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Construction of knock in mutants of saprophytic Leptospira biflexa and validation of leptospiral receptors

Grant number: 16/07680-5
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): July 11, 2016
Effective date (End): January 10, 2017
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Ana Lucia Tabet Oller Do Nascimento
Grantee:Luis Guilherme Virgílio Fernandes
Supervisor abroad: Mathieu Picardeau
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Local de pesquisa : Institut Pasteur, France  
Associated to the scholarship:12/24164-0 - Characterization of the interaction of Leptospira interrogans with prothrombin/thrombin system and possible implications in virulence, BP.DR

Abstract

The genus Leptospira encompasses both pathogenic and saprophytic species. Pathogenic Leptospira are the etiological agents of leptospirosis, while saprophytic bacteria are environment free-living organisms. The adhesion of pathogens to extracellular matrix components is considered to be essential in the initial stage of the infection, and thus far, we have reported several recombinant leptospiral proteins with in vitro binding properties. Also, we have recently described the capability of virulent leptospires to bind thrombin and other coagulation molecules, being some possible receptors described. The understanding of the mechanisms responsible for leptospirosis pathogenesis is yet poorly understood, mainly due the lack of genetic tools available for the pathogen; shuttle vectors that replicates in both Escherichia coli and saprophyte L. biflexa are being used for heterologous expression of proteins contained in pathogenic strains and validation of in vitro adhesion properties. The saprophytic strain L. biflexa is considered a "cleaner" model for the analysis of heterologous protein function, once this strain lacks several proteins involved in virulence mechanism displayed by pathogenic strains, which present a high degree of functional redundancy, denoted by the occurrence of several proteins with overlapping in vitro functions. For that reason, knock out of pathogenic strains might not produce any measurable effect. We aim to validate the binding abilities of some leptospiral recombinant proteins described in our laboratory regarding different host components, including coagulation molecules. For achieving this purpose, the selected genes will be cloned along with their promotor in the replicative plasmid, which will be inserted in the saprophytic L. biflexa. Binding assays will be performed to demonstrate the expected gain of function due to heterologous expression. At last, the technic will be fully implemented in our laboratory. (AU)