No description available.

No description available.

No description available.

No description available.

No description available.

Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.

<p>Type 4 pili (T4P) are fibrous appendages found on the surfaces of a wide range of bacteria. They are used for adherence to biotic and abiotic surfaces, twitching motility, and biofilm formation. Despite their ubiquitous distribution, identifying the core components required for T4P expression has been difficult due to conflicting data about the functions of orthologous components from the most common model organisms, <em>Neisseria</em> and <em>Pseudomonas</em>. By inactivating the retraction component of pilus function, genes essential for T4P assembly versus disassembly were discriminated in <em>P. aeruginosa</em>. In contradiction to data from the <em>Neisseria </em>system<em>,</em> we found that components of the inner membrane sub-complex consisting of PilN/O/P are not essential for surface pilus expression, while the highly conserved inner membrane protein, PilC is essential. The current model of T4P biogenesis suggests that PilC coordinates the activity of cytoplasmic extension (PilB) and retraction (PilT) ATPases via their interaction with its two large cytoplasmic domains. Hydrolysis of ATP by PilB or PilT is proposed to induce domain movements in PilC, resulting in the addition or removal of single pilin subunits from the base of the pilus. Using<em> </em><em>in vitro</em> co-affinity purification we showed that PilB is a potential interaction partner of the N-terminal cytoplasmic domain of PilC. Also, mutagenesis of the C-terminal cytoplasmic domain of PilC produced mutant proteins with a reduced capacity to support twitching motility, suggesting impairment of PilC-PilT interactions. The indispensability of PilC and its potential interactions with the ATPases PilB and PilT suggest that it is a core element required for function of the T4P system of <em>P. aeruginosa</em>.</p> / Master of Science (MSc)

<p>Peptidoglycan (PG) remodelling and cell division are two important cellular processes that are the major target of antibiotics. Due to rising resistance, the need for new antibiotics today has never been greater. Therefore it is important to fill the gaps in our understanding of these two important processes in order to discover new and promising antibiotic targets. Peptidoglycan synthesis and remodelling is a highly coordinated event that involves a wide number of enzymes and processes which are not well understood. N-acetylmuramoyl-L-alanine amidases, whose function is to cleave the amide linkage between the stem peptides and the lactyl moiety of N-acetylmuramic acid, is a major class of PG-active proteins. Their role in daughter cell separation during cell division is well established in <em>Escherichia coli</em> however little is known about it in other systems. Using enzymatic assays we characterize AmiC as a novel amidase in <em>Pseudomonas aeruginosa. </em>Through mutational analysis and microscopy we show that AmiB and AmiC are required for daughter cell separation. A deletion of both enzymes results in a cell chaining phenotype with abnormal cell morphology. Transmission electron microscopy reveals that the double mutant is arrested at the septal peptidoglycan separation step. In addition to cell chaining, the ∆<em>amiB/amiC</em> mutant exhibits a significant increase in susceptibility to antibiotics. We also demonstrate that the LysM motif of AmiB is not required for its role in cell separation. Furthermore, the <em>amiB</em> mutant has significantly shorter cells than the wildtype indicating an additional role for the enzyme in the cell. Lastly, through a novel bioinformatics strategy we identify PA5047 as a potential PG amidase.</p> / Bachelor of Science (BSc)

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

No description available.