Dados do Trabalho

Título

Expression and characterization of two Leucine-Rich Repeat Proteins of Leptospira interrogans

Introdução

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. Comparative genomic studies between pathogenic and saprophytic species of Leptospira have shown significant differences in the number of genes encoding proteins with Leucine Rich Repeat (LRR) domains, suggesting a potential role in virulence. LRR have been reported to be involved in bacterial host-pathogen interactions, invasion, and stimulation of host immune response.

Objetivo (s)

The objective of the project is the cloning, expression and purification of two proteins containing LRR domains from L. interrogans in order to characterize their immunogenic properties, and possible participation in pathogenicity.

Material e Métodos

The project was approved by CEUAIB, (4425161222). Coding sequences for the LIC11051 and LIC11505 were analyzed in silico and cloned into pAE expression vector. Escherichia coli was transformed and expression was performed by adding IPTG. The protein purification was performed by metal affinity chromatography and immunogenicity was evaluated in BALB/c mice. The collected sera were used for ELISA titration and detection of proteins in different fractions of the whole cell lysates L. interrogans. Recombinant proteins interactions with host macromolecules were evaluated by ELISA.

Resultados e Conclusão

Recombinant proteins were expressed in the soluble fraction in all tested E. coli strains, with the best yield achieved in BL21 Star (DE3) pLysS. Proteins rLIC11051 and rLIC11505 were successfully purified and both were immunogenic in BALB/c mice with titers of 160,000 and 640,000, respectively. Both recombinant proteins displayed significant interaction with plasminogen, whereas rLIC11505 had a broad spectrum of ligands, binding to plasma fibronectin, vitronectin, collagen 1 and glycosaminoglycans. Both native proteins counterparts were detected in whole cell lysates of L. interrogans along with secreted soluble fraction and SDS-soluble membrane proteins. rLIC11051 and rLIC11505 are immunogenic proteins and able to interact with host components. Functional characterization of their role in the pathogenicity of leptospirosis are currently under investigation.

Palavras Chave

leptospira; Leptospirosis; recombinant protein; leishmaniose