Human leptospirosis is an emerging neglected tropical disease caused by pathogenic bacteria of the genus Leptospira with more than 1 million cases reported worldwide. There are no effective vaccines for human. China, Cuba, France and Japan have an inactivated whole-cell (bacterin) vaccine for risk group workers with no long-term protection and limited use. The bacterin vaccine does not confer cross protection with serovars which are not included in the preparation. Some pathogenic mechanisms are known like the binding and degradation of host extracellular membrane cells mediated by Leptospira outer membrane proteins. Leptospira can spread in the host blood and are able to survive the immune system acquiring soluble plasminogen and converting into plasmin to degrade many blood plasma proteins and fibrin clots. Furthermore, the binding to components or regulators of complement system pathway allow Leptospira to evade immune system. Two proteins identified in the outer membrane of L. interrogans, LIC10774 and LIC13086 can bind different host components such as laminin, plasminogen, fibrinogen, thrombin, plasma fibronectin, C4BP and components of the terminal complement pathway exhibiting a multifunctional role during the infection process. Thus, we aim to produce mutants of these genes in pathogenic Leptospira spp. by allelic exchange to investigate the functional and pathogenic effect of this deletion in the genome. To perform the mutagenesis, a suicidal plasmid will be constructed containing the upstream and downstream flanking region of the gene fragment which encodes LIC10774 and LIC13086 with a spectinomycin resistance cassette replacing the genes. The plasmid will be transformed in E. coli to be used in conjugative transfer to pathogenic Leptospira strains. After this, with the successful allelic exchange, the mutants of LIC10774 and LIC13086 will be evaluated by the capacity of binding the same host components tested with the recombinant proteins. In addition, mutants will be tested to determine if they maintain its virulence in hamster model comparing with wild-type infection. Whether the animals infected with mutants survive, a more comprehensive analysis can be performed to determine the LD50, as well as a vaccine challenge using the mutants as live vaccine to immunize hamsters. Furthermore, the recombinant proteins LIC10774 and LIC13086 may be good vaccine candidates to a subunit vaccine, due to their multifunctional role in pathogenesis. Thereby, a challenge will be performed with three doses of the recombinant proteins as subunit vaccine in leptospirosis hamster model. Taken together, this study aims to elucidate the application of gene knock-out technique, to better understand pathogenicity mechanism and possibly, to develop an effective vaccine.
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