Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and is of particular concern due to its intrinsic resistance to many frontline antibiotics. To aid in infection, Pseudomonas encodes an arsenal of virulence factors, including type IV pili (T4P), hair-like adhesins involved in many processes, such as twitching motility and surface attachment. T4P are primarily composed of the major pilin, PilA, whose expression is tightly regulated by the PilS-PilR two-component system. The sensor kinase, PilS, monitors the inner membrane PilA inventory and modifies activity of the response regulator, PilR, to regulate pilA transcription. Here, we demonstrate that P. aeruginosa virulence in a roundworm infection model is reduced when the amount of T4P expressed at the cell surface increases, regardless of the ability of the bacteria to twitch. We propose that inappropriate increases in surface T4P expression may impair pathogenicity-associated systems which require intimate host-cell contact. New genes in the regulon of the PilS-PilR two-component system were also identified. A tool to fluorescently label and image T4P in real-time using microscopy was established in the lab. This work highlights the consequences of increased surface T4P expression, providing potential new targets for antipseudomonal therapeutics which act on components involved in T4P expression and function.

Type IV pili (T4P) are long, fibrous surface appendages involved in attachment, motility, biofilm formation and DNA uptake that are expressed by bacteria and archaea. They are an important virulence factor for a number of bacteria, including Pseudomonas aeruginosa, an opportunistic pathogen that is a common cause of nosocomial infections. T4P are composed mainly of monomers of the major pilin subunit, PilA, although several low abundance proteins called minor pilins are also present. These surface-exposed proteins are potential vaccine candidates, although a more complete understanding of their diversity and function is required for the rational development of a pilus-based vaccine. There are five distinct groups of P. aeruginosa major pilins, which vary based on their sequence and their associated accessory proteins, and two distinct sets of minor pilins, although the roles of the latter in pilus biology are poorly understood. This study focuses on the structural characterization of major and minor pilins and functional implications for pilus assembly and disassembly dynamics. The structural analysis of major pilins from groups III and V revealed specific differences in pilin structure that may affect subunit interactions within the pilus fibre and interactions with their specific accessory proteins and minor pilins. The minor pilins PilVWX were shown to form a putative subcomplex with the adhesin and anti-retraction protein PilY1, which is proposed to prime pilus assembly and thus traffic PilY1 to the bacterial surface. High resolution X-ray crystal structures of the minor pilins FimU and PilE were solved and functional characterization suggested that FimU and PilE are necessary for efficient pilus assembly to stably connect the priming subcomplex to the major pilin subunits. Together, this work has increased our understanding of pilin diversity and defined a concrete role for the minor pilins in pilus assembly. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a bacterium that can take advantage of a weakened immune system to cause lethal infections. The first step of infection involves attachment to the host using long sticky fibres called type IV pili. Each fibre is composed primarily of a single protein, the major pilin, but also contains low abundance proteins called minor pilins. Without these proteins, the bacteria can’t attach and cause infections, making pilins excellent vaccine candidates. This study focused on the characterization of major and minor pilins to understand the diversity of these proteins and how these differences might affect pilus assembly. We show that the molecular structure of the major pilin differs between strains although the core architecture is the same, and that the minor pilins are required for initiation of pilus assembly. This work furthers our understanding of the structures and functions of pilin proteins, and provides information helpful for the development of vaccines.

Clostridium perfringens is a Gram-positive anaerobe capable of causing disease in humans and many animals. C. perfringens is able to move across surfaces in a manner that is dependent on growth and type IV pili. Type IV pili are filaments that can be extended away from the cell by rapid polymerization, and retracted by depolymerization. Furthering the understanding of the initial and final energetic states of the pilins will reveal insights into possible mechanisms of type IV pilus assembly. Toward that end, a pilin was purified from the Gram-negative pathogen Pseudomonas aeruginosa and incorporated into an artificial membrane. The pilin was probed by a solid state nuclear magnetic resonance (ssNMR) technique that can determine the angle and depth of insertion of a helical peptide, as well as fluorescent and electron microscopy. All type IV pilus systems involve the action of an assembly ATPase to provide energy to polymerize the pilus. One proposed mechanism involves two primary proteins: an ATPase and an integral membrane core protein (IMCP). Other type IV pilus proteins are thought to play supportive roles in aiding the traversal of the cell envelope. In order to evaluate this model, the assembly ATPase PilB2 and IMCP PilC2 from C. perfringens were purified and examined for interactions. The evidence presented here suggest that PilB2 and PilC2 do not interact directly, and cannot function as a core assembly apparatus. The carbonic anhydrase (Cpb) from C. perfringens strain 13 was characterized both biochemically and physiologically. Cpb belongs to the type I subclass of the β class and is the first β class enzyme investigated from a strictly anaerobic bacteria. Kinetic analyses revealed a two-step, pingpong, zinc-hydroxide mechanism of catalysis. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semi-defined medium and an atmosphere without CO₂. The grew well in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases. / Ph. D.

