Shigella provoque la dysenterie bacillaire en envahissant les muqueuses du colon. Cette maladie diarrhéique est responsable d’un million de décès par an essentiellement dans les pays en voie développement. Les gènes nécessaires àl’entrée dans les cellules hôtes sont regroupés sur un fragment d’ADN plasmidique de 30-kb. Celui-ci contient deux types de gènes, les gènes ipa(B, C et D) et ipgcodant pour des protéines responsables de l’entrée de la bactérie dans les cellules, et les gènes mxi/spacodant pour un système de sécrétion appelétype III (SST3) nécessaire àla sécrétion des facteurs de virulence. Les gènes mxi/spaetipa/ipgsont exprimés à37°C et les protéines Ipa/Ipgrestent dans le cytoplasme jusqu’àce que le SST3 soit activéau contact de la cellule hôte. Ce contact induit l’internalisation de la bactérie par macropinocytose, suivie de sa dissémination intra-et intercellulaire. Des observations en microscopie électronique (ME) montrent que le SST3 est composéde trois parties: i) une aiguille dont la longueur est régulée à50 nm par la protéine Spa32, ii) un corps basal qui traverse les membranes interneet externe ainsi que le peptidoglycane, et iii) un bulbe cytoplasmique. Le SST3 est le dispositif principal de virulence et permet l’injection de facteurs de virulences du cytoplasme bactérien vers celui de la cellule cible. Shigelladoit sécréter ces protéines de manière ordonnée. Très peu de travaux ont abordécette question. L’objectif principal de ce travail de thèse a étéd’étudier les mécanismes moléculaires impliqués dans la hiérarchie de sécrétion.Nous avons principalement investiguéle rôle de 3 protéines: Spa32, Spa40 et MxiC dans la sécrétion. Nous avons montré, par des études génétiques, que contrairement aux études publiées sur les protéines homologues, Spa32 n’agit pas comme un «molecularruler»pour réguler la taille de l’aiguille. Nous avons montréque cette régulation nécessite l’interaction de Spa32 via ses résidus 206-246 au domaine C-terminal de Spa40 (Spa40C) (Botteaux et al. 2008a). Ayant identifiécette interaction avec Spa40, l’étape suivante de notre travail a portésur la caractérisation de la fonction du gène spa40par des méthodes génétique, biochimique et structurale (ME). Nos résultats montrent que Spa40 joue un rôle important dans l’assemblage du SST3. Des plus, nous avons mis en évidence de nouvelles interactions impliquant Spa40C et des composants du SST3 (Botteaux et al. in preparation).Parallèlement àces travaux, nous avons montréque l’inactivation du gène mxiCaboutit àune dérégulation spécifique de la sécrétion des effecteurs sans altérer celle des translocateurs IpaB et IpaC. Cette augmentation de sécrétion est due àune augmentation de transcription des gènes tardifs, conséquence de la sécrétion précoce de l’anti-activateur transcriptionnel, OspD1 (Botteaux et al. 2008b). De plus, nous avons montréque MxiC est un substrat de l’appareil de sécrétion et que cette sécrétion est associée àsa fonction. Finalement, la mise en évidence d’une interaction entre MxiC et Spa47, l’ATPase du SST3 nous permet de proposer un modèle régulant la hiérarchie de sécrétion des effecteurs.Dans une autre partie de notre travail, nous avons identifié, par des expériences de ME et d’immunomarquage, que l’invasineIpaD est localisée au sommet de l’aiguille du SST3 oùelle lui sert de bouchon. Enfin, de manière très intéressante, nous avons montréque des anticorps anti-IpaD neutralisent l’entrée de Shigelladans les cellules (Sani, Botteaux et al. 2007, dépôt de brevet). IpaD, étant conservée dans les isolats invasifs de Shigella, représente donc un réel candidat vaccinal pouvant pallier la diversitédes sérotypesbactériens.En conclusion, nos travaux représentent une contribution importante àla compréhension des mécanismes de virulence bactériens et dépassent le cadre de Shigellapuisque les systèmes de sécrétion sont hautement conservés parmi plusieurs pathogènes. L’identification de drogues pouvant interférer avec ces systèmes de sécrétion représente une voie d’avenir pour le développement de nouveaux agents anti-infectieux. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Clostridium difficile est un pathogène entérique qui cause d'importantes infections nosocomiales dont le traitement est parfois problématique. Il n'existe, à l'heure actuelle, que deux antibiotiques approuvés pour traiter les infections à C. difficile et le taux de rechute est assez important. Ce projet a initialement visé à isoler et caractériser des phages virulents contre C. difficile en vue de les utiliser en phagothérapies comme outils thérapeutiques alternatifs. Les eaux usées et les selles de patients infectés par C. difficile ont été utilisées pour isoler et détecter les phages virulents. Or, quatre phages différents (9MMPOI-O4) ont été isolés mais aucun de ces phages ne s'est révélé être virulent. Les quatre phages tempérés ont donc été caractérisés et leur impact a été évalué sur quelques phénotypes bactériens chez C. difficile dont la motilité et la production des toxines A et B. La caractérisation morphologique des phages (pMMPOl-04 a permis de déterminer que ceux-ci appartiennent à la familles des Myoviridae alors que la caractérisation génomique a permis de démontrer que certains de ces phages sont assez différents entre eux mais également par rapport aux autres phages tempérés, isolés et caractérisés dans la littérature. De façon générale, les phages (pMMPOl-04 ne semblent pas s'induire spontanément de manière plus importante mais suggère que la présence de certains antibiotiques pourrait augmenter l'induction de certains de ces phages. L'impact des phages (pMMPOl-04 sur la motilité chez C. difficile n'a pas démontré que ceux-ci avaient un rôle à jouer sur ce phénotype. Par contre, certains des phages (pMMP semblent augmenter ou diminuer la production en toxines A et B. Les résultats de nos travaux indiquent donc que certains des phages caractérisés présentent des différences importantes qui suggèrent une grande diversité parmi les phages tempérés chez C. difficile. De plus, certains des phages cpMMP auraient la capacité de participer aux transferts horizontaux de matériels génétiques et d'affecter la régulation de certains facteurs de virulence chez C. difficile tel que la production en toxines A et B. Évidemment, des travaux supplémentaires seront nécessaires pour confirmer la modification du phénotype de production en toxines par ces phages mais également sur d'autres phénotypes associés aux autres facteurs de virulence de cette bactérie. [Symboles non conformes]

