There are two main human pathogens in the family of Chlamydiaceae. Different serovars of Chlamydia trachomatis cause sexually-transmitted disease and eye infections whereas C. pneumoniae (TWAR) is a common cause of community-acquired respiratory infection. Chlamydia species are obligate, intracellular bacteria sharing a unique developmental cycle that occurs within a protected vacuole termed an inclusion. These microorganisms can be distinguished by two different forms: the infectious, metabolically inert elementary body (EB) and the reproducing non-infectious form, termed the reticulate body (RB). The cycle is terminated when re-differentiation of RBs back to infectious EBs occurs. Chlamydia possesses a type III secretion system (T3SS) essential for delivery of effector proteins into the host for host-cell interactions. This virulence system has been systematically characterized in several mammalian pathogens. Due to lack of a tractable genetic system for Chlamydia species, we have employed chemical genetics as a strategy to investigate molecular aspects of the T3SS. We have identified that the T3S-inhibitors INP0010 and INP0400 block the developmental cycle and interfere with secretion of T3S effector proteins in C. pneumoniae and C. trachomatis, without any cytotoxic effect. We have further shown that INP0010 decreases initiation of transcription in C. pneumoniae during the early mid-developmental cycle as demonstrated by a novel calculation, useful for measurement of transcription initiation in any intracellular pathogen. The mechanism regulating the signal(s) for primary as well as terminal differentiation of RBs has not been defined in Chlamydia. We show using T3S-inhibitors that INP0010 targets the T3SS and thereby arrests RB proliferation as well as RB to EB re-differentiation of C. pneumoniae as where INP0400 targets the T3SS and provokes a bacterial dissociation from the inclusion membrane presumed to mimic the natural occurrence of terminal differentiation. The effect of INP0010 on iron-responsive genes indicates a role for T3S in iron acquisition. Accordingly, our results suggest the possibility that C. pneumoniae acquires iron via the intracellular trafficking pathway of endocytosed transferrin. Moreover, we have for the first time presented data showing generalized bone loss from C. pneumoniae infection in mice. The infection was associated with increased levels of the bone resorptive cytokines IL-6 and IL-1beta. In addition, an increased sub-population of T-cells expressed RANKL during infection. Additionally, C. pneumoniae established an infection in a human osteoblast cell line in vitro with a similar cytokine profile as seen in vivo, supporting a causal linkage. Collectively, these data may indicate a previously unknown pathological role of C. pneumoniae in generalized bone loss.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in travellers and young children in developing countries. We previously characterized three vaccine candidates (MipA, Skp, and ETEC_2479) which effectively protected mice in an intranasal ETEC challenge model after immunization. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. In this study, we examined the potential of these antigens to affect the host intestinal microbiota and subsequently found no significant impact on healthy of host after vaccination. In addition, we also optimized the types of adjuvants and forms of antigens and evaluated the efficacy in a mouse intranasal challenge model. Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that cause global public health threads. EHEC possesses the potential to cause gastroenteritis, hemorrhagic colitis and hemolytic uremic syndrome (HUS), which may lead to renal failure. Type III secretion system (T3SS) is a hallmark of EHEC, characterized by the needle-like structure and a variety of effectors injected into host cells. NleB, one of T3SS effectors, is a glycosyltransferase with the ability to catalyze the transfer of N-acetyl-D-glucosamine (N-GlcNAc) to host proteins to suppress the activation of NF-kB signaling pathway. In this study, we employed luminescence-based glycosyltransferase assay and high-throughput screening using a chemical library of various compounds. A total of 128 chemicals was selected with significant inhibition on NleB glycosyltransferase activity for further pharmaceutical study as novel therapy against EHEC infection.

