Dados do Trabalho

Título

Characterization of a novel leptospiral protein reveals adhesion to endothelial cells in vitro.

Introdução

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp.. Humans are usually infected through contact with contaminated soil or water. More than 1 million of cases can occur each year, with approximately 60 thousand deaths. Leptospira pathogenesis is still little understood. Outer membrane proteins due to their location, are considered interesting target to investigate as they may be involved in pathogenicity mechanism.

Objetivo (s)

Cloning, expression and purification of putative lipoprotein LIC13355 of Leptospira interrogans and evaluation of its interactions with hosts purified components and endothelial cells in vitro.

Material e Métodos

The project was submitted to and approved by Butantan Institute Ethics Committee in Animal Usage (CEUAIB - 2840200422), that follows CONCEAs guidelines. The gene LIC13355 was amplified from L. interrogans sorovar Copenhageni strain L1-130 by PCR and cloned into pAE vector. Its heterologous expression in Escherichia coli strains was assessed by SDS-PAGE and Western Blotting, the recombinant protein was purified using IMAC. Its secondary structure was determined by circular dichroism. Protein interaction with host’s extracellular matrix, plasma components, and endothelial cells was evaluated by ELISA or Western Blotting.

Resultados e Conclusão

Sequencing analysis reveals that the gene LIC13355 was successfully cloned in pAE vector. Tests using E. coli as expression host under variable conditions showed that the protein is expressed in its insoluble form. Inclusion bodies were solubilized in urea and refolded with decreasing urea concentration in HisTrap nickel column, with subsequent elution. Fractions containing higher concentration of the purified protein were dialyzed in Tris-HCl buffer. Circular dichroism analysis indicates that the protein structure is composed of 31,6% alfa-helix, 21,8% of beta-sheet and 42,5% other structures. In ELISA assay, rLIC13355 has bound to laminin, type I collagen, cellular and plasmatic fibronectin, and plasminogen, exhibiting a dose-dependent profile with all components. rLIC13355 was able to capture plasminogen from human plasma, and convert into plasmin. rLIC13355 bound to Ea.hy926 and HMEC-1 in monolayer and suspension cells, indicating affinity to extracellular matrix and cell surface components. Binding to Ea.hy926 cells also presented a dose-dependent profile, with an estimated Kd of 24 µM.rLIC13355 is most probably a novel leptospiral adhesin that can participate in different steps of the infection.

Palavras Chave

Leptospira interrogans; protein expression; protein cell interaction; leishmaniose