"Structural, functional and immunogenic characterization of two proteins of Leptospira interrogans containing FecR domains"

• Grant number: 23/08486-1
• Support Opportunities: Scholarships in Brazil - Master
• Start date: November 01, 2023
• Status: Discontinued
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Ana Lucia Tabet Oller do Nascimento
• Grantee: João Pedro Gaspar Inácio
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 19/17488-2 - Advancing the understanding of pathogenesis and virulence of Leptospira interrogans through proteomics, structural, mutagenesis and immunological analyses, AP.TEM
• Associated scholarship(s): 24/14302-3 - Evaluation of Leptospira interrogans FecR proteins rLIC10902 and rLIC20185 interaction with human cells and heterologous expression in non-pathogenic Leptospira, BE.EP.MS

Abstract

Pathogenic Leptospira cause leptospirosis, a globally disseminated zoonosis. These bacteria colonize the renal tubules of animals and are excreted in urine. Humans are accidental and terminal hosts, exhibiting diverse clinical manifestations. The disease affects approximately 1 million people annually, resulting in about 60,000 deaths. The mechanisms of pathogenicity and infection of Leptospira spp. are not fully understood. One approach to understanding this function is to unravel the mechanisms performed by their outer membrane proteins. Proteins with FecR domains are associated with iron metabolism. The role of this domain in leptospira proteins is not yet understood. Therefore, in this study, the objective is to clone, express, and purify proteins containing the FecR domain: LIC10902 and LIC20185 from L. interrogans serovar Copenhageni, and evaluate their functional and immunogenic properties. For this purpose, the genes will be amplified by PCR, cloned into the pAE vector, and expressed in different Escherichia coli strains for solubility studies. Purification will be performed by metal affinity chromatography, and the protein structures will be evaluated by circular dichroism. The differential expression of the target proteins in pathogenic and attenuated strains of L. interrogans, as well as in the saprophytic species Leptospira biflexa, and the alteration of their expression in response to absence of iron, will be assessed by real-time quantitative PCR. The immunogenic evaluation of the proteins in BALB/c mice and the determination of the subcellular localization of the target proteins will be realized by ELISA. The immunological detection of the target proteins will be performed by western blotting, using hyperimmune mouse serum. The interaction of the proteins with cellular receptors and human components associated with the extracellular matrix and plasma will be tested for functional characterization. Obtaining and validating the immunological, structural, and functional aspects of these proteins will contribute to advancing the studies on the pathogenicity of leptospires.

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)

GASPAR, JOAO P.; TAKAHASHI, MARIA B.; TEIXEIRA, ALINE F.; NASCIMENTO, ANA L. T. O.. In silico analysis and functional characterization of a leucine-rich repeat protein of Leptospira interrogans. INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, v. 316, p. 11-pg., 2024-09-03. (19/17488-2, 19/05466-4, 16/11541-0, 23/08486-1, 17/26223-7)