Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.

No description available.

Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers. / Ph. D.

Les bactéries lactiques (BL), communément employées en industrie agroalimentaire, font à présent l’objet d’études visant à les utiliser pour de nouvelles applications telles que le développement de vaccins vivants ou la délivrance de molécules d’intérêt biothérapeutique chez l’hôte. Dans cette optique, différents systèmes de présentation de protéines à la surface des bactéries à Gram positif ont été développés. L’un d’entre eux est basé sur l’activité d’enzymes, les sortases, liant de façon covalente les protéines à la paroi bactérienne. Nous avons utilisé la BL modèle, Lactococcus lactis, afin d’étudier les sortases, jusqu’alors étudiées essentiellement chez les bactéries pathogènes. La sortase A (SrtA) est responsable de l’ancrage d’au moins cinq protéines à motif LPxTG à la surface. Une seconde sortase, de classe C (SrtC), a été identifiée et caractérisée. Nous avons mis en évidence la capacité de L. lactis à produire des pili à sa surface qui sont polymérisés par SrtC et ancrés à la paroi par SrtA. Ces pili résultent de la polymérisation de la piline majeure YhgE qui peut être surplombée par la piline mineure de coiffe YhgD. La production de pili chez L. lactis entraîne un changement de comportement des cellules résultant à des phénotypes particuliers. Nous avons pu l’associer à l’auto-agrégation des cellules en culture liquide, à la formation de biofilms hétérogènes et aériens, et à l’adhésion à la mucine gastrique de porc. Plus précisément, YhgE a été impliquée dans l’auto-agrégation et les biofilms atypiques, et une troisième piline, dont l’appartenance au pilus n’a pas été démontrée, semble aussi impliquée dans la production de biofilms atypiques. / Lactic acid bacteria (LAB), which are commonly used in food industry, are now being studied for their use in new applications such as biotherapeutic molecule delivery vehicules in human host or as live vaccines. Recently, surface protein delivery systems have been developed in Gram positive bacteria and one of them is based on enzymes, the sortases which covalently bind proteins to the cell wall. We used the LAB model, Lactococcus lactis, in order to study the sortases of these non-pathogenic bacteria. This work has functionally characterized the sortase A (SrtA) responsible for cell wall anchoring of at least five LPxTG proteins. A second sortase, from class C (SrtC), has been identified and characterized. We demonstrated the ability of L. lactis to produce pili on its surface that are polymerized by SrtC and cell wall anchored by SrtA. These pili result from polymerization of the YhgE major pilin and can be topped by the YhgD tip minor pilin. Pili production in L. lactis leads a change in cell behavior resulting in individual phenotypes. We were able to associate it with the self-aggregation of cells in liquid cultures, heterogeneous and aerial biofilm formation and bacterial adhesion onto pig gastric mucin. Specifically, YhgE was involved in both self-aggregation and atypical biofilm formation, while a third pilin, whose pilus membership has not been established, was also involved in the production of atypical biofilms.

<p>This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of <i>Neisseria gonorrhoeae, N. meningitidis</i> and <i>Streptococcus pyogenes</i> on host epithelial cells and <i>in vivo</i>. </p><p><i>N. gonorrhoeae</i> cause the sexually transmitted disease gonorrhoea and <i>N. meningitidis</i> is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, <i>N. meningitidis</i> generally induced an earlier response compared to <i>N. gonorrhoeae</i>, probably as a result of the meningococcal capsule. The role of <i>N. meningitidis</i> serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. </p><p><i>S. pyogenes</i> may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. <i>S. pyogenes</i> ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of <i>S. pyogenes</i> is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of <i>S. pyogenes</i>.</p>

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo. N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. S. pyogenes may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. S. pyogenes ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of S. pyogenes is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of S. pyogenes.

Antibiotics have been extensively used to treat bacterial infections since Alexander Fleming’s discovery of penicillin 1928. Disease causing microbes that have become resistant to antibiotic drug therapy are an increasing public health problem. According to the world health organization (WHO) there are about 440 000 new cases of multidrug-resistant tuberculosis emerging annually, causing at least 150 000 deaths. Consequently there is an immense need to develop new types of compounds with new modes of action for the treatment of bacterial infections. Presented herein is a class of antibacterial ring-fused 2-pyridones, which exhibit inhibitory effects against both the pili assembly system in uropathogenic Escherichia coli (UPEC), named the chaperone usher pathway, as well as polymerization of the major curli subunit protein CsgA, into a functional amyloid fibre. A pilus is an organelle that is vital for the bacteria to adhere to and infect host cells, as well as establish biofilms. Inhibition of the chaperone usher pathway disables the pili assembly machinery, and consequently renders the bacteria avirulent. The focus of this work has been to develop synthetic strategies to more efficiently alter the substitution pattern of the aforementioned ring-fused 2-pyridones. In addition, asymmetric routes to enantiomerically enriched key compounds and routes to compounds containing BODIPY and coumarin fluorophores as tools to study bacterial virulence mechanisms have been developed. Several of the new compounds have successfully been evaluated as antibacterial agents. In parallel with this research, manipulations of the core structure to create new heterocycle based central fragments for applications in medicinal chemistry have also been performed.

Corrigenda pasted onto front fly-leaf. / Bibliography: leaves 247-286. / xv, 286 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines an aspect of the pathogenesis of a model extracellular enteric pathogen, the causative agent of human cholera. The export of TcpA (Toxin-Coregulated Pilus) and assembly of the TCP is explored as a paradigm of macromolecular export in Gram negative bacteria. TcpA is examined in detail in an attempt to define strictly conserved regions between species. The TCP of the emergent 0139 (Bengal) serotype is demonstrated to be of El Tor type. The possibily that proteases such as the soluble haemagglutinin (SHA) may have a detachase role centring on TCP dispersal/TcpA degradation is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1997

Techn. Universiẗat, Diss., 2003--Berlin.