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No description available.

Enterohaemorrhagic Escherichia coli (EHEC) have recently emerged as significant zoonotic pathogens. O157:H7 is one of the most common EHEC serotypes associated with human disease, which is transmitted faeco-orally from a bovine reservoir. EHEC O157:H7 preferentially colonises the bovine terminal rectum (BTR). Injection of virulence factors by type-III secretion is necessary for colonisation of cattle and results in re-modelling of the host cytoskeleton. Flagella machinery is evolutionarily related to the Type III secretion apparatus and O157 strains lacking H7 flagella show reduced adherence to the BTR. Vaccination with FliC, the main component of H7 flagella, has the potential to protect cattle against E. coli O157:H7 infection. The focus of this work was to investigate the molecular basis for H7 flagella binding to the BTR, in order to understand the basis for FliCH7 being an immuno-protective antigen. H7 flagella were shown to adhere across the surface and penetrate into BTR epithelial cells. Both the FliC shaft and the FliD cap components of flagella filaments showed the capacity to adhere to BTR epithelial cells. Preliminary studies indicate that the current FliCH7 vaccination of cattle results in FliD-specific antibodies where oral challenge with O157:H7 does not. FliD is more conserved than FliCH7, which contains a predicted 88aa structural insertion, but variation occurs along the full length of the FliD protein. There was no evidence for post-translational modification of FliCH7. A number of actin binding proteins were identified as potential FliC and FliD binding partners from BTR epithelial cell lysates. From this, a panel of purified galectin-4, cofilin-1 and βγ-actin was used to compare binding of flagella from different pathogens. H7 flagella bound more to cofilin-1 than βγ-actin, whereas phase-1 and phase-2 flagella from Salmonella Typhimurium bound more to βγ-actin, than to cofilin-1. Size-exclusion chromatography indicated that cofilin-1 alters H7 flagella filament polymerisation dynamics. αβ-ctin polymerisation and depolymerisation experiments indicate that H7, phase-1 and phase-2 flagella interactions with actin affect actin dynamics.

Master of Science - Molecular and Cell Biology / The bacterial contamination profile of a typical commercial yeast factory was assessed by three replicate microbiological surveys. In order to detect low-level contamination in samples, this study made use of a preliminary incubation technique (24h at 37°C), which boosted bacterial counts for the identification of sources of contamination. Numbers of bacteria were quantified by standard pour- and spread-plate techniques and various selective media. Raw materials were negligible in contributing to the bacterial contamination of commercial yeast, with the exception of soda ash, used to control the pH of fermentations, which contained 2 log CFU/ ml Enterococcus and aerobic bacteria. It was found that the scale up of seed yeast biomass was the primary site for contamination with Enterococcus, which progressively increased in number as the product passed down the production line. Coliforms were present at low levels, with significant increases (P < 0.05) observed during the storage of yeast cream; extrusion of compressed yeast; and packaging of dry yeast. The environment surrounding the compressed yeast production line was identified as a potential source of airborne contamination. Although Salmonella spp. and S. aureus were not detected, L. monocytogenes was isolated from compressed and dry yeast products. In addition, Bacillus spp. commonly associated with the rope-spoilage of bread, were isolated from 67% of all dry yeast product samples. Shelf-life investigations, showed that cream and compressed yeast samples were spoiled with lengthened storage periods, and especially at higher temperatures (>10°C), whilst vacuum-packaged dry yeast remained bacteriologically stable. During shelf-life studies, isolates from spoiled cream and compressed yeast samples were predominantly Lactobacillus (up to 78%), while populations of Enterococcaceae predominated in vacuum-packaged dry yeast samples (up to 68%). The use of stainless steel surfaces, attached to processing equipment used in the manufacturing of Baker’s compressed yeast, in conjunction with SEM illustrated the accumulation of yeast and bacterial cells with early stages of biofilm formation, with time. Where populations of Gram-positive members of the lactic acid bacteria family, Lactobacillus and Enterococcaceae, were isolated in the highest proportion from processing equipment surfaces used in the manufacturing of Baker’s compressed yeast (81-100%).

