Genetic Approaches to Evaluation of the Roles of Leptospira interrogans Adhesins in Endothelial Interactions

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Abstract Leptospirosis is caused by several pathogenic species of spirochetes of the genus Leptospira and is the most widespread zoonosis worldwide. Pathogens in the genus Leptospira are maintained in zoonotic cycles, demanding that they be able to establish persistent, disseminated infection in immunocompetent host animals, but the genus also includes saprophytic, non-pathogenic species. Reservoir hosts do not develop severe disease, but the outcome of the Leptospira-host interaction depends on the host species, the Leptospira species, serological variant (serovar), and additional unknown factors. Disease in humans ranges from mild self-limited illness to acute life-threatening infection with kidney failure, and liver dysfunction, and widespread endothelial damage, increased permeability, and hemorrhage. Disruption of endothelial barriers is likely to facilitate dissemination and to contribute to hemorrhagic disease manifestations. We have focused on investigation of interactions between endothelial cell surface receptors and Leptospira in vitro, identified endothelial cell-surface receptors for L. interrogans, and identified candidate L. interrogans adhesins (denoted LIC11574 and LIC13411) that bind to VE-cadherin, the primary endothelial barrier determinant. We hypothesize that adhesion to host transmembrane receptors by multiple adhesins contributes to disruption of endothelial barriers and transmigration of the bacteria. In this proposal, we will take multiple approaches to determining the roles of previously identified adhesins in bacterial transmigration, disruption of VE-cadherin, and changes in transendothelial electrical resistance (TEER). We will employ Tn mutants, and gain of function derivatives of a saprophytic strain that produce VE-cadherin adhesins, to evaluate the potential roles of 7 adhesins. Innovation lies in use of both genetically modified Leptospira strain sets in our endothelial cell culture model. In Aim 1 we will characterize the phenotypes of L. interrogans transposon mutants in genes encoding known or candidate adhesins for adhesion to and transmigration across endothelial layers. In Aim 2 we will similarly characterize gain of function saprophytic strains producing L. interrogans adhesins that bind VE-cadherin. Our proposed work will constitute a major step toward resolving the biologically fundamental question of what are the functional determinants of Leptospira that separate the pathogens from the non-pathogens. The proposed work will also take steps toward resolving a long-standing controversy in the field: Is endothelial damage the result of bacterial interactions with the endothelium or to the host response, increasing impact.