Chlamydial infections have been associated with several chronic human diseases, including trachoma, pelvic inflammatory disease, chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. In Chlamydia-associated disease, the organisms are believed to exist in an atypical, persistent phase that is not well understood at the genetic level. The research presented in this thesis investigated chlamydial gene expression in in vitro cell culture models of persistence. The first set of studies analysed a continuous-infection model of persistence that has been recently developed for two C. pneumoniae isolates (TW-183 and CM-1). The spontaneous establishment and unique cyclical nature of continuous infections could be particularly relevant to in vivo events. An initial analysis using a semi-quantitative reverse transcriptase PCR (sqRT-PCR) approach provided evidence of differential gene expression in C. pneumoniae TW-183 continuous infections relative to acute control infections. Using a subsequently established fully quantitative real-time reverse transcriptase PCR (rtRT-PCR) assay, up-regulated expression profiles were confirmed for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in the continuous C. pneumoniae TW-183 infections. The omcB, pmp1 and porB genes, all of which encode membrane proteins, showed similar patterns of expression over both the acute and continuous time courses tested. Gene expression data for a second C. pneumoniae isolate, CM-1, revealed similar overall expression trends to those seen for C. pneumoniae TW-183 but also supported previous observations of different growth characteristics between the two isolates in the continuous-infection model. The rtRT-PCR assay was further optimised for use in gene expression studies of the gamma interferon (IFN-γ)-mediated model of C. pneumoniae A-03 persistence, in which altered growth and morphological traits typical of chlamydial persistence have been well characterised. Meanwhile, chlamydial genes such as euo, ftsK and hctB were emerging from the literature as reliable genetic markers of persistence. Therefore, a preliminary rtRT-PCR analysis of marker gene expression was used to assess the likely extent of persistence in individual IFN-γ-treated C. pneumoniae A-03 infections from a series of experiments that had been prepared for this persistence model. In this way, an appropriate pair of duplicate experiments was selected for further studies based on strong genetic evidence of persistence in IFN-γ-treated samples at 48 h post-infection (PI) in those experiments. Using rtRT-PCR, 14 genes of interest from the related peptidoglycan, aminosugars and lipopolysaccharide (LPS) biosynthetic pathways were analysed in the validated experiments of the IFN-γ-mediated C. pneumoniae A-03 persistence model. Selective up- and down-regulated expression trends were associated with IFN-γ-treatment at 48 h PI for genes encoding products that are located at specific enzymatic points in these pathways. Most strikingly, the expression of glmU, the product of which controls the amount of an essential precursor metabolite that enters both peptidoglycan and LPS biosynthesis, was strongly and reproducibly down-regulated in the 48-h PI IFN-γ-treated samples. This expression profile may contribute to a reduced rate of peptidoglycan biosynthesis in this persistence model and may therefore be related to the inhibited cell division and RB-to-EB differentiation that characterise chlamydial persistence. While most other genes in these pathways showed unchanged expression associated with IFN-γ treatment, murA and kdsB (from peptidoglycan and LPS biosynthesis, respectively) were selectively up-regulated in the 48-h PI IFN-γ-treated samples. Taken together, these data supported the concept of a persistence stimulon in C. pneumoniae that is regulated at key points in various metabolic pathways. In addition to the analysis of biosynthetic genes, the up-regulated gene set from continuous C. pneumoniae TW-183 infections was also analysed in the validated IFN-γ-mediated C. pneumoniae A-03 persistence experiments. The data revealed similarities and differences in gene expression patterns between these two in vitro persistence models. Furthermore, the profiles obtained for genes such as pmp1 and porB provided insights into the widely predicted phenomenon of late developmental gene shut-down during chlamydial persistence. A final investigation into an analogous IFN-γ-mediated persistence system for C. trachomatis serovar L2 focussed on one up-regulated (murA) and one down-regulated (glmU) gene from the validated IFN-γ-mediated persistent C. pneumoniae A-03 data set. Both genes were significantly down-regulated in persistent C. trachomatis, adding to a growing body of evidence for key differences among chlamydial species in their persistent gene expression patterns. This project has contributed significantly to our understanding of the molecular basis of the important persistent phase of chlamydial development.

