Thesis advisor: Mary F. Roberts / Thesis advisor: Jianmin Gao / The research reported in this thesis focused on three proteinaceous virulence factors of the intracellular bacterial pathogen Listeria monocytogenes: listeriolysin O (LLO), broad-range phospholipase C (PC-PLC), and phosphatidylinositol-specific phospholipase C (PI-PLC). Based on sequence homology of LLO with other cholesterol-dependent cytolysins (CDC), the protein has four domains of which domain 4 is thought to anchor the protein to cholesterol-containing surfaces while domain 3 mediates protein-protein binding on the membrane and contributes α-helices that convert to two β-strands that form the large β-barrel pore. It was previously assumed that the sequential and cooperative behaviors of domain 3 in each LLO monomer required D4 to bind to cholesterol-enriched membranes. By cloning and expressing a separate protein containing domains 1, 2, and 3 (D123) and the isolated domain 4 (D4) of LLO, I could uncouple some of the events in its membrane binding and pore-formation. Flow cytometry, used to investigate protein binding to vesicles and to red blood cells, showed that D123 had no membrane affinity on its own, but became membrane-bound when sub-lytic amounts of LLO were added. D123, not membrane-lytic by itself, became hemolytic when trace amounts of LLO were present to provide a membrane anchor for D123 proteins. FRET and fluorescence correlation spectroscopy were used to show that D123 and LLO formed oligomers at nanomolar concentration and could also associate with one another in the solution. These results suggest that D4 provides an initial membrane attachment but need not be present on all monomers to trigger the cooperative conformational change that leads to membrane insertion and pore formation. The gene for L. monocytogenes PC-PLC was obtained, expressed in E. coli and the product protein purified and characterized. The zinc content of this metalloenzyme was analyzed with ICP-MS. The dissociation constants of the three zinc ions proposed as necessary for PC-PLC activity ranged from 0.05 to 60 μM. Enzymatic activities of PC-PLC were analyzed for various substrates, include long-chain phospholipid in vesicles (LUVs, SUVs) and micelles (Triton X-100), and short-chain lipids (diC4PC, diC6PC, diC7PC) mono-dispersed in solutions. Key results include the following: (1) the L. monocytogenes PC-PLC has an acidic pH optimum (in contrast to other bacterial PC-PLC enzymes) consistent with its role in vacuole lysis upon acidification; (2) the preference of PC-PLC for longer chain monomeric substrates is not because of a higher kcat but a reduced Km suggesting some amount of hydrophobicity is important for substrate binding in the active site; (3) the apparent Kd of PC-PLC for Zn2+ derived from kinetics at pH 6.0 (1.94 ± 0.22 μM) is lower that that from ICP-MC; and (4) PC-PLC enzymatic activity is not enhanced by added LLO that generates pores in vesicles (likewise, PC-PLC does not affect the membrane lytic activity of LLO) indicating no synergism between the two virulence factors. These results should aid in understanding the function of PC-PLC in L. monocytogenes pathogenicity. The L. monocytogenes PI-PLC and a variant with reduced catalytic activity were expressed and are currently used in a collaborative project with the Portnoy laboratory at the University of California at Berkeley. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

