Study of the Antigenic Structure of Treponema Pallidum by Specific Agglutination2

Hardy, Paul H.; Nell, E. Ellen

doi:10.1093/oxfordjournals.aje.a119893

Cited by 29 publications

(31 citation statements)

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“…8, left (11,17). It has also been shown by immunoelectron microscopy that the surface of the virulent organism is resistant to the binding of antitreponemal antibodies in the absence of complement (13,14,25).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1988

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 1988

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“…As such, one might anticipate that the appearance of specific antibodies would herald the eradication of the invader and the control of syphilitic infection, yet as discussed above, clinical and experimental evidence to support this assumption has not been easy to garner. More than 5 decades ago, investigators began to note the poor surface reactivity of motile T. pallidum in serologic tests (55,86,89). Their studies fostered the notion that the spirochete is surrounded by a protective layer comprised of …”

Section: Discussionmentioning

confidence: 99%

“…Moreover, passive-transfer experiments have demonstrated that immune rabbit serum partially protects against intradermal challenge (97,125). On the other hand, investigators have experienced considerable difficulty in directly detecting antibodies bound to the surface of intact treponemes using a variety of techniques, including agglutination (55), radioimmunoassay (32), immunofluorescence (31,96), immunoelectron microscopy (61,103), and cell surface radioimmunoprecipitation (118). A number of years ago, we devised the gel microdroplet technique to probe the antigenic structure of T. pallidum under conditions that minimize damage to its fragile outer membrane during the immunolabeling procedure (31).…”

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confidence: 99%

Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins ofTreponema pallidum

Cox

Luthra²,

Dunham-Ems³

et al. 2010

Infect Immun

112

164

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Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely ␤-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of ␤-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and ␤-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.

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“…It does not contain lipopolysaccharide (8,9), and it can be disrupted under certain conditions (e.g., low concentrations of detergents) and by physical manipulations (e.g., centrifugation and resuspension) that cause little discernible damage to the outer membranes of conventional Gram-negative bacteria (8,(10)(11)(12). Perhaps of profound significance with respect to the ability of the organism to evade host immune defenses, virulent organisms in vitro bind only small amounts of the specific antibodies present in human or rabbit syphilitic sera (11)(12)(13)(14). This remarkable phenomenon traditionally has been attributed to a layer of host proteins and mucopolysaccharides external to the outer membrane (15,16).…”

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confidence: 99%

Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum.

Radolf

Norgard

Schulz

1989

Proc. Natl. Acad. Sci. U.S.A.

193

183

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Freeze fracture and deep etching were used to investigate the ultrastructural basis for the observation that anti-treponemal antibodies bind poorly to the surface of virulent Treponema pallidum. Fractures of T. pallidum outer membranes contained scarce, uniformly sized intramembranous particles (IMPs). IMPs on the convex faces often appeared to form linear arrays that wound in spirals about the organism. In contrast to the outer membrane, IMPs of the cytoplasmic membrane were randomly distributed, numerous, and heterogeneous in size. In Escherichia coli and T. pallidum cofractures, IMPs of the E. coli outer membranes were densely packed within the concave fracture faces, while the T. pallidum fractures were identical to the experiments lacking the E. coli internal controls. Outer membranes of two representative nonpathogenic treponemes, Treponema phagedenis biotype Reiter and Treponema denticola, contained numerous IMPs, which segregated preferentially with the concave halves. Examination of apposed replicas and deep-etched specimens indicated that at least some of the IMPs extend through the T. pallidum outer membrane and are exposed on the surface of the organism. The outer membrane of intact T. pallidum appears to contain a paucity of integral membrane proteins that can serve as targets for specific antibodies. These rmdings appear to represent an unusual parasitic strategy for evasion of host humoral defenses.Syphilis, a sexually transmitted disease caused by the bacterium Treponema pallidum subsp. pallidum, continues to be a major global public health problem. In untreated individuals, syphilis is a chronic infection that progresses through stages with characteristic clinical manifestations. The mechanisms that enable virulent treponemes to survive for years in the face of vigorous humoral and cellular immune responses by the host are among the most poorly understood aspects of syphilis pathogenesis.Like all spirochetes, T. pallidum morphologically consists of an outer membrane that surrounds the periplasmic endoflagella, the cytoplasmic membrane, and the protoplasmic cylinder (1). Investigators have presumed that, as with other bacterial pathogens, surface-exposed molecules mediate the interactions between treponemes and their human hosts (2). For this reason, characterization of the outer membrane of T. pallidum and analysis of the structure's protein constituents have become major goals of treponemal research. Complicating such investigations is the fact that T. pallidum remains one of the few important pathogens of humans that cannot be maintained by continuous in vitro cultivation. Recombinant DNA and monoclonal antibody methodologies have facilitated analyses of a number of treponemal immunogens, although the precise cellular locations of most ofthem remain uncertain (3-7).Despite these limitations, recent investigations have demonstrated that the T. pallidum outer membrane differs significantly from the analogous structure of conventional Gram-negative bacteria. It does not contain lipopolysa...

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Study of the Antigenic Structure of Treponema Pallidum by Specific Agglutination2

Cited by 29 publications

References 0 publications

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

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

Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins ofTreponema pallidum

Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum.

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