukaryotic cells caused cytoskeletal rearrangements consistent with an actin-binding protein. The core secretome represents a substantial resource of targets that will be mined for improved diagnostic assays and vaccines. Diagnostics that will detect early stages of disease to allow for more effective antimicrobial intervention are currently lacking. Furthermore, there is scope to design diagnostic assays with dual use such as to detect both melioidosis and infection of cystic fibrosis patients with the closely related opportunistic pathogen B. cepacia. The description of novel T3SS effector proteins is also of considerable value since T3SS proteins are often potent B- and T- cell antigens representing promising components of sub-unit vaccines. Such effector proteins commonly modulate cellular processes such as phagocytosis, inflammasome activation and cell cycle progression, hence the function of the predicted T3SS effectors will provide a series of future research opportunities.

Chlamydia trachomatis is a bimorphic, obligate intracellular parasite that develops entirely within a membrane-bound vacuole. Chlamydiae manipulate eukaryotic hosts by translocating effector proteins into the host cell. Chlamydia genomes encode a type III secretion system (T3SS) which is a likely mechanism for delivery of these effectors. The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early-cycle events. For example, the T3S-dependent chlamydial substrate Translocated actin-recruiting phosphoprotein (Tarp) is present in infected cells as early as 5 minutes post infection, and it is likely that EBs translocate other proteins in a T3S-dependent manner. Evidence is presented herein that the C. trachomatis protein CT694 represents an early-cycle-associated effector protein. CT694 is secreted by the heterologous Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK and the C-terminus of AHNAK2. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. In addition, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibers that correlated with the ability of CT694 to interact with AHNAK. Domain differentiation studies indicated that the C-terminus of CT694 is required to interact with AHNAK and a domain within the N-terminus localizes CT694 to the plasma membrane and triggers aberrant morphological changes in HeLa cells. CT694 function is conserved in other C. trachomatis serovars and C. muridarum, but not by similarly positioned genes of C. pneumoniae or C. caviae. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development unique to this species.

Enterohaemorrhagic Escherichia coli (EHEC) strains are associated with gastrointestinal and severe systemic disease in humans. EHEC O157:H7 is the most common serotype causing human infections in North America and the UK. Human infections mainly originate from cattle, through either direct contact with infected animals or indirectly through contamination of food or water with animal faeces. From the sequencing of EHEC O157 strains, it is clear that the genomes contain multiple prophages, many of them cryptic, which define this E. coli pathotype. These regions include the locus of enterocyte effacement (LEE) which is a critical horizontally acquired pathogenicity island and encodes a type III secretion system (T3SS). The T3SS translocates effector proteins into epithelial cells that enable tight attachment to these host cells and also modify innate responses and other cellular functions to promote persistence in the animal host. The T3SS is essential for the colonisation of cattle by EHEC O157 where it is localised to the terminal rectum. The regulation of T3S is complex with many regulators and environmental factors already identified. Previous work has demonstrated marked variation in the levels of T3S among EHEC O157 strains. The aim of this research was to further investigate the regulation of T3S towards two objectives: (1) to understand the localisation of EHEC O157 at the terminal rectum of cattle; (2) to understand the strain variation in T3S. (1) In relation to rectal and mucosal colonisation, established aerobic/anaerobic regulators were investigated including arcA, fnr, narX, narQ. Briefly, arcA, fnr, narX, narQ were deleted in an E. coli O157 strain ZAP198 by lambda red recombination. Apart from the fnr mutant which showed lower levels of T3S, the remaining mutants displayed similar T3S protein levels compared to the wild type strain. In addition, no significant changes in adherence and A/E lesion formation capacity were measured for the mutants following interaction with bovine epithelial cells. (2) Strain secretion variation was approached in two ways; the first was to control expression from the LEE1 operon, required for T3S expression, in order to both induce expression and examine the importance of downstream regulation. The second was to investigate variation in T3S between different phages types of EHEC O157. While attempts to construct an inducible T3SS were not successful, intermediate strains made in the process have been useful to dissect how regulators being studied in the laboratory control T3S. The main novel insights from the research have come from examining T3S in different EHEC O157 phage types. We found that the average level of T3S in PT 21/28 strains was lower than in PT 32 strains. Interestingly, most (90%) of PT 21/28 strains contained both Stx2 and Stx2c phages. In contrast, only 28% of PT 32 strains had both phages. Taken together, this raised the possibility that Stx phage integration might have a repressive impact on T3SS regulation in E.coli O157:H7. This hypothesis was addressed using a number of different approaches. Deletions of Stx phages were constructed and these had increased levels of T3S when compared to the parental strains. This phage regulation of T3SS was confirmed in an E. coli K12 background by examining an induced LEE1 reporter in the presence and absence of a transduced Stx2 phage. In addition, it was shown that deletion of the CII phage regulator led to increased T3S and may contribute to the Stx phage repression reported above. This work demonstrates for the first time that Stx phage integration represses T3S expression. It is proposed that this control may limit immune exposure of this critical colonisation factor and that the repression actually allows activation by prophage encoded regulators, including PchA/B, that co-ordinate T3S and non LEE-encoded effector expression to promote epithelial cell colonisation.

