Functional characterization of four thermolysins present in pathogenic species of Leptospira

Abstract

Leptospirosis, caused by pathogenic bacteria of the genus Leptospira, is an important medical and veterinary problem and is presumed to be the most widespread zoonosis in the world. Leptospires are currently classified in more than 300 serovars grouped in 21 species (pathogenic, intermediate and saprophyte). These spirochetes produce several proteases potentially capable of causing tissue damage. We have demonstrated that proteins secreted by pathogenic Leptospira strains are able to degrade several molecules from the host including extracellular matrix and coagulation cascade components, as well as complement proteins, thus contributing to invasion and immune evasion. Analysis of the available Leptospira genomes allowed identification of four thermolysins only in the pathogenic species encoded by the genes LIC13320, LIC13321, LIC13322 and LIC10715. Preliminary data indicate that at least one of the above-mentioned thermolysins (LIC13322) is secreted and displays proteolytic activity against fibrinogen, complement C3 and C6 and the proteoglycan decorin. Considering that enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions, the aim of this project is to extend our current knowledge on these Leptospira metalloproteases in the invasion and immune evasion processes. Two main goals will be pursued in this research project: 1) functional characterization of the thermolysins encoded by LIC13320, LIC13321 and LIC10715 from L. interrogans and 2) heterologous expression of the thermolysin encoded by LIC13322 from L. interrogans in L. biflexa. The implementation of tools allowing genetic manipulation of leptospires in our laboratory will be crucial for future projects, since the main focus of our group is to identify and characterize Leptospira virulence factors. (AU)