TREPONEMA-PALLIDUM IN GEL MICRODROPLETS - A NOVEL STRATEGY FOR INVESTIGATION OF TREPONEMAL MOLECULAR ARCHITECTURE

Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein im munogens. To address these issues and circumvent problems associated w ith prior efforts to localize treponemal surface antigens, we develope d a novel strategy for investigating T. pallidum molecular architectur e. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X- 100. Intact, encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a ca ndidate T. pallidum outer membrane protein (TpN50) with C-terminal seq uence homology to Escherichia coli OmpA or by human or rabbit syphilit ic serum. Each of these immunologic reagents, however, labelled encaps ulated treponemes co-incubated with detergent. In contrast, antibodies generated against isolated T. pallidum outer membranes labelled intac t organisms and the pattern of fluorescence was consistent with the di stribution of rare outer membrane proteins visualized by freeze-fractu re electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of t he topographical relationships among T. pallidum cell envelope constit uents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surface s of virulent treponemes.