CHARACTERIZATION OF OUTER MEMBRANES ISOLATED FROM TREPONEMA-PALLIDUM,THE SYPHILIS SPIROCHETE

Previous freeze-fracture electron microscopy (EM) studies have shown t hat the outer membrane (OM) of Treponema pallidum contains sparse tran smembrane proteins. One strategy for molecular characterization of the se rare OM proteins involves isolation of T. pallidum OMs. Here we des cribe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs we re isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical m arkers for periplasmic, cytoplasmic membrane, and cytosolic compartmen ts, and (iii) freeze-fracture EM demonstrating that the putative OMs c ontained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylam ide gel electrophoresis analysis revealed that the OMs contained a rel atively small number of treponemal proteins, including several which d id not appear to correspond to previously characterized T. pallidum an tigens. interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblottin g: and enhanced chemiluminescence. Compared with whole cells, T. palli dum OMs were deficient in cardiolipin, the major lipoidal antigen reac tive with antibodies in syphilitic sera, Also noteworthy was that othe r lipoidal constituents of OMs, including the recently discovered glyc olipids, did not react with human syphilitic sera. These latter observ ations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.