La tuberculose, causée par Mycobacterium tuberculosis, connaît actuellement une résurgence inquiétante, et l’OMS estime à plus de 10 millions le nombre de nouveaux cas cliniques en 2015 avec environ 1,8 millions de décès dus à la maladie. Environ un tiers de la population mondiale est exposée à M.tuberculosis, et après exposition, la plupart des individus sont infectés par la mycobactérie. La grande majorité (~90%) des individus infectés ne présentera jamais de symptomatologie clinique. Parmi les 10% qui développent la maladie, environ la moitié le fera dans les deux années suivant l’infection, ce qui est en général considéré comme une forme primaire de tuberculose. Les autres patients présenteront leur maladie à distance de l’infection primaire (parfois plusieurs dizaines d’années plus tard) ; il s’agit des formes pulmonaires classiques de l’adulte. Chez l’homme, le rôle de certains facteurs génétiques a été maintenant démontré dans le développement d’une tuberculose active, à la fois la tuberculose pulmonaire de l’adulte et les formes plus disséminées de l’enfant, et aussi dans le contrôle de l’infection tuberculeuse. Cependant, la plus grande part de ces facteurs génétiques reste à identifier. Le premier objectif de ma thèse était d'identifier les facteurs génétiques de l'hôte modulant les phénotypes immunologiques de production d'Interféron gamma in vitro (IGRA) après exposition à M. tuberculosis dans un échantillon de 590 individus ayant été en contact avec un cas avéré de tuberculose dans le Val de Marne, en région parisienne. Puis, dans un second temps, de voir si les facteurs trouvés pouvaient être répliquées dans un échantillon familial d'Afrique du Sud, zone de très forte endémie tuberculeuse. Pour cela, j'ai tout d'abord réalisé des analyses de liaison génétique à l'échelle du génome entier sur plusieurs phénotypes quantitatifs d'IGRA. Celles-ci ont permis de mettre en évidence 2 loci majeurs (p < 10-4) répliqués en Afrique du Sud et liés à la production d'interféron gamma induite pour l’un par le bacille du BCG, et pour l’autre, par la part spécifique de l'antigène ESAT6 de M. tuberculosis (absent de la plupart des mycobactéries environnementales et du BCG), indépendamment de la capacité intrinsèque de réponse aux mycobactéries. La seconde étape a consisté en la réalisation d'une étude d'association sur les régions de liaison ainsi identifiées. Un variant associé au phénotype spécifique de l’ESAT6 (p < 10-5) a ainsi été trouvé, variant contribuant de manière significative au pic de liaison précédemment découvert (p<0.001) et ayant été rapporté comme modulant l’expression du gène ZXDC. Le second objectif de la thèse concernait l’identification de variants génétiques rares sous-jacents à la déclaration d’une tuberculose pulmonaire chez les individus infectés par le bacille. A cette fin, j’ai comparé les exomes de 120 patients tuberculeux à ceux de 136 individus infectés par le bacille mais non malades, tous originaires du Maroc. Cette étude m’a permis d’identifier le gène BTNL2, en bordure de la région HLA, dans lequel près de 10% des patients comportaient un variant rare perte de fonction contrairement aux contrôles qui n’en présentaient aucun. / Tuberculosis remains a major public health concern, with approximately 10.4 million new cases and 1.8 million deaths due to the disease in 2015 according to WHO. While an estimated one third of the world population is estimated to be infected with Mycobacterium tuberculosis, only about 10% of infected individuals go on to develop a clinical disease. Among them, half will declare the disease in the 2 years following infection, which is generally considered as primary tuberculosis. The other patients will develop the disease more distant in time of primary infection, sometimes several tens of years latter; these are classical pulmonary forms in adults. In humans, the role of genetic factors have been demonstrated in the development of active tuberculosis, in pulmonary forms as in disseminated forms in childhood, et also in the control of M.tuberculosis infection. Nevertheless, most of these genetic factors remain to identify. The first aim of my PhD was to identify genetic factors controlling in vitro interferon-gamma production phenotypes (IGRA) after exposure to M.tuberculosis in a sample of 590 subjects who were in contact with a proven tuberculous patient in Val-de-Marne, Paris suburbs, and in a second time, to try to replicate the findings in a south African familial sample where the tuberculosis is highly endemic. For this purpose, I first performed genome-wide genetic linkage analysis for several quantitative IGRA phenotypes. They led to identify 2 major loci (p<10-4) replicated in South-Africa and linked to the interferon-gamma production induced by live BCG for the first one, and for the second one, by the specific part of the ESAT6 antigen of M.tuberculosis (absent from most of environmental mycobacteria and from BCG), independently of intrinsic ability to respond to mycobacteria. The second step was an association study in the identified linkage regions. A variant associated to the specific ESAT6 phenotype was found (p<10-5), which was significantly contributing to the linkage peak (p<0.001) and previously reported as eQTL of ZXDC gene. The second objective of my PhD was the identification of rare genetic variants underlying the development of pulmonary tuberculosis in infected individuals. To this end, I compared exome data from 120 tuberculous patients and 136 infected individuals without any clinical symptoms. All of them were from Morocco. This study resulted in the lighting of BTNL2 gene, very closed to the HLA region, in which around 10% of patients had a rare loss of function variant whereas the controls didn’t have any.