LEPTOSPIRA-HOST INTERACTION: I. Action of proteases secreted by L. interrogans on human plasma molecules II. Characterization of the SPOR domain of the MPL36 protein involved in the interaction with plasminogen

Abstract

Bacteria of the genus Leptospira are spirochetes that have efficient strategies for dissemination in the host. The mechanisms underlying the entry, dissemination, persistence and tissue damage resulting from infection by pathogenic leptospires are still poorly known, but studies carried out by our group have shown that extracellular proteases produced by these bacteria contribute to the different stages of the colonization process. Furthermore, outer membrane proteins, crucial molecules for pathogenicity and modulation of the host's immune response, are among the virulence factors presented by pathogenic leptospires, particularly those with domains exposed on the cell surface.This project was subdivided into 2 subprojects. In subproject 1 we will apply degradomics to study the effects of proteases secreted by leptospires on human plasma molecules. The plasma will be incubated with leptospires or with extracellular proteins secreted by the bacteria. The N-terminal groups of the degradation products will be initially marked using TAILS approach (Terminal Amine Isotopic Labeling of Substrates) with the TMT isobaric marker, reduced with DTT, alkylated with iodoacetamide, and digested with trypsin. Samples will be analyzed in the Orbitrap Exploris 480 mass spectrometer coupled to the Vanquish Neo liquid nano-chromatograph. This approach emerges as a promising and unbiased alternative as opposed to classical methodologies, which presuppose a targeted analysis of a subset of proteins or host pathways selected a priori. In subproject 2, the aim is to deepen knowledge on a Leptospira surface protein, called MPL36, already described as a plasminogen (Plg)-interacting protein. Previous data suggest that the SPOR domain of MPL36 is the region responsible for interaction with human Plg. In this subproject we will obtain the recombinant SPOR domain, assess its interaction with Plg, and will also evaluate the functional consequences of this interaction. In silico comparisons with SPOR domain proteins from other bacteria (Escherichia coli and Pseudomonas aeruginosa) will be performed as well. These analyzes may reveal relevant aspects to understand the function of these proteins in different bacteria.Studies targeting the Leptospira-host interface can generate knowledge on changes that occur in the plasma involving molecules of innate immunity, the coagulation cascade and other pathways, opening perspectives for the identification of new substrates and to improve our understanding on the mechanisms used by this bacterium to cause disease. (AU)