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The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.

Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T. pallidum have been isolated. Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T. pallidum is capable of eliciting antibody responses during natural infections. The p24 antigen of T. pallidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum. We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T. pallidum because (i) p24 was secreted by the recombinant E. coli cells; (ii) recombinant p24 in E. coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms. The possible role of the p24 protein in determining the growth characteristics of T. pallidum is suggested by the ability of recombinant p24 to induce growth changes in E. coli cells. All E. coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a filamentous growth pattern that were different from those of other E. coli cells. The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T. pallidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA. However, any possible relationship between the p24 antigen and the virulence of T. pallidum remains to be determined. In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T. pallidum organisms.

Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp. pallidum (T. pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M. V. Norgard, N. R. Chamberlain, M. A. Swancutt, and M. S. Goldberg, Infect. Immun. 54:500-506, 1986). To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties. Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T. pallidum DNA fragment. A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system. Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular masses were observed that were identical to those previously detected on Western blots of native T. pallidum antigens with monoclonal antibodies. Sarkosyl extraction of E. coli recombinant cell envelopes localized the 47-kDa protein to both the inner and outer membranes of E. coli. The absolute requirement of detergents (N-lauroylsarcosine, 3-[(3-chloramidopropyl)dimethylammonio]-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40) for solubilization of the antigen from E. coli cell envelopes and the observation that the recombinant protein partitioned into the detergent phase on Triton X-114 solubilization were consistent with the fact that it is a hydrophobic, integral membrane protein. Western blots of the 47-kDa antigen purified by immunoaffinity chromatography supported results of previous reports that the 47-kDa protein is specific to pathogenic treponemes.