There are three human pathogenic species of the genus Yersiniae: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. To cause disease, these strains inhibit several key innate defense mechanisms, including phagocytosis, the critical process for bacterial clearance. The ability of Yersinia to evade the immune defense is dependent on delivery of virulence effectors, Yersinia outer proteins (Yops), into the interacting cell by a mechanism involving the type III secretion machinery. We have shown that the virulence protein YopK plays an important role in the control of Yop effector translocation via a feedback mechanism involving another virulence protein, YopE. We also found that YopK participated in regulation of Yop effector translocation by modulating level and ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, using a yeast two-hybrid screen with YopK as a bait, the eukaryotic protein RACK1 was identified as a target for this virulence protein. We found that RACK1 was engaged upon Y. pseudotuberculosis-mediated β1-integrin activation, where it was recruited to phagocytic cups. Downregulation of RACK1 by RNAi resulted in a reduced ability of Y. pseudotuberculosis to block phagocytosis, indicating that RACK1 is required for efficient Yersinia-mediated antiphagocytosis. Based on our data, we suggest a model where Yersinia, via YopK, targets RACK1 to ensure a directed delivery of the Yop effectors to the “right place” where they bind to and inactivate their targets, resulting in efficient inhibition of phagocytosis.   A yopK mutant strain over-delivers Yop effectors, but is still avirulent in mice, indicating that YopK is important for the fine-tuning of effector protein delivery during infection. To analyse this, we investigated the importance of YopK during in vivo infection. We found that a yopK mutant colonized Peyer’s patches and the mesenteric lymph node more rapidly compared to wild-type Y. pseudotuberculosis, but was unable to spread systemically to liver and spleen and cause full disease in mice. Further, we showed that a yopK mutant was able to colonize liver and spleen and cause full disease in mice lacking the main phagocytes, polymorphonuclear leukocytes (PMNs). We also showed that YopK was important for Yersinia-mediated silencing of the PMN response. To summarize, we suggest that YopK is important for Yersinia to evade the PMN defense and thereby spread systemically and cause disease. YopK is proposed to do this by allowing a controlled, directed Yop effector delivery that is just sufficient to inhibit host immune defense mechanisms. The controlled and precise delivery of virulence effectors avoids inappropriate triggering of PMNs and thereby an enhanced immune response favoring the host.

No description available.

Pathogenic Gram-negative Yersinia species preferentially target and inactivate phagocytic cells of the innate immune defense by translocation of effector Yersinia outer proteins (Yops) into the cells via a type III secretion system. This indicates that inactivation and avoidance of the early innate immune response is an efficient way for Yersinia species to avoid elimination and to cause diseases ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y. pestis). In this project, we aimed to study the interaction between enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection. In situ interaction studies on infected intestinal tissues showed that Y. pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of the intestine during initial infection. After massive recruitment of polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt) bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to spread systemically. Interestingly, our interaction assay showed that these mutants not only interacted with DCs, but also with PMNs during the initial stage of infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-dependent manner, while both YopH and YopE were shown to be involved in the blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH has cell type-specific effects. To further investigate the role of DCs during initial stages of infection, a mouse DC depletion model (CD11c-DTRtg) was applied. However, the DTx-mediated depletion of DCs in CD11c-DTRtg mice induced neutrophilia and the model could not give a definite answer to whether DCs play an important role in either restricting or stimulating progression of Y. pseudotuberculosis infection. To investigate involvement of PMNs during early infection mice were injected with the depleting antibody α-Ly6G. In absence of PMNs wt, as well as yopH and yopE mutants became more virulent, which further supports the importance of these Yops for the ability of Y. pseudotuberculosis to disseminate from the initial infection sites in the intestine to cause systemic disease. In summary, our studies show that inhibiting internalization and maturation of DCs and avoiding phagocytosis by and interaction with macrophages and PMNs during the early stages of infection are important virulence strategies for Y. pseudotuberculosis to be able to colonize tissues, proliferate and disseminate systemically.

ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

Shigella spp. are Gram-negative, non-motile bacterial pathogens that are the causative agent of bacillary dysentery in humans. During infection, Shigella utilize a complex type three secretion system (T3SS) to inject effector proteins and take over host cell functions. With the rise of multi-antibiotic resistant Shigella strains, the T3SS is a promising alternative therapeutic target. While the needle and syringe-like apparatus of the T3SS has been extensively studied in Shigella, several components and mechanisms of this system remain unclear. The research presented here addresses two major knowledge gaps in the current understanding of the T3SS ATPase, Spa47, and the initial host-pathogen interaction at the tip of the apparatus. In this work, high resolution crystal structures of Spa47 guided the creation of an oligomer model which suggested ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Mutagenesis experiments targeting predicted active site residues and the oligomerization domain revealed that active site residues alone are not responsible for Spa47 oligomerization while protein oligomerization is crucial for ATPase activity. Together with in vivo experiments, we show that ATP hydrolysis and proper Spa47 oligomer formation is critical for T3SS apparatus formation, effector secretion, and overall Shigella virulence. Additionally, we have combined the Langmuir Blodgett technique with fluorescent microscopy to visualize the interaction between key T3SS tip proteins with defined artificial phospholipid membranes. These membranes were generated using Langmuir Blodgett which provided control over lipid phase and composition. Lipid phase and protein localization were monitored using lipophilic dyes and selective fluorescent protein labeling. These experiments suggest a differential interaction between the tip protein IpaB with the membrane components cholesterol and sphingomyelin based on IpaB oligomerization. IpaC, another T3SS tip protein, was found to destabilize membranes when alone, but was stabilized in the presence of IpaB. These experiments suggest that IpaB confers IpaC stability within membranes and that tip protein localization is dependent on lipid phase and composition. Overall, these new insights into the T3SS ATPase and tip proteins provide a more complete understanding of Shigella virulence that will aid in future endeavors to identify alternative therapeutic targets for treatment.

O cancro cítrico é considerado na atualidade uma das doenças mais perigosas e prejudiciais à citricultura brasileira e mundial, devido aos danos causados na produção e qualidade dos frutos, sendo a Xanthomonas axonopodis pv. citri (Xac) a bactéria fitopatogênica responsável por tais prejuízos. Nosso laboratório iniciou estudos de identificação e análise funcional das interações proteína-proteína de Xac envolvendo sistemas importantes para sua patogenicidade (Alegria et. al., 2004). Nosso objetivo principal foi o estudo funcional e fisiológico de interações já identificadas entre proteínas do sistema de secreção do tipo III (T3SS) da Xac. O foco de nossa pesquisa foi tentar desvendar a importância biológica, na patogenicidade de Xac, das interações proteína-proteína: HrpB2-HrcU; HpaA-HpaB-HrcV; HrpD6-HrpB1- HrpW. Com este intuito clonamos, expressamos e purificamos as proteínas recombinantes. Produzimos soros policlonais específicos contra cada uma das proteínas citadas acima. Estudamos a interação entre as proteínas in vitro por meio de técnicas como Far-Western Blot, Pull Down, fluorescência e dicroísmo circular. Outro enfoque do nosso trabalho foi monitorar a contribuição individual destas proteínas no desenvolvimento da doença in planta. Para isso produzimos cepas de Xac mutantes para os genes hrpB2, hrcU, hpaA, hpaB, hrpB1 e hrpG. Os nocautes não polares foram infiltrados em plantas de laranja pêra, assim como também as cepas de complementação correspondentes, e assim foi testada a habilidade de desenvolver o cancro cítrico e/ou reverter os sintomas da doença. Também foi monitorada a capacidade de multiplicação e sobrevida in planta das cepas Xac ΔhrpB2, ΔhrcU e ΔhpaB, assim como a secreção das proteínas HrpB2 e HpaA pelo T3SS de Xac. Estudamos com mais detalhe a possível função de HrpB2 no T3SS de Xac, desenvolvendo experimentos para determinar a região da proteína imprescindível para sua função permanecer inalterada. Realizamos mutações sítio dirigidas, a fim de introduzir códons de terminação em diferentes regiões da proteína e testar a habilidade desses fragmentos de reverter os sintomas da doença na planta. Monitoramos a capacidade de proteínas mutantes de reverter fenótipos de patogenicidade em citrus, ausentes na cepa Xac ΔhrpB2 e revertidos na cepa de complementação Xac ΔhrpB2+pUFR047_hrpB2. Desta maneira, determinamos que os últimos seis aminoácidos de HrpB2 estão envolvidos no desenvolvimento da/s função/ões em Xac. / Citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is a disease with significant economic consequences for the Brazilian and global citrus industry due to reductions in production and fruit quality. Our laboratory has initiated studies for the identification and functional analysis of protein-protein interactions involving Xac systems involved in pathogenicity (Alegria et. al., 2004). One objective has been to study functional and physiological interactions between proteins that make up the Xac Type III secretion system (T3SS). The focus of the present study is to unravel the biological significance in Xac pathogenicity of the following previously identified protein-protein interactions: HrpB2-HrcU; HpaA-HpaBHrcV; HrpD6-HrpB1-HrpW. With therefore cloned, expressed and purified the above-mentioned recombinant proteins. Specific polyclonal serum were produced and interactions between the proteins were studied in vitro using a variety of methods, including Far-Western Blot, Pull Down, fluorescence and circular dichroism. To monitor the individual contribution of these proteins in disease development in planta, we produced mutant Xac strains in which the hrpB2, hrcU, hpaA, hpaB, hrpB1 and hrpG genes were disrupted. The nonpolar knockouts as well as the corresponding complementation strains were infiltrated into Citrus sensensis plants and the development of citrus canker symtoms and bacterial proliferation in planta was evaluated. We also evaluated the T3SS-dependent secretion of proteins HpaA and HrpB2 by these Xac mutant strains. Structure-function relationships of the HrpB2 protein were studied in more detail. We developed experiments to determine the region of the protein essential for its function. We produced a series of hrpB2 mutants which were used to complement the hrpB2 knockout strain and evaluated their abilities to reverse the symptoms of the disease in the plant. The results demonstrate that the last six amino acids HrpB2 are important for its function in the development of disease symptoms by Xac.

Salmonella Gallinarum (SG) et Salmonella Enteritidis (SE) sont deux sérotypes génétiquement proches. Pourtant, chez la volaille, SG induit une infection systémique létale tandis que SE est responsable d’une infection systémique transitoire et d’un portage intestinal asymptomatique. De plus, SE est un sérotype ubiquiste tandis que SG est spécifique d’hôte. Outre ces différences in vivo, ces deux sérotypes présentent des différences in vitro. Nous avons montré que SG est in vitro moins invasif que SE. Ce phénotype est indépendant de l’origine aviaire ou non-aviaire des cellules en dépit du tropisme de SG pour la volaille. LeT3SS-1, le facteur d’invasion majeur chez Salmonella, est composé d’un appareil de sécrétion et des effecteurs transloqués par celui-ci. Nous avons montré que les composants du T3SS-1 sont autant exprimés chez SG que chez SE et que tous deux possèdent un appareil de sécrétion fonctionnel. Pourtant, l’étude des capacités d’invasion dépendantes du T3SS-1 suggère que ce facteur est impliqué dans le défaut d’invasion de S. Gallinarum. L’analyse des gènes codant les effecteurs transloqués par le T3SS-1 a montré qu’il existe des mutations chez SG pouvant être à l’origine de ce défaut d’invasion. / Salmonella Gallinarum and Salmonella Enteritidis are genetically closed. However, whereas SG induces lethal systemic infection in poultry, SE is responsible for transient systemic infection and asymptomatic intestinal carriage. Moreover, SE is ubiquitous whereas SG is poultry specific. These serotypes also present in vitro differences. We have shown that SG is in vitro less invasive than SE whatever the avian or non-avian cell origin, in spite of SG’s tropism for avian species. T3SS-1, the main invasion factor in Salmonella, is composed of a secretion apparatus and its translocated effectors. We have shown that T3SS-1 components a expressed in a similar way by SE and SG and both harbor a functional secretion apparatus. However, study of T3SS-1 dependent invasion abilities suggested that T3SS-1 is involved in SG’s invasion defect. Sequence analysis has revealed that SG possesses several mutations in genes encoding main T3SS-1 effectors, that could explain the low invasive phenotype.