Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfatepolyacrylamide gel electrophoresis results from different laboratoriesAs the first step in a cooperative effort to standardize the identification of the polypeptides of Treponerna pallidum subsp. pallidurn, the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 ofthe major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T.pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T.pal1idum proteins. Abbreviations: CIE, crossed immunoelectrophoresis; 2DE, two-dimensional gel electrophoresis; IRS, infected rabbit serum; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; RIP, radioimmunoprecipitation: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; T, Tween 20 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0173-0835/87/0202-0077 %02.50/0 S. J. Norris et al. Electrophoresis 1987,8, 77-92 Figure 16. Reactivity of rabbit antiserum directed against cloned antigenTmpC (1301, seeFig. 6)with the 2DEpatternof T.paNidum.Theprocedure wasas described in Fig. 15. (A) Reaction with the TmpC-specific antiserum. (B) Antigenic profile following IRS counterstaining. The antiserum reacted specifically with a single band on the SDS-PAGE pattern (far left) and a single spot at the acid end of the 2DE pattern.Figure 17. Localization of cloned antigen TpD (1301, see Fig. 6) in the 2DE profile of T.pallidum, utilizing a specific rabbit antiserum prepared by affinity chromatography. (A) Reactivity of TpD-specific antiserum. Due to prolonged incubation with the color reagent, background reactivities of the antiserum to TmpC and the major 61 polypeptide were also detectable in this case. (B) Antigenic profile following IRS counterstaining. The positions of TpD and TmpC in the 2D and single dimension SDS-PAGE profiles are indicated by the arrows.

Groups of 10 young male guinea pigs of inbred strains 2 and 13 and outbred strains Hartley A, Hartley B, and one deficient in the fourth component of complement (C4D) were infected intradermally with 80 X 10(6) Treponema pallidum (Nichols). The course of infection and production of antitreponemal antibody were examined. Strain C4D guinea pigs were the most susceptible to infection (100%); inbred strains 2 and 13 and outbred strain Hartley B showed 80-90% symptomatic infection; and the Hartley A strain was the least susceptible to infection (10%). Strain 13 animals responded with the highest antitreponemal antibody activity, and the Hartley A strain with the lowest. The results suggest that genetic factors or complement, or both, may influence the degree of susceptibility to infection with T pallidum in guinea pigs.

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