Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

Microcystin-LR is a hepatotoxin produced by cyanobacteria. Aeromonas hydrophila and Edwardsiella piscicida infections are leading causes of losses in market-sized channel catfish (Ictalurus punctatus). These older fish should have natural immunity in place and a predisposing factor is likely a prerequisite for these disease outbreaks. While microcystin-LR rarely causes mortality in warm-water aquaculture, we believe it may be a predisposing factor that leads to bacterial disease outbreaks during the summer months due to its ability to damage the liver. Our study investigated microcystin-LR’s effects on channel catfish susceptibility to these pathogens. We found that a sublethal dose of microcystin-LR induced substantial damage to multiple immune organs. In our challenges with both the toxin and bacteria, we saw a significant increase in mortality of fish. Our findings suggest that microcystin-LR increases channel catfish susceptibility to Aeromonas hydrophila and Edwardsiella piscicida infections.

Pseudomonas aeruginosa is a central nosocomial pathogen that can infect any tissue in the human body. A two-component system in P. aeruginosa that regulates many virulence factors is the AlgZ/R system. A previously unidentified regulator of algZ, that does not affect algR, has been identified via transposon mutagenesis, ‘PA2771’. The mechanism of regulation has not been previously studied, and novel evidence of PA2771 functioning as a diguanyalate cyclase was observed. When PA2771 is active, cyclic di-GMP levels are high, promoting the upregulation of the fimU operon and Type VI pili. In the PA2771 mutant, an upregulation in the expression of the flagellar genes and swarming phenotype was observed, and restored via complementation. PA2771's function in regulating algZ expression, is likely indirect and alters virulence gene regulation and phenotypic outputs in P. aeruginosa in the switch between twitching and swimming motility, and appears to be specific to PA2771.

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen that causes acute and chronic infections. Infection is typically initiated via motile and virulent strains. After exposure to stressors, acute infections make both genotypic and phenotypic switches to a chronic, sessile strain. This is due to intricate regulatory networks directing gene expression in response to stressors. One network, GacA/GacS, has been established to control virulence factors. The stringent response of bacteria is mediated by alarmones produced primarily by RelA which responds to starvation. To study the effect of the stringent response on the virulence switch. A series of experiments were run in both PAO1, a virulent strain, and PDO300, an acute strain, and RelA deletion mutants of each transcriptional fusions of GacA/GacA system were integrated in the wild-types and mutants. Alginate, swimming, twitching, and biofilm formation assays were performed on all. The preliminary data suggests that the stringent response influences the GacA/GacS system.

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

Colonization is prerequisite for infection and transmission of Streptococcus pneumoniae, or pneumococcus. Currently available pneumococcal conjugate and pneumococcal polysaccharide vaccines can provide protection against a limited number of capsular serotypes. Implementation of vaccines has decreased the frequency of invasive pneumococcal disease and their colonization rates, but only in a serotype-dependent manner. This has led to serotype replacement in pneumococcal ecology and increased invasive disease caused by non-vaccine serotypes. Development of conserved protein-based vaccine that can provide protection against all pneumococcal serotypes is needed. Numerous surface proteins are conserved in all serotypes, and some are known to be involved in the colonization process. Understanding how pneumococcal surface proteins interact with host cells and determining their roles in colonization will aid in vaccine development. In this dissertation, we characterized host cell receptors of pneumococcal surface proteins, and proteins involved in biofilm formation, and their effect in colonization. We utilized a novel protein expression vector, pOS1, which can express secreted proteins with no LPS, IPTG induction, or cell lysis requirement. These expressed recombinant proteins were used for further investigation. We identified that human Annexin A2 (ANXA2) interacts with pneumococcal surface adhesion A (PsaA) protein. ANXA2 transduced cells showed significant increase in binding with pneumococcus compared to non-transduced cells. We conducted proteomic profiling of planktonic and biofilm membrane proteins and identified that two lipoproteins (AmiA, SP_0148) were overexpressed during biofilm formation. Isogenic mutants lacking these individual proteins showed decreased in biofilm formation compared to their parental strain. Deletion of SP_0148 led to decreased adhesion of pneumococcus to human nasopharyngeal epithelial cells (Detroit 562). These results increased our understanding of pneumococcal surface proteins involved in biofilm-formation and colonization as well as identifying new host receptors ligands for these adhesins.

