Resumo

Introduction: Leptospirosis is a zoonosis widespread throughout the world, caused by pathogenic spirochetes of the genus Leptospira, which colonize the renal tubules of wild and domestic animals. Transmission occurs through direct contact with water and soil contaminated with urine of infected animals. Pathogenic leptospires invade host tissues by penetrating damaged skin or the mucous membranes. After entering the host, the progression of the infection involves the adhesion of bacteria to eukaryotic cells and extracellular matrix proteins followed by invasion of tissues. The mechanism involved in pathogen invasion through extracellular barriers is not well elucidated. Enzymes capable of degrading extracellular matrix proteins could contribute to motility and chemotaxis of bacteria during these events. Pathogenic bacteria synthesize and secrete different types of proteases that degrade host proteins. The experimental proof of the existence and functional characterization of these proteins may contribute to the understanding of the pathogenesis of leptospirosis. Objectives: This work aimed the cloning, expression and functional characterization of a probable collagenase from L.interrogans serovar Copenhageni. Methods: Coding sequences of the collagenase domain 1 (D1), collagenase domain 2 (D2), and both domains (Full) of the ColA gene were amplified by PCR from genomic Leptospira DNA and cloned into the pAE expression vector. The recombinant 6xHis-tagged protein fragments were purified by nickel affinity chromatography. The purified fragments were used to obtain the polyclonal antiserum, and their enzymatic activities were evaluated by zymography. Results and Discussion: D1, D2 and Full fragments of ColA protein were expressed in E. coli BL21-SI and purified from the insoluble fractions. The purified D1, D2, and Full fragments exhibited single major bands of 35.8, 44.9, and 74.8-kDa, respectively, in SDS-PAGE. Rabbit polyclonal antiserum against the recombinant protein fragments were produced with a high antibody level detected by ELISA. Western-blotting experiments demonstrated the presence of ColA protein in different pathogenic serovars of Leptospira. Zymography revealed that ColA protein is able to degraded gelatin. These results indicated that ColA is probably a leptospiral protein involved in invasion of host tissues.