Abstract: Project 2, Institut Pasteur Leptospira is a highly heterogeneous bacterial genus and leptospires are ubiquitous bacteria found as free-living saprophytes in environmental water and soil or as pathogens that can cause disseminated infections, from asymptomatic carriage in rats to lethal acute infection in both humans and animals. Despite recent progress, the genus Leptospira remains understudied, and little is known about the ability of the pathogen to adapt to the host and cause disease. During dissemination, pathogens must circumvent host defense mechanisms and adapt to different forms of stresses. The work proposed in this program project seeks to identify and characterize bacterial determinants that promote mammalian host adaptation and enable leptospires to establish acute infection. In Aim 1, we will identify bacterial markers of disease severity. Genome sequencing of well-characterized Leptospira strains from our collection and clinical isolates collected in project 4 (Duke) will reveal new insights on the diversity of strains and species and their pathogenicity. We will perform the first Leptospira intra-genus large-scale evolutionary study to identify stepwise ancestral events at different nodes of evolution that have drastic consequences on the pathogens as we know them today and understand what could have been their contribution to the ecological niche adaptation and enhanced virulence of some of the pathogenic species. We will particularly focus our attention on the node of evolution in the pathogenic subclade P1 that separated the species infecting humans (P1hv) from the species not described as human pathogens (P1lv). The putative virulence factors will be inactivated in representative strains by targeted mutagenesis or gene silencing, or mutants will be selected from our library of random transposon mutants. We will collaborate with the Coburn and Haake labs to assess phenotypes of mutants. We will perform diagnosis for samples collected from project 4 (Duke) and we will provide technical support for field studies. Our work should also contribute to the identification of new antigens and/or markers for the development of novel strategies for the diagnosis of this neglected emerging disease. Aim 2 will characterize the regulation of gene expression in adaptation to the host environment. Successful host colonization by Leptospira requires sensing and response to face of changing conditions. A Dual RNA-seq approach in the acute hamster model will reveal the molecular mechanisms of interactions between L. interrogans and its host. Analysis of the transcriptome of Leptospira strains in vivo and different in vitro conditions will enhance our knowledge of how the pathogen can adapt to the host environment and the roles played by transcriptional regulators such as the Peroxide Stress Regulators PerRA and PerRB and non-coding small RNA in global gene expression. We will also investigate the role of epigenetic modification in global...