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Study of Type II secretion system and the molecular bases involved c-di-GMP signaling on Leptospira interrogans Serovar Copenhageni

Grant number: 16/08414-7
Support type:Regular Research Grants
Duration: April 01, 2017 - June 30, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Cristiane Rodrigues Guzzo Carvalho
Grantee:Cristiane Rodrigues Guzzo Carvalho
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated scholarship(s):17/26851-8 - Cloning and expression of the type II secretion system of Leptospira interrogans Copenhageni, BP.TT

Abstract

Leptospira interrogans serovar Copenhageni is a human pathogen that causes leptospirosis, a worldwide zoonosis. About 10% of leptospiral infections may progress to multiple organ failure and pulmonary haemorrhages leading to death. As with many other bacterial pathogens, environmental changes are sensed by L. interrogans during the course of infection and may trigger transitions between different cellular states, such as biofilm formation and activation of pathogenicity mechanisms. Such transitions in bacteria are commonly regulated by intracellular levels of c-di-GMP and L. interrogans possesses a wide array of GGDEF family c-di-GMP producing enzymes, c-di-GMP receptors and EAL domains, involved in the degradation of c-di-GMP.The aim of this proposal is the study of a group of proteins belongs to the c-di-GMP signaling as well as the proteins involved in the type II secretion system machinery present in the L. interrogans. The specific main objectives are: 1- Structural and functional studies of proteins involved in synthesis, degradation and binding of the bacterial second messenger c-di-GMP and 2- Structural and functional studies of type II secretion system found in Leptospira interrogans Copenhageni, the only secretion system found in its genome.This proposal is a continuation of present project in the laboratory (FAPESP number 2013 / 18664-2). In two years of project we have determined the three dimensional structure of LIC13137 GAF domain in complex with the cyclic nucleotide cAMP, functional assays were performed and we have just submitted an article to JBC journal, and we are waiting for the referee responses. In addition, we did collaboration with Dr. Ray Owens Group, Head of Facility Oxford Protein Production (Oxford Protein Production Facility_OPPF-UK) in the UK. In this collaboration most of the interested genes as well as fragments encoding some domains were cloned into expression vectors and screening for good candidates to structural studies. At the moment, we obtained crystals of PAS domains from LIC11127 and LIC11128 proteins. This proposal includes well establish techniques in our research group and novel techniques that I have just learnt with Professor Gabriel Waksman in England (cryo-electron microscopy). The results of this project will contribuit to the scientific community in general with projection of publications in a high impact scientific journals. (AU)

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