Abstract: Project 3: Medical College of Wisconsin Leptospirosis is the most widespread zoonotic disease worldwide, and varies in severity from mild illness to fatal hemorrhagic disease with multiple organ failure. Since endothelial damage and increased vascular permeability are prominent features of leptospirosis, we focus on identification of L. interrogans adhesins that mediate attachment to human endothelial cells in culture, identification of endothelial cell receptors to which the bacteria bind, and on the consequences of Leptospira-endothelial cell interaction on the integrity of the endothelial layer. Pathogenic L. interrogans crosses endothelial layers (transendothelial migration) efficiently, while the non-pathogenic L. biflexa does not. The major contributor to endothelial cell-cell adherens junctions (AJs) and barrier function, VE-cadherin, is a receptor for L. interrogans, and L. interrogans causes disruption of AJs, while L. biflexa does not. We identified two L. interrogans adhesins that bind to purified VE-cadherin with high affinity. We will collaborate with the Pasteur and UCLA groups to assess phenotypes of existing transposon mutant clones, as well as knock-in (gain of function) and knock-down mutants in cell culture and in animal models to identify and better characterize Leptospira proteins that contribute to invasion of endothelial barriers. In these experiments we will also generate samples for analyses of 1) bacterial and 2) host gene expression, and 3) host response biomarker production in a relatively simple cell culture model. Datasets 2 and 3 will be compared to those generated by the Duke group to identify and characterize the responses generated during human infection. Our hypothesis is that endothelial damage in leptospirosis results from collaboration between bacterial virulence determinants and endothelial cell responses. Aim 1 will assess human endothelial cell responses to, invasion dynamics, and in vivo tissue tropism of, diverse Leptospira isolates. Endothelial AJ integrity and bacterial transendothelial migration will be compared after infection with clinical and environmental isolates belonging to high-virulence and low-virulence pathogenic species, and saprophytic species, at various times post-inoculation. Supernatants will be used to quantify cytokines and other potential biomarkers of Leptospira infection. Cell layers and supernatants will be used for RNA-Seq to identify changes in host and bacterial gene expression. Statistical analyses of associations between pathogenicity, endothelial damage, and host responses will identify potential biomarkers as indicators of leptospirosis in patient blood samples, which could inform improved diagnostics in conjunction with the results obtained by the Duke group. Aim 2 will determine the roles of known and candidate L. interrogans adhesion proteins in adherence to, and invasion of, endothelial cell layers. Some of the candidate adhesins of interest include a family...