There is an emerging threat of Neisseria gonorrhoeae strains that are resistant to all antibiotics. Because of this, the purpose of this research is to isolate, analyze, and partially characterize a new inhibitor(s) of N. gonorrhoeae. Since there is an unknown molecule secreted by Candida albicans that inhibits N. gonorrhoeae, this molecule can be partially characterized using 1H NMR Spectroscopy to assist in the development of a new antibiotic compound. It was hypothesized that quorum-sensing molecules, trans, trans- farnesol, tyrosol, phenylethyl alcohol, and tryptophol, could be possible candidates for the inhibitor. Because of this, 1H NMR spectra for these quorum-sensing molecules were obtained to serve as controls. Column chromatography and fractionation was used to isolate the inhibitor in large scale from C. albicans grown in salts-based media. Attempts to isolate the inhibitor in large scale, however, was unsuccessful since no inhibition of N. gonorrhoeae was observed. Because of this, analysis of growth media was conducted to test the media effect on producing the inhibitor. C. albicans was grown in liquid chocolate, liquid white chocolate, salts-based, and YPD media in aerobic and candle jar environments. Analysis of growth media in different environments suggests that liquid chocolate and salts-based media retain the inhibitory activity. 1H NMR spectra were obtained for the isolated molecule in liquid chocolate and salts-based media in both aerobic and candle jar environments. Analysis of this 1H NMR suggested that the inhibitor could be isolated from either the aerobic or candle jar environment for both liquid chocolate and salt-based media because a clear peak between 3.5 and 4.0 ppm was observed in all spectra. Comparison of 1H NMR spectra from quorum-sensing molecules with spectra from the isolated molecule suggests that the inhibitor is not a quorum-sensing molecule. The peaks represented by the inhibitor cannot be fully characterized and thus, either correspond to a single molecule or a complex molecular structure. It can be concluded that the inhibitor secreted by C. albicans to inhibit N. gonorrhoeae is a new unknown compound.

<p><strong><br /></strong></p> / <p><strong>In Canada the incidence of yeast infections have increased over the past decade, which in turn has resulted in the increased mortality and morbidity rates among the immuno-compromised patients. Yeasts are ubiquitous in nature and constitute a healthy portion of human skin and gut flora. Factors such as the urban settings and food have been previous shown to influence the yeast flora people harbour. This makes us believe that to effectively tackle the rising yeast infections in Canada we need to not only conduct epidemiological yeast studies in clinical settings but should also understand the diversity and distribution of them in the urban environment. This thesis constitutes of an epidemiological fungemia study and an urban environmental yeast profiling study conducted in the city of Hamilton.</strong></p> <p><strong> </strong></p> <p><strong>In the first chapter of the thesis I discuss the results of the epidemiological candidemia study. We noticed that over the past decade the mean age of the population with candidemia in hospitals within Hamilton has increased by 10 years. DNA fingerprinting analysis suggested that 33% of the blood stream</strong><strong><em>Candida</em></strong><strong> </strong><strong>isolates from January 2005 to February 2009 belonged to 18 clusters, some of which were shared between wards and hospitals. we found that for each of the four species, strains isolated closer to each other temporally were overall genetically more similar to each other as well, which suggested that nosocomial sources likely caused repeated candidemia infections. The study is the first of its sort in Canada and the results of this chapter are expected to aid infection control practitioners in the Hamilton hospitals and make the stay of patients in hospitals safer.</strong></p> <p><strong> </strong></p> <p><strong>In the second chapter, we discuss the diversity and distribution of yeasts prevalent on trees in and around Hamilton. We identified a total of 88 environmental yeasts belonging to 20 species (based on ITS sequence data). The yeast populations were highly heterogeneous in both species and genotype composition. Among the 14 tree species sampled, yeasts were frequently found on cedar, cottonwood and basswood. Interestingly all the</strong><strong> <em>Candida parapsilosis</em> </strong><strong>strains were found from pine tree only. Some of the potential environmental factors shaping the distribution of yeast populations in Hamilton are discussed.</strong></p> / Master of Science (MSc)

Surveys of the four major soilborne pathogens of strawberry (Fusarium oxysporum f. sp. fragariae, Macrophomina phaseolina, Phytophthora spp., and Verticillium dahliae) to determine their relative prevalence were conducted in Watsonville-Salinas, CA in 2021 and in Santa Maria, CA in 2022. All four major pathogens were detected at relatively similar prevalence in Watsonville-Salinas, between 22% and 31% of sampled fields. In Santa Maria, M. phaseolina was far more prevalent at 52% of sampled fields, the other three falling between 14% and 17%. Additionally replicated greenhouse and field trials were conducted to evaluate the effects of wheat as a single season cover crop on Macrophomina root rot of strawberry and the soil microbiome. Greenhouse trials and the first year of the field trial are described here and demonstrate a lack of substantial disease mitigation or pathogen reduction in the soil following wheat growth compared to no-treatment control. Significant changes were seen in the soil microbiome following wheat growth, including the significant amplification of several bacterial species known to be antagonistic to plant-pathogenic fungi.

Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.