Shigella are bacteria that are responsible for millions of infections and hundreds of thousands of deaths every year. The emergence of antibiotic resistant Shigella adds to the potentially devastating effect that these bacteria can have on human health. Shigella flexneri utilize specialized molecular machinery called the Type III secretion system to infect humans and cause disease. Research of this machinery promises to provide the knowledge, tools, and direction for the development of new avenues to combat shigellosis. This dissertation presents studies of two Shigella proteins, invasion plasmid antigens C and D (IpaC and IpaD). These proteins are part of a syringe and needle like protein structure that allows Shigella to secrete proteins directly into the host that hijack host cells to benefit support Shigella infections. IpaC and IpaD are part of a protein tip complex that is directly involved in these Shigella-host (e.g. human) interactions. We have advanced the biochemical tools for the in vitro study of IpaC by utilizing a new way to isolate it. This purification methodology allowed us to look at one of IpaC’s main roles, to interact with the host cell membranes. We examined IpaC’s role and tried to identify the parts of IpaC responsible for some specific interactions. We found that the parts of IpaC we believed were responsible were not but that the composition of the membrane IpaC is interacting with is more important than we previously believed. Finally, we examined a rare part of IpaD structure to determine its role. We determined that this rare feature is required for IpaD to sense Shigella’s host environment and prepare the bacteria to infect, making a promising target for anti-infective treatments against Shigella infections. Our findings advance the understanding of key molecular mechanisms that are required for Shigella virulence. We expect that our findings will aid future researchers as the pursuit for new treatments for shigellosis continues.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.

The gram-negative marine bacterium Vibrio parahaemolyticus is a pathogen and a common worldwide cause of seafood-associated gastroenteritis. When grown on a surface, V. parahaemolyticus undergoes a dramatic differentiation to an elongated, highly flagellated swarmer cell from the short rod typical of swimming cells. Swarming motility is a complex form of adaptation to growth on a surface, and we developed a set of microarray experiments to examine the global gene expression changes that occur upon differentiation to the swarmer cell. We hypothesized that growth on a surface would elicit a specific response involving genes for motility and surface colonization and not the broad changes in physiology suggested by others to be co-regulated with swarming motility. By taking advantage of the two known signals required for swarmer cell induction (inhibiting polar flagellar rotation and limiting iron), the swarming response was artificially induced in liquid and used to define the set of genes associated with surface sensing by transcriptome analysis. This approach avoided the confounding physiological differences between growth in liquid and growth on a surface. Fifteen microarrays performed with different strains and growth conditions were used to define a concise set of about 70 genes that comprise the core set of surface-induced genes. This set includes genes encoding the surface motility system lateral flagella and virulence factors including a type three secretion system (T3SS1). I showed a biological consequence of the increased expression of T3SS1 genes, as surface-induced cells were more toxic in a tissue culture infection than either liquid-grown or surface-grown non-swarming mutants. I explored the role of calcium signaling in regulating the surface sensing network, as calcium seemed a pertinent signal to a marine organism and low calcium is a known inducing signal for T3SS in other organisms. Calcium was shown to enhance swarming motility and lateral flagellar gene expression. Microarrays were used to analyze the transcriptome response to growth with EGTA (a cation chelator commonly used to generate low calcium) or calcium. Surprisingly, both low and high calcium induced T3SS1 gene expression. The EGTA effect was determined to be the result of iron limitation, which was thus shown to be a new inducing signal for T3SS1. I overexpressed the master transcriptional regulator of the T3SS system, encoded by exsA, to define the entire set of T3SS1-associated genes. I found that ExsA was also a new regulator of the surface sensing regulon, which was repressed when exsA was overexpressed. Microarray analysis showed that calcium is a global regulator, controlling transcription of about 50 genes under the conditions tested. I characterized a new calcium-regulated transcription factor that we named CalR, and showed that CalR repressed swarming motility and T3SS1 gene expression. The transcription factor OpaR was previously known to repress swarming genes and control colony opacity. It is homologous to the output regulators of the quorum sensing pathway in other Vibrio species. I used microarray analysis and mutant strains to explore the functionality of the quorum sensing cascade in V. parahaemolyticus and define the OpaR regulon during growth on a surface. I showed that the quorum sensing regulator LuxO when active silences opaR as it does in other Vibrios, using a translational reporter fusion in opaR. I used microarray analysis to show that 323 genes are induced or repressed by OpaR. The surface-sensing regulon is repressed by OpaR. Many genes encoding proteins involved in virulence, signal transduction, and modulation of the signaling molecule cyclic dimeric GMP are regulated by OpaR. The quorum sensing controlled network of gene expression in V. parahaemolyticus is quite distinct from other Vibrios, with respect to both the specific nature as well as the direction of regulation of genes controlled by OpaR.

