Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114

Abstract

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.

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis

“…These organisms are also capable of several biological functions in vitro that correlate with pathogenesis in vivo; they attach to a number of cell types in culture (9) and can penetrate endothelial cell monolayers through tight junctions (30). Our thin-section electron microscopy studies have shown that freshly extracted organisms have intact cross-sectional morphology characteristic of that of the spirochetes (6). Thus, the organisms used in this study represent, by all available biological parameters, virulent, structurally intact T. pallidum.…”

“…Therefore, a quantitative assessment of the degree of OM release was not possible. In our laboratory, Cunningham et al showed by thin-section electron microscopy that 1% Triton X-114 removed the OM of T. pallidum but left the IM morphologically intact (6). After phase partitioning of the Triton-soluble material, a set of eight polypeptides remained in the detergent phase, characteristic of integral membrane proteins.…”

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog

“…After centrifugation, Triton X-114 (TX-114; Calbiochem) was added to the supernatant to a final concentration of 1%. The solution was phase partitioned, and the detergent phase was washed three times with cold 50 mM Tris-HCl (pH 8.0) as described previously (20,43,53). The detergent phase was then diluted to 10 ml with 50 mM Tris-HCl (pH 8.0)-0.1% hydrogenated TX-100 (hTX-100) (Calbiochem Corp., San Diego, Calif.).…”

Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete Borrelia hermsii

“…Phenylmethylsulfonyl fluoride (Fisher), prepared as a 50-mg ml Ϫ1 stock solution in isopropanol, was added to a final concentration of 0.5 g l Ϫ1 , and the cells were harvested by centrifugation (11,228 ϫ g; 4°C), washed with PBS, and immediately resuspended in SDS solubilizing solution containing PMSF (0.5 g l Ϫ1 ). Triton X-114 extraction and phase partitioning was performed as described by Cunningham et al (10). Briefly, exponential-phase cells at an optical density at 600 nm of 0.5 were harvested by centrifugation, resuspended in 1% Triton X-114 (in PBS), and incubated at 4°C overnight with gentle rocking.…”