Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli

Abstract

We have previously described the construction in Escherichia coli K-12 of a hybrid plasmid colony bank of Treponema pallidum (Nichols strain) genomic DNA. By screening a portion of this bank with an in situ immunoassay, we identified six E. coli clones that express T. pallidum antigens. In this study, the recombinant plasmids from each of these clones have been analyzed in E. coli maxicells and have been found to encode a number of proteins that are not of vector pBR322 origin and are, therefore, of treponemal origin. In each case, several of these proteins can be specifically precipitated from solubilized maxicell extracts by high-titer experimental rabbit syphilitic serum. Certain of these proteins are also precipitated by high-titer latent human syphilitic sera (HSS). The T. pallidum DNA inserts in these plasmids range in size from 6.2 to 14 kilobase pairs, and from the restriction patterns of the inserts and the protein profiles generated by each plasmid in maxicells, it is apparent that we have recovered a total of four unique clones from our colony bank. Recombinant plasmids pLVS3 and pLVS5 were of particular interest. Plasmid pLVS3 encodes three major protein antigens with molecular weights of 39,000, 35,000, and 25,000. These three proteins, which were not recognized by pooled normal human sera, were efficiently precipitated by most secondary HSS, latent HSS, and late HSS tested. These proteins were also precipitated, although somewhat inefficiently, by most primary HSS tested. Plasmid pLVS5 encodes a major protein antigen with a molecular weight of 32,000 and several minor protein antigens that, although efficiently precipitated by experimental rabbit syphilitic serum, were generally not recognized by the various HSS tested. Evidence is presented indicating that the protein antigens encoded by plasmids pLVS3 and pLVS5 are specific for pathogenic treponemal species. We have also demonstrated that immunoglobulin G antibodies directed against these protein antigens can be detected in rabbits experimentally infected with T. pallidum Nichols as early as 11 days postinfection.