Comprendre l’émergence des maladies dans les agroécosystèmes nécessite d’étudier l’histoire évolutive des populations bactériennes associées aux plantes. L’objectif de ce travail était de déterminer les évènements évolutifsconduisant à l’émergence des lignées pathogènes ou pathovars dans l’espèce Xanthomonas arboricola. Une analyse de génétique des populations a été menée sur un panel de souches phytopathogènes et commensales et complétée par l’inférence des gains et pertes de facteurs de virulence. Cette espèce possède une structure de population épidémique ; les clones épidémiques ont émergé suite à l’acquisition de facteurs de virulence à partir d’un fond recombinant de souches commensales. Une analyse de génomique des populations et la reconstruction de scénarios de divergence entre ces clones et le réseau de souches recombinantes, a montré la persistance d’un flux de gènes asymétrique entre ces deux groupes, dans le sens souches pathogènes vers souches commensales. Enfin, l’histoire évolutive du principal facteur de virulence des Xanthomonas, le système de sécrétion de type 3, a été retracée au sein du genre, et a montré que celui-ci avait été acquis ancestralement puis perdu dans certaines souches commensales. En conclusion, l’ancêtre commun de X. arboricola possédait des facteurs de virulence et au sein des souches commensales, certaines ont perdu ces facteurs, tandis que d’autres ont conservé le répertoire ancestral. Ces dernières diffèrent peu de certains agents pathogènes, et pourraient représenter un risque pour de nouvelles émergences. Des travaux de génomique fonctionnelle permettraient de valider ces hypothèses. / Deciphering the evolutionary history of bacterial populations associated to plants is necessary to understand diseaseemergence in agroecosystems. The aim of this study is to unveil the evolutionary events responsible for pathogeniclineages or pathovar emergences in Xanthomonas arboricola. This species is composed of both plant pathogenic andcommensal strains Population genetics analyses and gain and loss inferences of virulence factors showed that X. arboricola exhibits an epidemic population structure, within which epidemic clones emerged from a recombinogenic background population following virulence factor acquisition. Population genomics and inference of divergence scenarii between epidemic clones and the network of recombinant strains showed persistence of homologous recombination along divergence of these two groups, with an asymmetric gene flux from pathogenic strains to commensal ones. Finally, evolutionary history of the type three secretion system (T3SS), the main virulence factor in Xanthomonas genus, was studied at genus scale and showed that T3SS was ancestrally acquired and lost in commensal strains. Altogether these analyses allowed us to show that the common ancestor of X.arboricola had virulence factors, and that within commensal strains, some lost these virulence factors whereas others kept the ancestral repertoire. These latter strains have a similar repertoire to that of some pathogenic strains, and could represent a risk for new disease emergence. Functional genomics could allow us to validate these hypotheses.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Microbial pathogens have evolved secretion systems to deliver arsenals of virulence proteins (effectors) to disrupt host homeostasis and manipulate host immune defenses. The best-characterized system mediating effector delivery into host cells is type III secretion system (T3SS) expressed by Gram-negative bacteria, including enteric pathogens enteropathogenic/enterohemorrhagic Escherichia coli (EPEC/EHEC), Shigella, Yersinia, and Salmonella. Pathogen-host cell protein interactions within the host cell alter host cell signaling and ultimately subvert pathogen-induced inflammatory response. In the first project, we identified the Salmonella Secreted Effector L (SseL) that deubiquitinated ribosomal protein S3 (RPS3) to inhibit its nuclear translocation. RPS3 guides the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) subunits to specific B sites and plays an important role in the innate response to bacterial infection. Two