Evaluation of the functional role of HtrA-like proteins present in pathogenic strains by creating mutants of Leptospira biflexa and Leptospira interrogans

• Grant number: 18/21959-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2019
• End date: February 28, 2022
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Ana Lucia Tabet Oller do Nascimento
• Grantee: Brenda Daroz
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 14/50981-0 - Search for surface proteins among the genome sequences of Leptospira interrogans: functional and immunological characterization to understanding mechanisms involved in the bacterial pathogenesis, AP.TEM

Abstract

Leptospires are divided into saprophytic, free-living species, such as L. biflexa, and pathogenics, such as L. interrogans, which causes Leptospirosis. This disease is a zoonosis of global importance recently included in the list of Neglected Tropical Diseases. Rodents play the major reservoirs of the disease in urban centers by allowing the colonization of leptospires in the proximal renal tubules and excreting them alive in the urine. There is no effective vaccine so far. The identification of conserved outer membrane proteins among pathogenic strains is the main research targets to elucidate pathogenicity mechanisms. These are the proteins probably involved in the interaction of leptospires with the external environment, and may also serve as targets for the host immune system. In addition, some spirochetes utilize outer membrane proteases as a mechanism for dissemination in host tissues. In this sense, the project proposes the creation of Leptospira mutants with the genes LIC11111, LIC1112, LIC20143 and LIC11037 overexpressing them in the case of L. biflexa (saprophytic) or silencing them, in the case of L. interrogans (pathogenic). These proteins are classified as HtrA (High temperature requirement A), belonging to an important family of proteases, identified by bioinformatics in the L. interrogans genome sequences. It is intended to characterize these proteins in vitro for reactivity against host components and to evaluate the enzymatic activity by degrading components of the extracellular matrix such as fibronectin. In order to validate the function and importance of these proteins, the project proposes to obtain leptospiral mutants, knock-in, in the case of saprophytes and knock-out, in the case of pathogens. The idea is to evaluate the acquisition or loss of function of these mutants compared to those observed in vitro. Mutants will be used to evaluate (a) cell localization of proteins using the immunofluorescence technique in the case of knock-in; (b) reactivity of recombinant bacteria to sera from patients diagnosed with Leptospirosis - comparing knock-in and knock-out and; (c) interaction of the bacteria expressing the proteins with host macromolecules and their proteolytic potential on them (both knock-in and knock-out mutants). (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)

DAROZ, BRENDA BEVILAQUA; VIRGILIO FERNANDES, LUIS GUILHERME; TEIXEIRA, ALINE FLORENCIO; TABET OLLER NASCIMENTO, ANA LUCIA. In Silico Structural and Functional Characterization of HtrA Proteins of Leptospira spp.: Possible Implications in Pathogenesis. TROPICAL MEDICINE AND INFECTIOUS DISEASE, v. 5, n. 4, DEC 2020. (18/21959-8, 17/06731-8, 16/11541-0, 14/50981-0)

DAROZ, BRENDA B.; FERNANDES, LUIS G. V.; CAVENAGUE, MARIA F.; KOCHI, LEANDRO T.; PASSALIA, FELIPE J.; TAKAHASHI, MARIA B.; NASCIMENTO FILHO, EDSON G.; TEIXEIRA, ALINE F.; NASCIMENTO, ANA L. T. O.. A Review on Host-Leptospira Interactions: What We Know and Future Expectations. FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, v. 11, p. 13-pg., 2021-11-25. (18/06201-1, 18/08131-0, 17/01102-2, 17/26223-7, 16/11541-0, 18/09652-4, 18/21959-8, 19/17488-2, 17/06731-8)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

DAROZ, Brenda. Functional role evaluation of proteins from the HtrA family of Leptospira interrogans. 2022. Doctoral Thesis - Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) São Paulo.