Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

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Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be divided into three subgroups: saprophytes, pathogens, and a middle group of unknown pathogenicity. The most widely used and studied species are L. biflexa (a free-living, non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have decided to initially focus on L. biflexa in order to develop the microbial and genetic expertise we plan to transfer to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. Only one report of targeted gene inactivation in L. interrogans has been published and no successful shuttle vector system currently exists for the pathogen. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon mutants. We have therefore decided to focus on increasing and improving the molecular genetic tools available to manipulate leptospires, as we have experience in developing a genetic system for another spirochete, Borrelia burgdorferi, the causative agent of Lyme disease. The long-term objective of this project is to use the improved tools and techniques to understand the mechanisms of infection and pathogenecity of L. interrogans and accelerate the development of preventative measures against Leptospirosis.