E. coli effectors block RPS3 nuclear translocation. Non-locus-of-enterocyte-effacement (non-LEE) encoded effector NleH1 inhibits RPS3 phosphorylation by IKK-, an essential aspect of the RPS3 nuclear translocation process. NleC proteolysis of p65 generates an N-terminal p65 fragment that competes for full-length p65 binding to RPS3, thus also inhibiting RPS3 nuclear translocation. Thus, E. coli has multiple mechanisms by which to block RPS3-mediated transcriptional activation. With this in mind, we considered whether other enteric pathogens also encode T3SS effectors that impact this important host regulatory pathway. In this study, we report that SseL, which was previously shown to function as a deubiquitinase and inhibit NF-B signaling, also inhibits RPS3 nuclear translocation by deubiquitinating this important host transcriptional co-factor. RPS3 deubiquitination by SseL was restricted to K63-linkages and mutating the active-site cysteine of SseL abolished its ability to deubiquitinate and subsequently inhibit RPS3 nuclear translocation. Thus, Salmonella also encodes at least one T3SS effector that impacts RPS3 activities in the host nucleus. In the second project, we attempted to identify a cofactor involved in the interaction between E. coli effector NleH1 and host kinase the IB kinase- (IKK). The EHEC NleH1 effector inhibits NF-B pathway by reducing the nuclear translocation of RPS3. NleH1 prevents RPS3 phosphorylation by IKKIKK is a central kinase in the NF-B signaling pathway, yet the EHEC NleH1 effector only restricts the phosphorylation of a subset of the IKK substrates. We hypothesized that a protein cofactor might dictate the inhibitory specificity of NleH1 on IKK. We used mass spectrometry and determined that heat shock protein 90 (Hsp90) interacts with both NleH1 and IKK, and that inhibiting Hsp90 activity reduces RPS3 nuclear translocation. In the third project, we focused on the crystal structures of Salmonella secreted effector SseK1 and SseK2 from Salmonella typhimurium SL1344, and non-LEE encoded effector NleB2 from E. coli O145:H28 and propose catalytic residues for arginine glycosylation. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like glycosyltransferases, although they differ in protein substrate specificity. The bacterial effectors SseK and NleB1 glycosylate host cell death domain target proteins on arginine residues that inhibits death receptor signaling. We report crystal structures of SseK1, SseK2, and NleB2 and found they are highly similar to each other and comprises three domains including helix-loop-helix (HLH), lid, and catalytic domain. His-Glu-Asn (HEN) motif in the active site is essential for enzyme catalysis. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are important for enzyme recognition.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD. / PHD / Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for causing severe infections in immunocompromised individuals. Acute Pseudomonas aeruginosa infections are characterized by immediate adverse effects. An initial acute infection may become chronic, leading to long-term morbidity and mortality in affected individuals. During the initial stages of infection P. aeruginosa uses the type three secretion system, a syringe-like structure, to puncture the host cell and inject potent toxins. The activation of the genes required for forming this structure is tightly controlled by an activator protein, ExsA. When P.aeruginosa is not invading a host, ExsA is inhibited by another protein called ExsD, to prevent the needless production of the secretion apparatus. The presented work explores the mechanism of how ExsD achieves this inhibition of ExsA. This information is of potential biomedical interest because a clear understanding of the molecular basis for the interaction could inform the development of a small-molecule mimic of ExsD to be used in therapy. In Chapter 2 we report the structure of the domain of ExsA that is known to bind ExsD. Also, in this chapter and more so in Chapter 3, we performed a detailed analysis of potential interacting regions and ultimately succeeded in identifying key interacting regions in both ExsA and ExsD.

