The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 ,um to 0.1-0.15 ,im. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4°C were divided into aqueous and hydrophobic phases after incubation at 37°C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organism. Full release of the 47-and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.

Three monoclonal antibodies (MAbs) were prepared against an arthritogenic strain of Mycoplasma hominis isolated from the joint aspirates of a patient with chronic septic arthritis. Immunoblots of polyacrylamide gel-electrophoresed proteins before and after surface proteolysis showed that the predominant antigenic determinants were on surface-exposed polypeptides. These polypeptides have extensive hydrophobic characteristics, as demonstrated by Triton X-114 phase partitioning. The electrophoresed proteins from cells grown in medium containing [14C]palmitate were blotted onto nitrocellulose which was both reacted with the MAbs and exposed to X-ray film. Superimposable bands on both the immunoblots and the exposed film suggested that the proteins might be acylated. The MAbs were further tested for reactivity with 16 other strains of M. hominis isolated from patients with septic arthritis (1 strain), septicemia (10 strains), or nongonococcal urethritis (1 strain); from the cervix (1 strain), rectum (1 strain), or surgical wound (1 strain) of patients; and from a contaminated cell culture. No single protein was consistently recognized from strain to strain, although a 94-kDa protein from 16 of the 17 strains tested was bound by at least one of the MAbs. The apparent antigenic heterogeneity among strains of M. hominis, including those isolated from the same tissue source and/or from patients with the same type of clinical disease, might be misleading in that all strains express epitopes associated with a discrete number of proteins to which one, two, or all three MAbs bind. The expression of the epitopes on multiple proteins from the same or different strains may reflect a mechanism for generating antigenic diversity.

A cloned Treponema pallidum antigen, designated 4D, was purified from Eschenichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190and 90-kd forms of 4D were stable at 68°C but converted to 19and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.

A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy.

Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.