Etude par transcriptomique et génomique comparative de la pathogénicité de Coxiella burnetii : une approche puce à ADN

The objective of this thesis was to increase knowledge of obligate intracellular bacteriaand specifically, the causative agent of Q fever C. burnetii. In this regard, we have improvedstrategies to purify RNA for the transcriptional studies. We also used the technologymicroarrays to analyze the transcriptome and genomic diversity of C. burnetii.The use of clinical samples in the transcriptional studies is limited by the amount ofmaterial available and thus the transcriptional profiles of the pathogen and its host duringinfection can not be simultaneously analyze. We developed, with a model of eschars obtainedfrom Mediterranean spotted fever patients, a strategy based on subtractive hybridization toseparate RNA eukaryotic and prokaryotic cells in order to perform microarray experiments.Analysis of the survival strategies used by this bacterium to adapt to new environmentalconditions is critical for our understanding of C. burnetii pathogenicity. Here, we report theearly transcriptional response of C. burnetii under temperature stresses. Our data show thatC. burnetii exhibited minor changes in gene regulation under short exposure to heat or coldshock. While small differences were observed, C. burnetii seemed to respond similarly to coldand heat shock. The expression profiles obtained using microarrays produced in-house wereconfirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentiallyexpressed in at least one condition, with a fold change of up to 4. Globally, the differentiallyexpressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis,wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycansynthesis. These findings could be associated with growth arrest and witnessed transformationof the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes weredifferentially expressed. These clusters do not belong to operons or genetic networks; theyhave no evident associated functions and are not under the control of the same promoters. Wealso found undescribed but comparable clusters of regulation in previously reportedtranscriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeriamonocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stressespermits the recognition of unpredicted clusters of regulation for which the trigger mechanismremains unidentified but which may be the result of a new mechanism of epigenetic regulation.Different typing methods have been previously developed to classify C. burnetii isolatesin order to explore its pathogenicity. Here, we report a comprehensive genomotyping methodbased on presence or absence of genes using microarray. The genomotyping method was thentested on 52 isolates obtained from different geographic areas, different hosts and isolated frompatient with different clinical manifestations. The analysis reveals the presence of10 genomotypes organized in 3 groups with a topology congruent with that of Multi SpacerTyping. We also found out 4 genomotypes especially associated with acute Q fever whereas allthe genomotypes could be associated to chronic human infection. Serendipity, genomotypingreveals that hard ticks isolates including Nine Mile belong to the same genomotype.Overall, the data we obtained confirm that DNA microarrays are a suitable tool forexploring pathogenicity of C. Burnetti and other obligate intracellular bacteria. However newtechnologies such as DNAseq or RNAseq seem more promising but still need to optimize andalso are still expensive compared to microarray.