Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo

Abstract

We radiolabeled Leptospira proteins with [35S]methionine. Solubilized extracts of radiolabeled L. interrogans serovar hardjo strain hardjoprajitno were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. We compared the protein profile obtained in this manner to the protein profiles of various [35S]methionine-labeled Leptospira spp. The profiles of the pathogenic L. interrogans strains were very similar but not identical and exhibited no obvious relationship to those of the two nonpathogenic species. We used solubilized, radiolabeled hardjoprajitno extracts and a sensitive radioimmunoprecipitation procedure to identify protein antigens recognized by immunoglobulin G antibodies present in various rabbit anti-hardjo sera. Homologous hyperimmune rabbit serum efficiently precipitated a large subset of proteins, the majority of which were between 30,000 and 66,500 daltons. Radioimmunoprecipitations with sera prepared against each of four recent hardjo isolates cultured from infected cattle produced similar results. Immunoprecipitations done with various radiolabeled Leptospira extracts and anti-hardjoprajitno serum demonstrated that the pathogenic leptospires possessed a number of cross-reactive major and minor protein antigens. By cell fractionation procedures, we found that most of the major protein antigens were present in the outer envelope. These proteins were exposed on the leptospiral cell surface because intact radiolabeled leptospires bound antibodies directed against them.

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis

“…Although there have been extensive studies on Leptospifal antigens employing monoclonal [4,6,7,9] or polyclonal rabbit immune sera [21,22], little information is available about the specific antigens which are immunodominant in natural human leptospirosis; only Chapman [5,26] de-scribed some antigens recognized by the human response to L. hardjo infection. In this study we planned to identify the immunodominant antigens recognized by humans after infection by leptospires of the serogroup Australis.…”

“…It cannot be ruled out that the human natural response protein antigen also contributes to the antigenic mosaic of the serovar. Nunes-Edwards et al stressed the importance of some protein antigens for protection [22] and considered them to be localized on the outer envelope. Further work is planned in an attempt to characterize this antigen.…”

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes

“…The samples were labeled for 60 min, and the cells were pelleted, washed, and solubilized. Immunoprecipitations with normal rabbit serum (NRS) and rabbit anti-E. coli RecA serum were performed as previously described, and the results were analyzed by SDS-PAGE and fluorography (20). Radioactivity in individual protein bands was quantitated with an AMBIS Radioanalytic Imaging System (Ambis Systems, San Diego, Calif.).…”

In vitro association of leptospires with host cells

“…Leptospires were used at passage 5 or earlier and were hamster infective. For some experiments, leptospires were radiolabeled by a modification of the method of Nunes-Edwards et al (9). Briefly, 5 to 10 ,uCi of [35S]methionine (specific activity, 1,051 Ci/mmol; ICN Biomedicals, Inc., Costa Mesa, Calif.) per ml was added to early-log-phase cultures, and incubation was continued at 32°C until a density of 5 x 107 to 8 x 107 organisms per ml was reached.…”