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. 2006 May;74(5):2659-66.
doi: 10.1128/IAI.74.5.2659-2666.2006.

LfhA, a Novel Factor H-binding Protein of Leptospira Interrogans

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Free PMC article

LfhA, a Novel Factor H-binding Protein of Leptospira Interrogans

Ashutosh Verma et al. Infect Immun. .
Free PMC article

Abstract

The early phase of leptospiral infection is characterized by the presence of live organisms in the blood. Pathogenic Leptospira interrogans is resistant to the alternative pathway of complement mediated-killing, while nonpathogenic members of the genus are not. Consistent with that observation, only pathogenic leptospires bound factor H, a host fluid-phase regulator of the alternative complement pathway. Ligand affinity blot analyses revealed that pathogenic L. interrogans produces at least two factor H-binding proteins. Through screening of a lambda phage expression library, we identified one of these as the novel membrane protein LfhA. Ligand affinity assays and surface plasmon resonance analyses of recombinant LfhA revealed specific binding of both factor H and factor H-related protein 1. Serological examination of infected humans and horses demonstrated that LfhA is expressed by L. interrogans during mammalian infection. LfhA may therefore contribute to the resistance of pathogenic leptospires to complement-mediated killing during leptospiremic phases of the disease.

Figures

FIG. 1.
FIG. 1.
Indirect immunofluorescence analysis of L. interrogans serovar Lai and L. biflexa incubated with purified human factor H, anti-factor H monoclonal antibody, and fluorescently conjugated secondary antibody. Magnification, ×100.
FIG. 2.
FIG. 2.
Factor H ligand affinity analyses. Recombinant B. burgdorferi ErpA and ErpC proteins were included as positive controls (1, 30, 32, 41, 56), and carbonic anhydrase, soybean trypsin inhibitor, and lysozyme were included as negative controls. A. A lysate of L. interrogans serovar Pomona strain JEN4 was subjected to SDS-PAGE and examined for proteins that bind purified human factor H. Two such protein bands were identified, with apparent molecular masses of approximately 30 and 50 kDa. B. Ligand affinity blot analysis demonstrating that recombinant LfhA binds purified human factor H. Positions of molecular mass standards are indicated to the left of each panel (in kilodaltons).
FIG. 3.
FIG. 3.
Surface plasmon resonance analyses of factor H and FHR-1 binding to recombinant LfhA. A. Analyses of factor H binding, utilizing 200, 100, 50, 25, and 12.5 nM factor H. B. Analyses of FHR-1 (200 nM) binding to recombinant LfhA. Binding of proteins to the immobilized ligand is reported as relative resonance units per time.
FIG. 4.
FIG. 4.
Cellular localization of LfhA by cellular fractionation with Triton X-114. Fractions of the detergent phase (outer membrane fraction), aqueous phase (periplasmic fraction), and protoplasmic cylinder (inner membrane plus cytoplasm fractions) were separated by SDS-PAGE and analyzed by immunoblotting using affinity-purified rabbit antiserum directed against LfhA (top panel). As a control for fractionation purity, fractions were also analyzed using rabbit antiserum to Treponema pallidum FlaB, which also recognizes the L. interrogans endoflagellum (42) (bottom panel). Spirochete flagella are anchored to the inner membrane only and thus purify with the protoplasmic cylinder.
FIG. 5.
FIG. 5.
Southern blot analysis of the presence of lfhA genes in pathogenic serovars of L. interrogans, other Leptospira spp., and Leptonema illini.
FIG. 6.
FIG. 6.
Immunoblot analyses of LfhA with serum samples from humans and horses naturally infected with L. interrogans. Strip blots containing recombinant LfhA were probed with sera from clinically confirmed cases of human and equine leptospirosis. Normal human and equine sera and rabbit polyclonal antiserum specific for LfhA were included as controls.

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