title: In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin.
abstract: LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.
authors.affiliation: Centro de Biotecnologia/Laboratório de Bacteriologia, Instituto Butantan, 05503-900 São Paulo, SP, Brazil.
authors.forename: Pricila
authors.initials: P
authors.lastname: Hauk
authors.forename: Felipe
authors.initials: F
authors.lastname: Macedo
authors.forename: Eliete Caló
authors.initials: EC
authors.lastname: Romero
authors.forename: Sílvio Arruda
authors.initials: SA
authors.lastname: Vasconcellos
authors.forename: Zenaide Maria
authors.initials: ZM
authors.lastname: de Morais
authors.forename: Angela Silva
authors.initials: AS
authors.lastname: Barbosa
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D005780:Gelatin / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D016056:Immunodominant Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007919:Leptospira / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007922:Leptospirosis / Q000097:blood / Q000276:immunology; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D024141:Collagen Type IV; D005353:Fibronectins; D007106:Immune Sera; D016056:Immunodominant Epitopes; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D005780:Gelatin; D006493:Heparin