INTRODUCTION:Honey bees, Apis mellifera, have a diverse community of pathogens. Previous research has mostly focused on bacterial brood diseases of high virulence, but milder diseases caused by fungal pathogens have recently attracted more attention. This interest has been triggered by partial evidence that co-infection with multiple pathogens has the potential to accelerate honey bee mortality. In the present study we tested whether co-infection with closely related fungal brood-pathogen species that are either specialists or non-specialist results in higher host mortality than infections with a single specialist. We used a specially designed laboratory assay to expose honey bee larvae to controlled infections with spores of three Ascosphaera species: A. apis, the specialist pathogen that causes chalkbrood disease in honey bees, A. proliperda, a specialist pathogen that causes chalkbrood disease in solitary bees, and A. atra, a saprophytic fungus growing typically on pollen brood-provision masses of solitary bees.RESULTS:We show for the first time that single infection with a pollen fungus A. atra may induce some mortality and that co-infection with A. atra and A. apis resulted in higher mortality of honey bees compared to single infections with A. apis. However, similar single and mixed infections with A. proliperda did not increase brood mortality.CONCLUSION:Our results show that co-infection with a closely related fungal species can either increase or have no effect on host mortality, depending on the identity of the second species. Together with other studies suggesting that multiple interacting pathogens may be contributing to worldwide honey bee health declines, our results highlight the importance of studying effects of multiple infections, even when all interacting species are not known to be specialist pathogens.

Shigella flexneri is a Gram negative facultative anaerobe that infects millions world-wide each year. The route for infection of a host is through the intestinal and rectal epithelium layers, but it also can survive in the environment. Different genes have been found to be up regulated depending upon its presence in the intracellular or extracellular environment, as shown in previous work in the lab. This thesis seeks to examine the role these upregulated genes, bioA and lysA, play in the intracellular activity of S. flexneri. Knock-out mutations in the bioA and lysA genes were created using P1 transduction. To test the effects of these mutations on S. flexneri, plaque, invasion, and attachment assays were performed. It was found that the bioA mutation resulted in fewer plaques being formed, while the lysA mutation resulted in slower forming and incompletely lysed plaques being formed. / text

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<p>Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.</p><p> The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.</p> / Dissertation