CHARACTERIZATION OF LEPTOSPIRA INTERROGANS LSA31 PROTEIN BINDING ADHESIVE MATRIX MOLECULES

Denize Monaris (IBu); Amane Paldes Gonçales (FMVZ-USP); Zenaide Maria Morais (FMVZ-USP); Silvio Arruda Vasconcellos (FMVZ-USP); Angela Silva Barbosa (IBu); Patricia Antonia Estima Abreu (IBu)

Introduction: Leptospirosis is an infectious disease caused by pathogenic leptospires that are transmitted to humans through direct contact with infected animals, or through indirect contact with water or soil contaminated with the urine from infected animals. Leptospires enter the body by penetrating mucous membranes or broken skin and disseminate via the bloodstream to colonize the renal tubules of hosts. The colonization and survival of leptospires in the host require several types of surface-exposed proteins, such as lipoproteins, porins, adhesins and others. The protein chosen for this work is an outer membrane lipoprotein of Leptospira interrogans serovar Copenhageni that displays extracellular matrix binding properties. Objectives: The aim of this work was to study the capacity of Lsa31 protein to mediate attachment to extracellular matrix molecules (ECM). Methods:  The gene was cloned into the expression vector pAE in Escherichia coli. The recombinant 6xHis-tagged protein was purified from insoluble fraction by nickel affinity chromatography, and characterized by circular dichroism spectroscopy (CD). The capacity of recombinant purified protein to bind to ECM components was evaluated by ELISA and Western blotting-based methods. To examine its interaction with ECM components, BSA, fetuin and protein encoded by the gene LIC11030 were used as negative controls. The recombinant proteins were subjected to SDS-PAGE, and then transferred to nitrocellulose membranes. For analyses of laminin and fibronectin binding, membranes were blocked with BSA or skim milk, and then incubated with laminin or fibronectin. Following were incubated with antibodies specific for laminin or human fibronectin. Finally, the membranes were incubated with antibodies peroxide-conjugated. Bound antibodies were detected using chemiluminescence substrate. Results and Discussion: The purified Lsa31 protein exhibited a single major band of  31.8-kDa in SDS-PAGE. The structural integrity of Lsa31 recombinant purified protein was assessed by CD spectroscopy, which revealed a predominant b-sheet secondary structure, and showed successful refolding process. Furthermore, recombinant Lsa31 was able to bind strongly to laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin and matrigel. Lsa 31 is probably a leptospire antigen involved in adherence to host tissues.