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Evaluation of the proteolytic activity of proteases secreted by pathogenic leptospires in phagocytosis by human and murine phagocytes

Grant number: 17/10208-9
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): October 01, 2017
Effective date (End): May 31, 2021
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal researcher:Lourdes Isaac
Grantee:Thais Akemi Amamura
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Leptospirosis is a zoonosis caused by bacteria spirochetes belonging to the genus leptospira. Very common in tropical and subtropical areas, the disease represents a serious public health problem. Pathogenic leptospires have the ability to escape from the complement system, being able to survive when in contact with normal serum. In an earlier study conducted by our group, several pathogenic leptospires species were able to secrete proteases cleaving complement proteins as the C3 core molecule, as well as its fragments, C3b and iC3b, as well as alternativa (Factor B), classical and lectin pathway (C4b and C2). During my master's degree, we found that these proteases were also able to cleave the purified C6, C7, C8 and C9 proteins from the MAC, whereas the non-pathogenic proteins did not show significant proteolytic activity on these substrates. Assays using protease inhibitors revealed an inhibition of proteolytic activity by 1, 10-phenanthroline, indicating the involvement of metalloproteases. Among these, we have been able to prove the proteolytic activity of the recombinant thermolysin LIC13322, a metalloprotease exclusively expressed by pathogenic strains of leptospira. This recombinant thermolysin was able to cleave the C6 component, either alone or as part of the SC5b-9 complex. In addition, we have found that the MAC proteins are capable of interacting with the recombinant thermolysin, indicating that this protease may exert an inhibitory effect additional on these molecules by a possible mechanism of inhibition by direct interaction. Few studies have so far sought to uncover why pathogenic leptospires are more refractory to phagocytosis than non-pathogenic species. Our working hypothesis is that proteases secreted by pathogenic leptospira could interfere with their internalization and death within phagocytes. In this project, we will evaluate if such enzymes will be able to cleave receptors and opsonins present on the surfaces of phagocytes, thus affecting the recognition of the bacteria by these cells. Opsonization is one of the most important mechanisms that occur during the Complement activation phase, with fragments C3b, iC3b and C4b responsible for covalently binding to the surface of different pathogens. This phagocytosis process can be facilitated by the interaction of these opsonins with specific receptors (eg Fc³R, CR1 and CR3) present on the surfaces of phagocytic cells such as neutrophils and macrophages. Other receptors are quite important for the recognition of pathogens by macrophages and neutrophils, among them: TLR that, after interaction with PAMPs, triggers the release of intracellular signals that culminate in the induction of transcription factors of genes important for the immune response; mannose receptor that recognizes certain terminal sugars present in the carbohydrates of the microbial surface; and scavenger receptor that were initially identified for their ability to recognize and remove modified lipoproteins. The evasion of the immune response of the host allows the dissemination of the pathogen, favoring the establishment of the infection. Therefore, we consider it important to investigate the role of proteases secreted by pathogenic leptospires on phagocytes. We believe that the identification of biological targets of leptospira proteases is of great importance for a better understanding of the pathogenesis of the disease, and may also contribute to the development of therapeutic and/or preventive strategies in the infection by these pathogens. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
SANTIESTEBAN-LORES, LAZARA ELENA; AMAMURA, THAIS AKEMI; DA SILVA, TIAGO FRANCISCO; MIDON, LEONARDO MOURA; CARNEIRO, MILENA CARVALHO; ISAAC, LOURDES; BAVIA, LORENA. A double edged-sword - The Complement System during SARS-CoV-2 infection. Life Sciences, v. 272, MAY 1 2021. Web of Science Citations: 0.

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