This thesis focuses on the use of a genomic approach to identify novel molecular markers to differentiate Streptococcus suis (S. suis) isolates into two populations, i) disease-associated and ii) non-disease associated. S. suis is a Gram-positive coccus that is considered one of the most important zoonotic bacterial pathogens of swine responsible for significant economic losses to swine production worldwide. Importantly, S. suis is not only an invasive pathogen but also a very successful coloniser of mucosal surfaces; often endemic in swine populations sampled. The widescale use of antibiotics to control and prevent the various clinical manifestations caused by S.suis has become unsustainable, due to increases in antibiotic resistance and government pressures. Other popular control strategies, such as the development of efficacious vaccines, are hindered by differences in virulence not only between but also within S. suis serotypes, as well as, the lack of a detailed understanding of the role in pathogenesis of many proposed virulence-factors. As a result, the detection of S. suis in asymptomatic swine herds is of little practical value in predicting the likelihood of future clinical relevance. This thesis aims to further understanding of the role the S. suis genome has in pathogenesis. The value of future surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. The first results chapter of this thesis (chapter 3) describes the use of genome-wide associations studies, a so-far unexploited method for S. suis, to identify genetic markers associated with the observed clinical phenotype i) invasive disease or ii) asymptomatic carriage on the palatine tonsils of swine. Chapter 4 then describes the analyses used to select three genetic markers to pathotype S. suis - differentiate isolates of the same species based on their ability to cause disease; going on to describe the design and evaluation of a multiplex-PCR tool targeting the three newly defined "pathotyping markers" in comparison to existing methods used to characterise S. suis. These findings were taken further by using the pathotyping markers to screen material scrapped from the palatine tonsils of swine with no obvious signs of streptococcal disease. This produced an interesting result - the production of both invasive disease-associated and non- disease associated multiplex-PCR amplicons from the same experimental sample. Unsurprising in itself, what was surprising is the frequency with which this observation was found. Picking single colonies from solid agar plates is a crippling bottleneck of existing S. suis diagnostics, and its removal has the potential to improve the sensitivity of surveillance and preventive health management programs. Chapter 5 describes investigation of this surprising observation and indicates that classic culture-based methods of detection are not sensitive enough to confidently report the presence (or absence) of invasive disease-associated S. suis strains. This thesis concludes with the description of efforts to address the lack of a comprehensive understanding of S. suis virulence/'virulence-associated' factors. Chapter 6 describes the design of an isogenic mutant knocking out the invasive disease-associated pathotyping marker, SSU1589 (also known as virA). That is then evaluated in simple in vitro and in vivo experimental models in order to understand the role Type I restriction modification proteins have in S. suis pathogenesis. In conclusion, this thesis furthers our understanding that differences in the S. suis genome are an important factor in S. suis pathogenesis, and describes the identification and evaluation of novel genetic markers for the detection and control of invasive disease-associated S. suis strains in intensive pig production systems.

