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Characterization of the low-molecular-mass proteins of virulent Treponema pallidum
1Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill 27599-7400.
Abstract:
We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.
Insights
Treponema pallidum synthesizes low-molecular-mass proteins, identified as 15.5-kDa and 21.5-kDa antigens, which are located on or near the cell surface. The 15.5-kDa protein is specifically cell surface-exposed, offering potential targets for Treponema pallidum research.
Area of Science:
- Microbiology
- Immunology
- Protein Biochemistry
Background:
- Treponema pallidum synthesizes extracellular, low-molecular-mass proteins in vitro.
- These proteins are distinct from previously identified 15- and 17-kDa detergent-phase proteins.
- Metabolic radiolabeling allows for high specific activity characterization of these antigens.
Purpose of the Study:
- To further characterize the low-molecular-mass, extracellular antigens synthesized by Treponema pallidum.
- To determine the localization and surface exposure of these novel antigens.
- To differentiate these antigens from known T. pallidum proteins.
Main Methods:
- Metabolic radiolabeling of Treponema pallidum cells.
- Preparation of polyclonal antisera against low-molecular-mass proteins.
- Immunoprecipitation assays using rabbit IgG antibodies.
- Selective solubilization with Triton X-114 and proteinase K accessibility assays.
Main Results:
- Two low-molecular-mass proteins, 15.5-kDa and 21.5-kDa, were identified and precipitated by specific antisera.
- These proteins remain associated with treponemal cells in the absence of heat-inactivated normal rabbit serum (HINRS).
- The 15.5-kDa protein was confirmed as cell surface-exposed, while the 21.5-kDa protein's localization was less defined.
Conclusions:
- Treponema pallidum expresses novel cell surface-associated low-molecular-mass proteins.
- The 15.5-kDa protein is a potential cell surface-exposed antigen of Treponema pallidum.
- Further investigation into these antigens may reveal new diagnostic or therapeutic targets.