Treponema pallidum is the etiologic agent of syphilis. Virtually nothing is known concerning the virulence determinants of this organism, due to the inability to successfully cultivate this spirochete in vitro for sustained periods. Although T. pallidum can be cultivated in rabbits and purified in small quantities, such procedures usually result in loss of virulence. Hence, it is difficult and expensive to obtain sufficient quantities of treponemes for meaningful analyses. In our laboratory, we are seeking express in Escherichia coli the genetic information encoding the important proteins of T. pallidum. Our long term objective is to produce these treponemal proteins in sufficient quantity such that their role in syphilis and possibly other human treponematoses can be defined, and their applicability as vaccinogens and/or improved serodiagnostic reagents can be investigated. For the project period, we propose to do the following: (i) Identify key treponemal proteins as targets for our cloning efforts. We are concentrating on those cellular and extracellular proteins that are in direct contact with the tissues of the infected host. We have developed a protocol that allows us to radiolabel treponemal proteins in vitro with S35-methionine to a very high specific activity. This permits us to effectively work with small quantities of material. We have tentatively identified a number of surface-exposed and extracellular protein antigens of T. pallidum. These will be further characterized. (ii) We will attempt to identify E. coli clones expressing specific T. pallidum proteins of interest. We have constructed two libraries of T. pallidum genomic DNA using two different vector systems. We have developed techniques to screen for clones expressing treponemal antigens. One specific 39K surface protein of T. pallidum has been cloned, expressed in E. coli in large quantity, and purified, demonstrating the feasibility of this research project. (iii) We will initiate a variety of studies aimed at determining the biological significance of the specific treponemal proteins under investigation.