Shigella provoque la dysenterie bacillaire en envahissant les muqueuses du colon. Cette maladie diarrhéique est responsable d’un million de décès par an essentiellement dans les pays en voie développement. Les gènes nécessaires àl’entrée dans les cellules hôtes sont regroupés sur un fragment d’ADN plasmidique de 30-kb. Celui-ci contient deux types de gènes, les gènes ipa(B, C et D) et ipgcodant pour des protéines responsables de l’entrée de la bactérie dans les cellules, et les gènes mxi/spacodant pour un système de sécrétion appelétype III (SST3) nécessaire àla sécrétion des facteurs de virulence. Les gènes mxi/spaetipa/ipgsont exprimés à37°C et les protéines Ipa/Ipgrestent dans le cytoplasme jusqu’àce que le SST3 soit activéau contact de la cellule hôte. Ce contact induit l’internalisation de la bactérie par macropinocytose, suivie de sa dissémination intra-et intercellulaire. Des observations en microscopie électronique (ME) montrent que le SST3 est composéde trois parties: i) une aiguille dont la longueur est régulée à50 nm par la protéine Spa32, ii) un corps basal qui traverse les membranes interneet externe ainsi que le peptidoglycane, et iii) un bulbe cytoplasmique. Le SST3 est le dispositif principal de virulence et permet l’injection de facteurs de virulences du cytoplasme bactérien vers celui de la cellule cible. Shigelladoit sécréter ces protéines de manière ordonnée. Très peu de travaux ont abordécette question. L’objectif principal de ce travail de thèse a étéd’étudier les mécanismes moléculaires impliqués dans la hiérarchie de sécrétion.Nous avons principalement investiguéle rôle de 3 protéines: Spa32, Spa40 et MxiC dans la sécrétion. Nous avons montré, par des études génétiques, que contrairement aux études publiées sur les protéines homologues, Spa32 n’agit pas comme un «molecularruler»pour réguler la taille de l’aiguille. Nous avons montréque cette régulation nécessite l’interaction de Spa32 via ses résidus 206-246 au domaine C-terminal de Spa40 (Spa40C) (Botteaux et al., 2008a). Ayant identifiécette interaction avec Spa40, l’étape suivante de notre travail a portésur la caractérisation de la fonction du gène spa40par des méthodes génétique, biochimique et structurale (ME). Nos résultats montrent que Spa40 joue un rôle important dans l’assemblage du SST3. Des plus, nous avons mis en évidence de nouvelles interactions impliquant Spa40C et des composants du SST3 (Botteaux et al., in preparation).Parallèlement àces travaux, nous avons montréque l’inactivation du gène mxiCaboutit àune dérégulation spécifique de la sécrétion des effecteurs sans altérer celle des translocateurs IpaB et IpaC. Cette augmentation de sécrétion est due àune augmentation de transcription des gènes tardifs, conséquence de la sécrétion précoce de l’anti-activateur transcriptionnel, OspD1 (Botteaux et al., 2008b). De plus, nous avons montréque MxiC est un substrat de l’appareil de sécrétion et que cette sécrétion est associée àsa fonction. Finalement, la mise en évidence d’une interaction entre MxiC et Spa47, l’ATPase du SST3 nous permet de proposer un modèle régulant la hiérarchie de sécrétion des effecteurs.Dans une autre partie de notre travail, nous avons identifié, par des expériences de ME et d’immunomarquage, que l’invasineIpaD est localisée au sommet de l’aiguille du SST3 oùelle lui sert de bouchon. Enfin, de manière très intéressante, nous avons montréque des anticorps anti-IpaD neutralisent l’entrée de Shigelladans les cellules (Sani, Botteaux et al., 2007, dépôt de brevet). IpaD, étant conservée dans les isolats invasifs de Shigella, représente donc un réel candidat vaccinal pouvant pallier la diversitédes sérotypesbactériens.En conclusion, nos travaux représentent une contribution importante àla compréhension des mécanismes de virulence bactériens et dépassent le cadre de Shigellapuisque les systèmes de sécrétion sont hautement conservés parmi plusieurs pathogènes. L’identification de drogues pouvant interférer avec ces systèmes de sécrétion représente une voie d’avenir pour le développement de nouveaux agents anti-infectieux.

Tularemia is a zoonotic disease caused by the bacterial pathogen Francisella. A major virulence determinant of Francisella is the ability to survive and multiply within macrophages. Previous research identified a genetic element of approximately 30 kb in length, which possessed characteristics typical of a pathogenicity island. In F. novicida, the Francisella pathogenicity island (FPI) is composed of 18 genes. Initial studies revealed that several FPI-encoded genes are required for intramacrophage growth. The FPI contains several homologues of a newly described type six secretion system (T6SS). I developed a chicken embryo infection model to provide a simple, low-cost assay to evaluate the virulence of Francisella strains. The results demonstrate that this assay is able to discriminate large differences in virulence among Francisella strains. Further, this system can facilitate large-scale experiments to quickly survey mutant collections for virulence, while reducing animal suffering. Next, I adapted a genetic technique called co-transformation for use in Francisella. This technique facilitates the introduction of mutant or wild type DNA into the chromosome, without requiring the introduction of antibiotic resistance markers or negative selection markers. I also developed two new Francisella shuttle vectors for use in complementation studies. I demonstrated that these vectors are compatible with other pFNL-10-based Francisella shuttle vectors. They also permit tri-parental mating, allowing researchers to circumvent the restriction modification system in F. novicida. Finally, conjugation removes the need for electroporation equipment, which can create aerosols. These aerosols can represent a potential health risk for researchers studying highly virulent Francisella strains. The FPI gene pdpC was investigated for its role in virulence and intramacrophage growth. We found that pdpC was dispensable for growth in macrophages but required for virulence in two animal models. Microscopy studies using epitope tagged pdpC suggest that the protein may be secreted during macrophage infection. Quantitative microscopy provides evidence that PdpE (the gene immediately downstream of PdpC) is secreted in a T6SS dependent manner. Additional mutations in the pdpC gene revealed an effect upon the expression of the Igl proteins located in the minor FPI operon. The mechanism linking pdpC to iglA-D expression is unknown, but it is unlikely to be post-translational in nature. The genetic basis for this effect has been difficult to define, but we have developed a working hypothesis. We propose that two genetic mutations in pdpC are required; the first consists of a defined deletion in the N-terminal-half of the gene, while the second consists of an undefined region located at the C-terminal end. / Graduate / 0410

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [101]-104).

