Identity of <i>Treponema pallidum</i> subsp. <i>pallidum</i> polypeptides: Correlation of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results from different laboratories

Norris, Steven J.; Alderete, John F.; Axelsen, N. H.; Bailey, Mark; Baker‐Zander, Sharon A.; Baseman, Joel B.; Bassford, P J; Baughn, Robert E.; Hanff, Philip A.; Hindersson, Peter; Larsen, Sandra A.; Lovett, Michael; Lukehart, Sheila A.; Miller, James N.; Moskophidis, Matthäus; Müller, F.; Norgard, Michael V.; Penn, Charles W.; Stamm, Lola V.; Embden, J D van; Wicher, Konrad

doi:10.1002/elps.1150080202

Cited by 77 publications

(53 citation statements)

References 48 publications

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“…Based upon its molecular mass and the fact that it can be visualized only by two-dimensional NEPHGE, we believe that this is indeed a novel T. pallidum membrane protein (50,52). In two prior freeze-fracture analyses (11,59), we noted the size homogeneity of outer membrane particles and proposed that there may be a rather limited number of proteins within the T. pallidum outer membrane, perhaps only a single protein species.…”

Section: Discussionmentioning

confidence: 97%

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane

et al. 1997

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Venereal syphilis is a chronic, multisystem infectious disorder caused by the spirochetal bacterium Treponema pallidum subsp. pallidum (T. pallidum). Like all spirochetes, T. pallidum is an elongated, highly motile organism that consists of a fragile outer membrane surrounding a periplasmic space, a peptidoglycan-cytoplasmic membrane (PG-CM) complex, and a protoplasmic cylinder (36). There is now a substantial body of evidence that the outer membranes of T. pallidum and enteric gram-negative bacteria differ considerably with respect to composition and molecular architecture (52, 57). One of the key differences concerns the relative abundance of proteins with membrane-spanning domains. Whereas outer membranes of gram-negative bacteria contain high densities of such polypeptides (48), freeze-fracture electron microscopy and cell fractionation studies have shown that they are sparse in T. pallidum, hence the designation rare outer membrane proteins (52,57,59,77).Based on the assumption that T. pallidum rare outer membrane proteins are important in disease pathogenesis, molecular characterization of these polypeptides has become a major objective of contemporary syphilis research. One strategy recently developed to accomplish this goal is to isolate T. pallidum outer membranes for partial amino acid sequencing of candidate rare outer membrane proteins (9, 60). Using this approach, Blanco et al. (6) identified a 31-kDa protein (Tromp1) in isolated outer membranes which formed ion-conducting channels in planar lipid bilayers. More recently, the same investigators reported that recombinant Tromp1 expressed in Escherichia coli is surface exposed and that the recombinant protein possesses porin-like properties (7). However, to date, no evidence for either outer membrane location or surface exposure of native Tromp1 within motile or intact treponemes has been presented (6, 7). Moreover, Tromp1 has extensive sequence homology with the periplasmic substratebinding proteins of known ATP-binding cassette (ABC) transporters and the tromp1 gene is transcriptionally linked to open reading frames which encode homologs for ABC transporter components which, in gram-negative bacteria, are cytoplasmic membrane associated (30,33). The apparent discrepancy between the studies of Blanco and coworkers (6, 7) and these newer genetic data prompted efforts to clarify the physicochemical properties and cellular location of Tromp1 in T. pallidum. Here we present evidence that Tromp1, rather than being an outer membrane-spanning protein, actually is anchored by an uncleaved signal sequence to the T. pallidum cytoplasmic membrane.

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Section: Discussionmentioning

confidence: 97%

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane

et al. 1997

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“…Unlike gram-negative bacteria such as Escherichia coli, the outer membrane of T. pallidum is very fragile (12) and is easily disrupted by physical manipulation. This characteristic has resulted in misidentification of highly immunogenic periplasmic lipoproteins as surface exposed antigens (24), although these molecules are now thought to be anchored to the periplasmic leaflet of the cytoplasmic membrane (11,(25)(26)(27). Freeze fracture electron microscopy studies reveal a very limited number of surface-exposed transmembrane proteins localized in the outer membrane (28,32).…”

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ThetprKGene Is Heterogeneous amongTreponema pallidumStrains and Has Multiple Alleles

et al. 2000

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We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-defined tprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.

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“…In the case of syphilis, efforts to elucidate this phenomenon revealed that the outer membrane of T. pallidum is a fragile phospholipid bilayer with a paucity of integral membrane proteins (23, 25,35). The major membrane immunogens of T. pallidum, molecules formerly thought to be surface exposed (1,17,21), are now believed to be lipoproteins anchored by fatty acids to the periplasmic leaflet of the cytoplasmic membrane (8,22,23,28,33). The recognition that the outer membrane of B. burgdorferi is similarly labile (2) and therefore easily disrupted during experimental manipulation led us to consider the possibility that the major lipoprotein immunogens of B. burgdorferi, outer surface proteins (Osps) A and B (7), also have been incorrectly characterized as exclusively surface-exposed molecules (3,4,18).…”

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Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi

et al. 1991

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Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracelhularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.Lyme disease and syphilis are infectious disorders caused by the spirochetal pathogens Borrelia burgdorferi and Treponema pallidum, respectively. These diseases share many clinical and microbiological features, including the abilities of their respective pathogens to persist for prolonged periods in individuals with high titers of specific antibodies (19,31). In the case of syphilis, efforts to elucidate this phenomenon revealed that the outer membrane of T. pallidum is a fragile phospholipid bilayer with a paucity of integral membrane proteins (23,25,35). The major membrane immunogens of T. pallidum, molecules formerly thought to be surface exposed (1,17,21), are now believed to be lipoproteins anchored by fatty acids to the periplasmic leaflet of the cytoplasmic membrane (8,22,23,28,33). The recognition that the outer membrane of B. burgdorferi is similarly labile (2) and therefore easily disrupted during experimental manipulation led us to consider the possibility that the major lipoprotein immunogens of B. burgdorferi, outer surface proteins (Osps) A and B (7), also have been incorrectly characterized as exclusively surface-exposed molecules (3,4,18). Interestingly, a reevaluation of published data provides some support for this contention. Periplasmic constitutents, namely, endoflagella (15) washing to remove this material could disrupt or even remove the outer membranes. We therefore focused our analyses on intact cells and protoplasmic cylinders, reasoning that obvious reductions of putative outer membrane proteins should be detectable in the cylinder fractions following selective removal of the outer membranes.Three preparations of B. burgdorferi B31 (high passage) were investigated. One consisted of viable organisms pelleted directly from BSK II medium at 13,700 x g for 10 min. In the other two, organisms were incubated on ice for 30 min with or without 0.1% Triton X-114; this incubation was followed by three successive centrifugations (each at 13,700 x g for 10 min at 4°C) and resuspensions (washes) in phosphate-buffered saline (PBS), pH 7.4. Portions of each preparation were removed for SDS-polyacrylamide gel electrophoresis (PAGE) or prepared for whole-mount and ultrathin-section electron microscopy.Nearly all of the organisms taken directly from BSK II medium were intact ( Fig. la and b). Repeated centrifugation and resuspension, manipulations comparable to those used in surface localization studies (3,4,18), disrupted many of the organisms (Fig. lc and ld); scattered membrane vesicles also were seen (data not shown). Outer membranes were uniformly absent from the detergent-incubated organisms, and membrane vesicles were not seen despite extensive ...

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Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results from different laboratories

Cited by 77 publications

References 48 publications

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane

ThetprKGene Is Heterogeneous amongTreponema pallidumStrains and Has Multiple Alleles

Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi

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