In frogs, part of the important immune defence system of their skin is the secretion of antimicrobial peptides from granular glands. This study investigated the immune function of skin peptides in protection against bacterial pathogens associated with infectious bacterial dermatosepticemia under a number of environmental conditions and at certain stages of the life cycle of frogs. The natural peptide mixture of skin peptides was collected from skin secretions of three semi-aquatic Litoria frog species L. aurea, L. raniformis and L. ewingii and aquatic Xenopus laevis and assayed for activity against the bacterial pathogens: Aeromonas hydrophila, Chryseobacterium meningosepticum, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Serratia liquefaciens. The peptide mixtures of three frog species Xenopus laevis, Litoria aurea and Litoria raniformis showed activity against C. freundii, C. meningosepticum, K. pneumoniae and P. aeruginosa in vitro indicating a likely protective function. One Litoria species, L. ewingii, had a peptide mixture that did not have activity against any pathogen. Subsequently, in experimental exposure of animals to the pathogen K. pneumoniae, this species was found to be susceptible to disease while the other sympatric species L. raniformis was found to be resistant. A strong correlation was shown between composition of skin peptides and resistance to disease. A comparison of the production and activity of skin peptides from four frog species showed the aquatic X. laevis to have more effective immune defence against bacterial pathogens than three tested Litoria species. X. laevis produced significantly greater amount of bioactive peptide mixture than three tested Litoria species. Three pathogens A. hydrophila, P. mirabilis and S. liquefaciens are abundant components of the skin microbiota of healthy frogs and were found to be resistant to the peptide mixtures of all four frog species tested. It was shown that one pathogen, A. hydrophila, had the ability to secrete proteases which could inactivate skin peptides. Thus while skin peptides could function against several pathogens, some pathogens might have co-evolved to resist skin peptides. A comparison of the peptide mixtures from skin secretions of adults, metamorphs and larvae of L. ewingii using liquid chromatography-mass spectrometry analyses showed that peptide mixtures of post metamorphic animals, adults and metamorphs, had a species-specific profile that included the antimicrobial peptide uperin 7.1, while the larval peptide mixture did not contain uperin 7.1 or any other known species-specific peptide. This finding indicates the absence of a secretory mechanism that could compensate for the absence of granular glands in larvae. Analyses of the production and activity of skin peptides of L. raniformis after exposure to two different environmental stressors, low environmental temperature and pesticide carbaryl, showed that the total amount of bioactive peptide was significantly reduced which could consequently increase susceptibility to disease. Thus suppression of skin peptides could be a possible mechanism for synergism between the important stressors and pathogens in disease development.

Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system. The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation. Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis. This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.

Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system. The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation. Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis. This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.

Les biofilms sont des communautés structurées de micro-organismes enrobées dans une matrice extracellulaire. Les biofilms sont impliqués dans la persistance de plusieurs maladies infectieuses et la matrice extracellulaire du biofilm protège les bactéries contre les cellules du système immunitaire de l'hôte, les antibiotiques et les désinfectants. Récemment notre laboratoire a démontré que le zinc inhibe la formation de biofilm chez Actinobacillus pleuropneumoniae, une bactérie pathogène du porc. Le but de cette étude est d'évaluer l'effet du zinc sur la croissance et la formation du biofilm chez différentes bactéries pathogènes du porc, telles que Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus et Streptococcus suis. Les bactéries ont été cultivées dans des plaques de 96 puits sous condition optimale de formation de biofilm et les biofilms ont été colorés au cristal violet. La présence du biofilm a été confirmée par microscopie confocale à balayage laser à l’aide du marqueur fluorescent FilmTracerTM FM ® 1-43. À des concentrations micromolaires, le zinc inhibe faiblement la croissance bactérienne et bloque d'une manière dose-dépendante la formation de biofilm d’A. pleuropneumoniae, Salmonella Typhimurium et H. parasuis. De plus, la formation de biofilm de E. coli, S. aureus et S. suis a été faiblement inhibée par le zinc. Nos résultats indiquent que le zinc a un effet inhibiteur sur la formation de biofilm de la plupart des pathogènes bactériens d'origine porcine. Cependant, le mécanisme sous-jacent de l'activité anti-biofilm du zinc reste à être caractérisé. / Biofilms are structured communities of microorganisms enclosed in a self-produced extracellular matrix. Biofilms are responsible for the persistence of most infectious diseases, because the biofilm matrix acts as a form of protection for the bacteria against the host immune system, antibiotic and disinfectants. Recent work in our laboratory demonstrated that zinc could inhibit biofilm formation of Actinobacillus pleuropneumoniae, a swine pathogen. The aim of this study was to evaluate the effect of zinc on growth and biofilm formation of other bacterial swine pathogens, such as Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus, and Streptococcus suis. Bacteria were grown on 96-well plates under optimal biofilm forming conditions and the biofilms were stained with crystal violet. The presence of biofilms was confirmed by confocal laser scanning microscopy with FilmTracerTM FM® 1-43. At micromolar concentrations, zinc weakly inhibited bacterial growth and effectively blocked biofilm-formation by A. pleuropneumoniae, Salmonella Typhimurium, and H. parasuis in a dose-dependent manner. Additionally, biofilm formation of E. coli, S. aureus and S. suis was slightly inhibited by zinc. Our results indicate that zinc has an inhibitory effect on biofilm formation of most bacteria of porcine origin. However, the mechanism behind the antibiofilm activity of zinc has yet to be characterized.