Mycobacterium marinum est une mycobactérie pathogène des ectothermes génétiquement proche de M. tuberculosis. Ce modèle peut être utilisé pour mieux comprendre la formation des granulomes tuberculeux et déterminer le rôle des glycoconjugués pariétaux dans la régulation de la réponse immunitaire. Dans ce contexte, nos travaux ont porté sur la purification des glycolipides polaires et apolaires extraits de la paroi de M. marinum et leur caractérisation en utilisant la résonance magnétique nucléaire et la spectrométrie de masse. Nos résultats concernant l’analyse structurale de la famille des lipooligosaccharides polaires (LOSs, comprenant les LOS-I à LOS-IV) ont démontré la présence de monosaccharides rares, dont le caryophyllose et un monosaccharide N-acylé, spécifique du LOS-IV. Nous avons également observé que tous les LOSs inhibent la sécrétion macrophagique d’une cytokine pro-inflammatoire, le TNF-α. En revanche, seul le LOS-IV stimule l’expression d’antigènes (ICAM-1 et CD40) à la surface des macrophages et la production de chimiokine (IL-8). Cet effet inducteur du LOS-IV pourrait être lié à la présence du monosaccharide terminal N-acylé. Par ailleurs, les autres glycolipides polaires analysés, incluant les phosphatidyl-myo-inositol mannosides (PIM), le lipomannane (LM) et le lipoarabinomannane (LAM), possèdent des structures similaires à celles de M. tuberculosis.L’étude des glycolipides apolaires a permis de préciser la structure des glycolipides phénoliques (PGL) et des tréhaloses di-mycolates (TDM). De plus, une famille de glycolipides méconnue comprenant le Di-Mycolyl-Di-Arabinoglycérol (DMDA) a été identifiée. Le DMDA présente une structure très proche de la partie terminale du mycolyl-arabinogalactane-peptidoglycanne (mAGp), macro-complexe pariétal majoritaire des mycobactéries. Des études complémentaires effectuées chez M. bovis BCG ont démontré que ce glycolipide était lié au métabolisme du mAGp, ce qui ouvre de nouvelles pistes pour la compréhension du mode d’action de certaines drogues anti-tuberculeuses telles que la thiacétazone. / Mycobacterium marinum is a natural pathogen of ectotherms genetically close to M. tuberculosis. This pathogen model is useful for deciphering the role of mycobacterial cell wall glycolipids in granulomatous infection. In this context, our work focused on the purification of both polar and apolar glycolipids extracted from M. marinum cell wall and their structural characterization using nuclear magnetic resonance and mass spectrometry. Analysis of the polar lipooligosaccharide family (LOSs, including LOS-I to LOS-IV) demonstrated the presence of several rare or even unique monosaccharides including caryophyllose, derivatives and a N-acylated monosaccharide specific of LOS-IV. Biological activity assays showed that LOSs exert an important pro-inflammatory effect by decreasing the TNF-α secretion from macrophages. Moreover, LOS-IV was found to stimulate the expression of the chemokine IL-8 and cell surface antigens (CD40 and ICAM-1) on macrophages. This specific immunostimulatory property was related to the presence of the terminal N-acylated monosaccharide in LOS-IV. In addition, other polar glycolipids analyzed, including phosphatidyl-myo-inositol mannosides (PIM), lipomannane (LM) and lipoarabinomannan (LAM), possess similar structures than M. tuberculosis. The study of apolar glycolipids permitted to precise the structure of phenolic glycolipids (PGL) and trehalose di-mycolates (TDM). Moreover, a family of unusual glycolipids, including Di-Mycolyl-Di-Arabinoglycérol (DMDA), was identified. DMDA structure is very close from the terminal part of peptidoglycan-arabinogalactan-mycolyl (mAGp), the mycobacterial cell wall macro-complex. Additional studies performed in M. bovis BCG showed that this glycolipid is related to mAGp, providing new insights about the mode of action of anti-tuberculous drugs such as thiacetazone.

<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