No description available.

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.

<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.

Type IV pili (T4P) are protein filaments found on the surface of a variety of bacterial species and mediate biofilm formation, adhesion, and flagellum-independent twitching motility. The biogenesis of T4P is dependent on a cell envelope-spanning, multiprotein complex that localizes to the poles in rod-shaped cells. How these proteins localize and cross the peptidoglycan (PG) layer in the absence of dedicated PG-hydrolyzing enzymes is unknown. In P. aeruginosa, PilMNOP interact to form the alignment subcomplex, connected via PilP to PilQ, which forms the outer membrane secretin. We hypothesized that polar localization and integration of the T4P machinery was driven by ordered recruitment to future sites of cell division, placing assembly system components at division septa in the correct position before daughter-cell separation. To determine which T4P components are essential for localization of the complex, we fused the T4P inner membrane assembly protein PilO to the fluorescent protein mCherry to monitor its localization. mCherry-PilO localized to the cell poles and midcell in wild type bacteria. However, it was delocalized in a strain lacking PilQ. A PilQ-mCherry fusion localized to the cell poles, likely through its putative septal PG binding AmiN domains, suggesting that PilQ binds PG and thus localizes its partners to future sites of cell division. In the absence of the associated pilotin protein (PilF), which is required for PilQ multimerization in the OM, T4P components were polarly localized, implying that localization is not dependent on secretin formation. The results of this research support a pre-installation mechanism for integration of protein complexes in the gram negative cell envelope without PG hydrolysis, which may be applicable to other systems. / Thesis / Master of Science (MSc)

No description available.

Type IV pili (T4P) are long adhesive surface filaments produced by bacteria and are a key virulence factor for many pathogens. T4P are produced by a dynamic intracellular nanomachine that facilitates the assembly (extension) and disassembly (retraction) of pili. Pilus dynamics are enabled by the motor subcomplex of the nanomachine, where cytoplasmic ATPases power pilus assembly (PilB) and disassembly (PilT and PilU). In many, but not all, T4P expressing bacteria – including our model organism Pseudomonas aeruginosa – two retraction ATPases are required for functional retraction, which can be assessed by measuring twitching motility. Deletion of pilT results in loss of twitching and phage susceptibility (another hallmark of pilus function) while deletion of pilU results in loss of twitching but retention of phage susceptibility, indicating pili can still be retracted. We hypothesized that PilU adds to the force of pilus retraction, facilitating disassembly when the fiber is under tension. We mutated ΔpilU and pilU::Tn5 strains with ethyl methanesulfonate and screened for gain-of-twitching mutants. Whole genome sequencing revealed multiple point mutations in the major pilin protein PilA or the pilus adhesin, PilY1. These point mutations were recapitulated in a ΔpilU strain and restored twitching to varying degrees. Complementation of pilA point mutants with pilU in trans influenced the twitching zone of only one mutant, and in trans expression of wild-type pilA resulted in a significant reduction in twitching in most. The contribution of PilU to the force of pilus retraction was further investigated by a polyacrylamide micropillar assay, where no pulling events could be detected for either ΔpilT or ΔpilU mutants. Exopolysaccharide production, a proxy for surface sensing, was uncoupled from twitching motility in the pilA point mutants. These results are a significant step forward to understanding what PilU does and, provides insight to the dynamics of the pilus fiber. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes serious infections. P. aeruginosa uses adhesive, “grappling hook” filaments called Type IV pili (T4P) to stick to its hosts. T4P can be repeatedly extended and retracted, allowing the bacteria to crawl on surfaces (twitching) but making them susceptible to bacteriophages, viruses that attach to pili then kill the bacterial cells. The motor proteins PilT and PilU are required for twitching, but only PilT is essential for phage killing, implying that pili are retracted even when PilU is missing. Here we hypothesized that PilU is important for twitching because it helps generate force for retraction when pili are under tension. We isolated multiple mutations in pilus components that restored twitching in the absence of PilU, and propose that these mutations allow for easier retraction of pili. This information helps us understand how T4P help the bacteria to spread during infection.