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.

Membrane Topology and Cellular Location of theTreponema pallidumGlycerophosphodiester Phosphodiesterase (GlpQ) Ortholog

“…Final topological assignment and conclusion. The many difficulties inherent in the molecular characterization of T. pallidum OM proteins (50) have spawned a variety of experimental approaches for accomplishing this important but elusive objective (10,11,31,54,60). In this regard, it is striking that two dissimilar strategies, OM isolation (54) and differential genomic library screening to identify potential T. pallidum opsonic targets (60), yielded the same candidate OM protein, nGlpQ.…”

“…Radiolabeling of isolated T. pallidum outer membranes with ([ 125 I]TID). Outer membranes isolated from 10 10 T. pallidum organisms as previously described (54)…”

Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

“…Like LPS, LipL36 was completely solubilized by 0.1% Triton X-100, implying that LipL36 is found exclusively in the outer membrane. In contrast, LipL41 appears to be located in both leptospiral membranes, a pattern of distribution found for other spirochetal lipoproteins (5,33,39). Previous surface immunoprecipitation studies indicate that LipL41 is surface exposed, while LipL36 is not (38).…”

TheMycobacterium tuberculosisrecombinant LprN protein ofmce4operon induces Th-1 type response deleterious to protection in mice

“…Lipoproteins are efficiently immunogenic and affect both innate and adaptive immune responses (Infante-Duarte & Kamradt, 1997;Oftung et al, 1997). Lipoproteins have been identified as major antigens in M. tuberculosis, Treponema pallidum, Mycoplasma hyorhinis, Leptospira interrogans, and Borrelia burgdorferi (Bricker et al, 1988;Radolf et al, 1995;Simon et al, 1995;Haake et al, 1999). Several mycobacterial lipoproteins have been shown to be an important trigger to the activation of humoral and cellular immune responses to mycobacteria (Young & Garbe, 1991).…”