PROJECT SUMMARY Leptospirosis is an emerging infectious disease and the leading zoonotic cause of morbidity and mortality worldwide, with its greatest burden on subsistence farmers and urban slum populations. Leptospirosis causes life- threatening disease and has emerged as a major cause worldwide of pulmonary hemorrhage syndrome (LPHS) and acute kidney injury. To date, there is no effective prevention and control for leptospirosis in resource-poor settings. In the US and other industrialized countries, leptospirosis is a major cause of disease among inner-city populations, military personnel, and individuals engaged in swimming and water sports. Leptospirosis is caused by environmentally transmitted spirochetes belonging to more than 300 serovars among seventeen pathogenic species within the genus Leptospira, a genus that also comprises 26 non-pathogenic species and 21 intermediate species, the latter with undefined role as disease causative agent. The hallmark of infection with Leptospira species is its rapid hematogenous dissemination after the organism penetrates through mucous membranes, disseminating to multiple organs throughout the host. Despite the sizable burden of disease associated with leptospirosis, the biological and genetic mechanisms of pathogenesis associated with Leptospira remain poorly understood. This crucial gap of knowledge has impaired the development of better diagnostic and control tools. In this exploratory proposal, we hypothesize that shifts in gene expression, together with divergent and convergent evolution of gene function, have led to diversity in ability of Leptospira to survive and thrive outside of the host and facultative pathogenicity. To identify genes responsible for pathogenicity, we will use a comparative systems biological approach, in which we profile transcription across species using in vitro models of infection that are in vivo surrogates for gene expression. Those in vitro models mimic the epidemiological life cycle of the bacteria and the biological processes essential to establish disease. Through access to our extensive library of himar1-based mutants, we will identify strains with gene disruption for some of those targets. Through known functional phenotypes using well-characterized in vitro assays of adherence and translocation, as well as ability to survive and thrive in environmental matrices such as water and soil, we will determine and characterize the role of those genes in pathogenicity and transmission. Acquiring knowledge of the identity of genes responsible for pathogenicity and transmission is a major priority for the molecular understanding of the mechanisms of leptospiral pathogenesis that will directly facilitate the advance of public health measures through the development of improved diagnostic and prevention methods.