Dados do Trabalho

Título

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

Introdução

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

Objetivo (s)

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

Material e Métodos

<p>The project was approved by CEUAIB, (4425161222). Coding sequences for the LIC11051 and LIC11505 were analyzed in silico and cloned into pAE expression vector. <em>Escherichia coli</em> 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 <em>L. interrogans</em>. Recombinant proteins interactions with host macromolecules were evaluated by ELISA.</p>

Resultados e Conclusão

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

Palavras Chave

leptospira; Leptospirosis; recombinant protein; leishmaniose