title: Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.
abstract: Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe 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 were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations 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.
authors.affiliation: Department of Internal Medicine, U.T. Southwestern Medical Center, Dallas 75235, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
authors.forename: E J
authors.initials: EJ
authors.lastname: Robinson
authors.forename: K W
authors.initials: KW
authors.lastname: Bourell
authors.forename: D R
authors.initials: DR
authors.lastname: Akins
authors.forename: S F
authors.initials: SF
authors.lastname: Porcella
authors.forename: L M
authors.initials: LM
authors.lastname: Weigel
authors.forename: J D
authors.initials: JD
authors.lastname: Jones
authors.forename: M V
authors.initials: MV
authors.lastname: Norgard
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D015748:Borrelia burgdorferi Group / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D005614:Freeze Fracturing; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008854:Microscopy, Electron; D008970:Molecular Weight; D013587:Syphilis / Q000382:microbiology; D014210:Treponema pallidum / Q000737:chemistry*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins