Functional role evaluation of proteins from the HtrA family of Leptospira interrogans

Author(s)

Brenda Daroz

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2022-02-04

Examining board members

Ana Lucia Tabet Oller do Nascimento; Tania Aparecida Tardelli Gomes do Amaral; Regina Lúcia Baldini; Michelle Darrieux Sampaio Bertoncini; Andrea Balan Fernandes; Rita de Cassia Cafe Ferreira

• Advisor: Ana Lucia Tabet Oller do Nascimento; Luís Guilherme Virgilio Fernandes

Abstract

Leptospirosis is a zoonosis of global importance included in the list of Neglected Tropical Diseases, caused by pathogenic bacteria of the genus Leptospira. Rodents are the main reservoir of the disease in urban centers by allowing colonization of leptospires in the proximal renal tubules and excreting them alive in urine. According to the World Health Organization, the number of annual clinical cases in the world remains underestimated, mainly because of the wide spectrum of nonspecific symptoms that the patient can present, in addition to an inefficient diagnosis, especially in the initial phase of the disease. To date, there is no effective vaccine. The identification of outer membrane proteins conserved among pathogenic strains is the main research goal to elucidate the mechanisms of pathogenicity. These proteins are probably involved in the interaction of leptospires with the external environment and may also serve as a target for the host\'s immune system. Some spirochetes use outer membrane proteases as a mechanism for spreading through host tissues. In this sense, the project proposes the cloning and expression of three proteins predicted to be of the HtrA family (High Temperature Requirement A), an important family of proteases, identified by bioinformatics in the genome sequences of L. interrogans. The genes encoding these proteins, LIC11111, LIC11112 and LIC20143, were amplified from genomic DNA and the insert were cloned into pAE expression vector in Escherichia coli; then the proteins were purified and used for the experiments. We evaluated (a) immunogenicity activity of proteins in an animal model, (b) reactivity of recombinant proteins against sera from patients diagnosed with leptospirosis, (c) interaction of these proteins with host macromolecules (d) conservation against different strains of Leptospira and (e) enzymatic activity of proteins. Constructs from LIC11111, LIC11112 and LIC20143 fused with the strong and constitutive promoters of the L. interrogans genes lipL32 and lipL21 were obtained and used to transform L. biflexa serovar Patoc and evaluate the possible gain of function in the saprophytic bacteria. (AU)

• FAPESP's process: 18/21959-8 - Evaluation of the functional role of HtrA-like proteins present in pathogenic strains by creating mutants of Leptospira biflexa and Leptospira interrogans
• Grantee: Brenda Daroz
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)