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Doctoral Thesis
DOI
https://doi.org/10.11606/T.87.2022.tde-29112022-102012
Document
Author
Full name
Brenda Daroz
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2022
Supervisor
Committee
Nascimento, Ana Lucia Tabet Oller do (President)
Amaral, Tania Aparecida Tardelli Gomes do
Baldini, Regina Lúcia
Bertoncini, Michelle Darrieux Sampaio
Fernandes, Andrea Balan
Ferreira, Rita de Cassia Cafe
Title in Portuguese
Avaliação do papel funcional de proteínas da família HtrA de Leptospira interrogans
Keywords in Portuguese
Leptospira
HtrA
leptospirose
patogênese
Abstract in Portuguese
A leptospirose é uma zoonose de importância global recentemente incluída na lista das Doenças Tropicais Negligenciadas, causada por bactérias patogênicas do gênero Leptospira. Os roedores desempenham o papel de principais reservatórios da doença em centros urbanos por permitirem a colonização das leptospiras nos túbulos renais proximais e as excretarem vivas na urina. Segundo a Organização Mundial da Saúde, o número de casos clínicos anuais no mundo permanece subestimado, principalmente por causa do amplo espectro de sintomas inespecíficos que o paciente pode apresentar além de um ineficiente diagnóstico, sobretudo na fase inicial da doença. Não há vacina eficaz até o momento. A identificação de proteínas da membrana externa conservadas entre estirpes patogênicas é o principal alvo de pesquisa para elucidar os mecanismos de patogenicidade. São estas proteínas que, provavelmente, estão envolvidas na interação das leptospiras com o ambiente externo, podendo também servir de alvo para o sistema imune do hospedeiro. Algumas espiroquetas se utilizam de proteases de membrana externa como um mecanismo de disseminação por tecidos do hospedeiro. Neste sentido, o projeto propõe a clonagem e expressão de três proteínas preditas como sendo do tipo HtrA (do inglês High Temperature Requirement A), uma importante família de proteases, identificadas por bioinformática no genoma de L. interrogans. Os genes que codificam estas proteínas, LIC11111, LIC11112 e LIC20143, foram amplificados a partir do DNA genômico e o inserto clonado em vetor de expressão em Escherichia coli; então as proteínas foram purificadas e utilizadas para realização dos experimentos. Foram avaliados (a) atividade de imunogenicidade das proteínas em modelo animal, (b) reatividade das proteínas recombinantes frente a soros de pacientes diagnosticados com leptospirose, (c) interação dessas proteínas com macromoléculas do hospedeiro (d) conservação frente a diferentes estirpes de Leptospira e (e) atividade enzimática das proteínas. Construções da LIC11111, LIC11112 e LIC20143 fusionadas com os promotores fortes e constitutivos dos genes lipL32 e lipL21 de L. interrogans foram feitas para transformar L. biflexa sorovar Patoc e avaliar o possível ganho de função na bactéria saprofítica.
Title in English
Functional role evaluation of proteins from the HtrA family of Leptospira interrogans
Keywords in English
Leptospira
HtrA
leptospirosis
pathogenesis
Abstract in English
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.
 
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Publishing Date
2022-12-07
 
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