<p>The Gram-negative bacteria Chlamydia trachomatis is a common sexually transmitted pathogen that can cause severe sequelae including cause pelvic inflammatory disease and sterility. This obligate intracellular pathogen effectively manipulates host cellular functions by secreting virulence factors across its membrane bound vacuole. Identifying these virulence components and how they help in establishing an environment conducive for bacterial growth is central to understanding chlamydial pathogenesis. This is experimentally challenging due to a lack of tools to perform molecular genetic studies. In the absence of genetic tools, we developed a yeast model system to identify and characterize chlamydial proteins involved in virulence mechanisms. In this study we describe the identification of twenty-eight proteins potentially involved in modulating host cellular functions and the secretion of virulence factors into the host. Since the delivery of virulence proteins by a type three secretion (T3S) system is a critical step for Chlamydia, we identified the proteins that interacted with the T3S apparatus by yeast two-hybrid analysis. We discovered several novel interactions between and determined that the C. trachomatis T3S apparatus displayed a similar architecture to that of other T3S systems. Furthermore with these approaches we identified networks of proteins that interacted with the secretion apparatus including a novel secretion chaperone protein. We characterized Ct260/Mcsc one of the putative secretion and demonstrated that it represents a novel class 1B secretion chaperone protein. Unlike other known chaperones, Mcsc directly interact with a conserved component of the T3S apparatus cytoplasmic domain, CdsQ. These finding represents a novel mechanism by which the secretion chaperone protein Ct260 may increase the secretion efficiency of its effector cargo and may reveal new facets of secretory cargo recognition by T3S systems.</p> / Dissertation

The growing bacterial resistance against antibiotics creates a limitation for using traditional antibiotics and requests development of new approaches for treatment of bacterial infections. Among the bacterial infections that are most difficult to treat, biofilm-associated infections are one of the most hazardous. Consequently, the prevention of biofilm formation is a very important issue. One of the techniques that are widely investigated nowadays for this purpose is surface modification by polymer brushes that allows generating antifouling antibacterial surfaces. Previously, it was reported that salicylidene acylhydrazides (hydrazones) are good candidates as antivirulence drugs targeting the type three secretion system (T3SS). This secretion system is used by several Gramnegative pathogens, including Pseudomonas aeruginosa, to deliver toxins into a host cell. Furthermore, the chemical structure of these substances allows formation of complexes with metal ions, such as Fe3+ and Ga3+. The antibacterial activity of Ga3+ is well known and attributed to its similarity to the Fe3+ ion. It has also been shown that Ga3+ ions are able to suppress biofilm formation and growth in bacteria. In this thesis the chemistry of antibacterial and antivirulence Ga3+-Hydrazone complexes in solution was studied. First, to get insights in the solution chemistry, the protonation and the stability constants as well as the speciation of the Ga3+-Hydrazone complexes were determined. Additionally, a procedure for anchoring one of the hydrazone substances to antifouling polymer brushes was optimized, and the resulting surfaces were characterized. Results showed that the complexation with Ga3+ ions stabilizes the ligand and increases its solubility. Ga3+ ion binds to the hydrazone molecule forming a strong chelate that should be stable at physiological conditions. The different biological assays, such as Ga3+ uptake, antivirulence and antibiofilm effects, indicated very complex interaction of these complexes with the bacterial cell. Negatively charged and zwitterionic surfaces strongly reduced protein adsorption as well as biofilm formation. Therefore, the antifouling zwitterionic poly-[2-(methacryloyloxy)ethyl]dimethyl-3- sulfopropyl)-ammonium hydroxide (pMEDSAH) brushes were post-modified and successfully functionalized with bioactive substances via a block-copolymerization strategy. However, in order to maintain the availability of the bioactive substance after functionalization, the hydrophobic polyglycidylmethacrylate (pGMA) top block is probably better to functionalize with a lipophilic molecules to reduce diblock copolymer brush rearrangement.