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed July 10, 2007). PDF text: 188 p. : ill. UMI publication number: AAT 3252444. Includes bibliographical references. Also available in microfilm and microfiche formats.

The pathogenicity of food-borne intracellular bacterium Listeria monocytogenes is greatly associated with its abilities to invade non-phagocytic cells, counteract the host innate immune system, resist bactericidal antibiotic-mediated killing, and breaking the physical barriers. In the last 30 years of research on L. monocytogenes, several virulence factors, such as Listeriolysin O (LLO), InlA, InlB, ActA, PI-PLC, and PC-PLC have already been characterized as important players that help this bacterium to achieve the key stage of infection. There are approximately 3,000 open reading frames in Listeria’s genome; however, only few virulence factors are functionally characterized. Thus, it is important to identify new virulence factors and understand how new virulence factors in Listeria help this opportunistic pathogen to counteract the host innate immune system, resist antibiotic-mediated killing, colonize vital organs, and finally successfully develop life-threatening listeriosis. In this study, inrame deletion mutagenesis was used to generate the deletion mutants of novel listerial virulence factors and a series of biochemical, in vitro and in vivo experiments were conducted to characterize the roles of these virulence factors during the infection process. In the first part of this study, an AlkD-like protein (Adlp, LmoF2365_0220) was identified and the protein is associated with oxidant tolerance and aminoglycoside antibiotic resistance. In the second part of this study, a new internalin-like protein (LmoH7858_0369) was shown to be involved in invasion of Hep-G2 cells and organ colonization in mice. The third part of this study showed that listeriolysin O (LLO) mediates cytotoxicity on brain endothelial cells, suggesting that LLO may contribute to the invasion of the central nervous system by L. monocytogenes. In summary, we identified and characterized two novel virulence factors, Adlp and LmoH7858_0369 that contributed to bacterial infection and revealed a new invasion mechanism of CNS cells that is mediated by LLO. Results from these studies provide a better understanding on the pathogenicity of L. monocytogenes and can be used as therapeutical targets or vaccine candidates

Enteric pathogens have a substantial impact on human health as they can cause outbreaks and severe disease outcomes. These pathogens employ many virulence strategies to evade host defenses and cause disease. While some virulence strategies have been carefully studied, other mechanisms remain largely uncharacterized. In addition, there are a number of putative virulence factors that have yet to be phenotypically or biochemically characterized. In order to facilitate the development of novel and effective treatment strategies for enteric pathogens, an understanding of how these putative virulence-associated proteins contribute to pathogenesis is required. In this work, the characterizations of two proteins implicated in the processes of motility and type III secretion are presented. The Escherichia coli O157:H7 protein Z0021 is found in an O-island unique to the most virulent serotypes of Shiga-toxin producing E. coli. Z0021 was found to encode for a repressor of motility that exerted its regulatory effect prior to the activation of class II promoters in the flagellar cascade. This work provided the first identification and characterization of a fimbrial operon-encoded motility repressor in E. coli O157:H7. The second protein, SsaN, from Salmonella enterica is a putative type III secretion system ATPase. This work examined the role of SsaN in virulence and effector secretion, and moreover provided insight into the mechanism by which SsaN binds to a chaperone to facilitate effector secretion. Together, these findings contribute to the understanding of the virulence strategies of two enteric pathogens that have had, and continue to have, significant impacts on human health worldwide. / Thesis / Doctor of Philosophy (PhD)

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