Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum

Weigel, Linda M.; Brandt, Mary E.; Norgard, Michael V.

doi:10.1128/iai.60.4.1568-1576.1992

Cited by 61 publications

(30 citation statements)

References 38 publications

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“…A principal objective of the present study was to resolve conflicting data concerning the cellular location{s) of the T. patlidum lipoprotein immunogens. In contrast to our previous investigations, which mainly employed syphilitic sera {Radoif et aL, 1988; Cox ef a!., 1992), here we have exploited newly available, high-titre monospeeific antisera directed against four different recotiibinant lipoproteins, including the highly abundant 47 kDa antigen (Akins et a!., 1993;Weigel et aL, 1992), The use of polyclonal antisera eliminated the possibility that a negative result inereiy r'efiected the inaccessibility of a single epitope on the protein. Antibody binding to the surfaces of intact treponemes was not detected with any of the four antisera, while aritibodies bound readily under conditions that selectively exposed periplasmic constituents.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of non-lipidated forms of the 15,17. and 47 kDa T. pallidum lipoproteins (Tppi 5, Tppi 7. and Tpp47) and OspA and OspB of S. burgdorferi as fusions with GST was described previously (Weigel et aL.. 1992: Akins et a/., 1993: Radolf etaL, 1994. A lusion of GST with the 34kDa T. patlidum lipoprotein (Tpp34) was produced by cloning a DNA fragmeni encoding the mature (i.e., processed) portion of the poiypeptide (Swancutt et aL, 1989) into the SamHI and EcoRI sites of pGEX-2T (Pharmacia) (Smith and Johnson, 1988).…”

Section: Expression Of T Paiiidum and B Bur'gdorferi Proteins As Fumentioning

confidence: 99%

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Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Cox¹,

Akins²,

Porcella³

et al. 1995

Molecular Microbiology

106

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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 immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. 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 candidate T. pallidum outer membrane protein (TpN50) with C-terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co-incubated with detergent. In contrast, antibodies generated against isolated T. pallidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze-fracture 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 the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.

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Section: Discussionmentioning

confidence: 99%

Section: Expression Of T Paiiidum and B Bur'gdorferi Proteins As Fumentioning

confidence: 99%

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Cox¹,

Akins²,

Porcella³

et al. 1995

Molecular Microbiology

106

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“…The resulting GST fusions were purified by affinity chromatography on an agarose-glutathione matrix according to the manufacturer's instructions (Pharmacia LKB Biotechnology). Alternatively, the BbK2.10, BbK2.11, and OspF portions of the GST fusion proteins were cleaved from the GST moiety while still bound to the agarose-glutathione matrix as previously described (Weigel et aL, 1992).…”

Section: Production Of Glutathione S-transferase (Gst) Fusion Proteinsmentioning

confidence: 99%

Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue

Akins¹,

Porcella²,

Popova

et al. 1995

Molecular Microbiology

148

220

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Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi-infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.10-reactive antibodies produced by the B. burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera. Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.

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“…We constructed Tp genes expressing three forms of recombinant proteins: mature antigens, antigens containing signal sequences, and antigens fused with GST at the NH 2 terminal of the mature antigen. The amino acid sequences of the antigens containing signal sequences were as follows (12)(13)(14)(15)(16)(17):…”

Section: Preparation Of R-tp Antigensmentioning

confidence: 99%

“…The major immunoreactive antigens of these membrane proteins have been reported to have a MW of 47, 42, 17, and 15KDa based on WB analy-sis (8)(9)(10). The putative amino acid sequences of 47, 42, 17, and 15KDa have been demonstrated (11)(12)(13)(14)(15)(16)(17). Here, we describe several forms of expressed r-Tp antigens related to the membrane proteins mentioned above and the detection of anti-Tp antibodies by means of indirect ELISA, and compare the ELISA results with those of the conventional TPHA method.…”

Section: Introduction: Forms Of Expressed R-tp Antigensmentioning

confidence: 99%

Reactivity of recombinant treponema pallidum (r‐Tp) antigens with anti‐Tp antibodies in human syphilitic sera evaluated by ELISA

Fujimura

Ise

Ueno

et al. 1997

J. Clin. Lab. Anal.

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We evaluated the immunoreactivity of recombinant Treponema pallidum (r-Tp) antigens with human sera by indirect enzyme-linked immunoabsorbant assay (ELISA). We expressed antigens with a molecular weight (MW) of 17KDa, 15KDa, 47KDa, and 42KDa, which are believed to be major immunoreactive membrane proteins of Tp cells. The expressed proteins were described by adding the prefix M, S, and G to the corresponding Tp antigens, namely, mature antigens, signal sequence containing antigens, and glutathione s-transferase (GST)-fused antigen in this report. A rather high expression occurred for M47 and S42 proteins in the Escherichia coli system, whereas for M15 and M17 proteins, a poor expression was observed. However, a fairly high expression occurred for G15 and G17. Thus expressed proteins were purified by means of chromatographies to a level of > 95%, and the purified proteins were found to be reactive with TPHA positive serum by Western blotting (WB). An ELISA performed with a serum of 1/1000 dilution using these purified antigens for coating on the solid phase showed that G17 antigen was more effective in detecting syphilis antibodies in human serum than M47, S42, and G15. There was a good consistency between ELISA and TPHA, whereby the cutoff indexes (CI) on ELISA showed a correlation coefficient of 0.7276 in logarithmic TPHA titers.

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Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum

Cited by 61 publications

References 38 publications

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue

Reactivity of recombinant treponema pallidum (r‐Tp) antigens with anti‐Tp antibodies in human syphilitic sera evaluated by ELISA

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