CHARACTERIZATION OF THE LOW-MOLECULAR-MASS PROTEINS OF VIRULENT TREPONEMA-PALLIDUM

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-m olecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically r adiolabel T. pallidum cells to high specific activity to further chara cterize 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. Imm un. 56:490-498, 1988). The low-molecular-mass proteins did not incorpo rate H-3-labeled fatty acids and were not precipitated by rabbit immun oglobulin G (IgG) antibodies directed against glutathione S-transferas e fusions to the nonlipidated 15- and 17-kDa proteins. We prepared pol yclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG an tibodies present in the sera of both rabbits precipitated a 21.5-kDa p rotein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an addi tional 15.5-kDa low-molecular-mass protein only from solubilized extra cts of supernatant. While investigating the effect of eliminating HINR S 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 cell s by the addition of HINRS or rabbit serum albumin, suggesting that th ey are located on or near the treponemal cell surface. The 15.5- and 2 1.5-kDa low-molecular-mass proteins were not washed off treponemal cel ls with buffer containing I M KCI. Experiments employing selective sol ubilization of the T. pallidum outer membrane with 0.1% Triton X-114 a nd proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.