Molecular characterization of receptor binding proteins and immunogens of virulent Treponema pallidum.

Baseman, Joel B.; Hayes, Edward C.

doi:10.1084/jem.151.3.573

Cited by 123 publications

(87 citation statements)

References 29 publications

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“…Several cytadhesins have been identified by mild extraction of pathogenic bacteria with nonionic detergents such as OGP, by using formaldehyde-or glutaraldehyde-fixed host cell cultures (7)(8)(9). Formaldehyde-fixed mouse L cells have been used to study the adherence of Chlamydia psittaci (11).…”

Section: Discussionmentioning

confidence: 99%

A heat-labile protein of Chlamydia trachomatis binds to HeLa cells and inhibits the adherence of chlamydiae.

Joseph

Bose

1991

Proc. Natl. Acad. Sci. U.S.A.

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From highly purified elementary bodies (EBs) was found to exhibit cytadherence activity. Extracts containing this protein, the chlamydial cytadhesin (CCA), were also used to study inhibition of adherence of viable EBs to HeLa cells. The effects of exposure to heat and to trypsin on the cytadherence and protection activity of this protein were examined. MATERIALS AND METHODS Organisms. C. trachomatis serovars B, E, and LGV440(Li) were grown in mycoplasma-free HeLa 229 or McCoy cells; the EBs were purified as described (1), and samples were stored at -800C. Mixing experiments using mockinfected HeLa cells labeled with a mixture of [3H]amino acids and unlabeled chlamydia-infected cells had shown that less than 0.02% of the host-derived protein copurified in the final EB preparation (1).Radioactive Labeling of EUs. Metabolic labeling of the chlamydial proteins was effected with Tran35S-label (ICN) in the presence of emetine, an irreversible inhibitor of eukaryotic protein synthesis, as described in detail (1, 5). Extrinsic labeling of the purified EBs with 1251 was performed using N-chloro-bezenesulfonamide-derivatized beads (IodoBeads, Pierce). Briefly, to 100 ul of the purified EB suspension (protein content, 100-500 AMg) in phosphate-buffered saline (PBS), two Iodo-Beads, and 250-350 p.Ci of Na125I (1 Cj = 37 GBq) were added and incubated for 10 min on ice. Unreacted Na125I was removed by centrifuging the reaction mixture through a desalting gel (Bio-Gel P-6DG, Bio-Rad) equilibrated with 2% (wt/vol) bovine serum albumin in PBS (6). The iodinated EBs were recovered by sedimentation. Adherence of these EBs to unfixed as well as to glutaraldehyde-fixed HeLa cells at 40C was a saturable process (1).Extraction of Chlamydial Proteins. The pelleted EBs were resuspended in 100 1.l of OGP buffer [2% (wt/vol) OGP in PBS, pH 7.5/10 mM DL-dithiothreitol/1 mM phenylmethylsulfonyl fluoride/aprotinin (10 pAg/ml)/leupeptin (10 ,ug/ml)/ N-a-tosyl-L-lysine chloromethyl ketone (TLCK; 10jug/ml)].The suspension was kept on ice for 1 hr and then sedimented in a microcentrifuge for 15 min at 40C. The pellet was reextracted and the pooled supernatants (100 A.l each) were dialyzed at 40C for 2-3 hr against PBS. Protein concentrations in the extracts were determined with the Bio-Rad reagent, using bovine serum albumin as the standard. Samples of the dialyzed OPG extract (OGPE) were stored at -800C.Glutaraldehyde Fixation of HeLa Monolayers. HeLa cells were plated in 96-well cell culture dishes at 6 x 104 cells in 200 ,ul of growth medium and incubated overnight at 370C to achieve confluence. The monolayers were fixed with glutaAbbreviations: EB, elementary body; OGP, n-octyl f-D-glucopyranoside; OGPE, OGP buffer extract of the elementary bodies; CCA, chlamydial cytadhesin; MOMP, major outer membrane protein.*To whom reprint requests should be addressed. 4054The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ...

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Section: Discussionmentioning

confidence: 99%

A heat-labile protein of Chlamydia trachomatis binds to HeLa cells and inhibits the adherence of chlamydiae.

Joseph

Bose

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Three treponemal proteins with fibronectin-binding properties were discovered and partially characterized over 20 years ago (18,310), but until recently the identity of potential T. pallidum adhesins was unknown. Cameron et al (41) used computer analysis to search the T. pallidum genome for adhesin candidates.…”

Section: Attachmentmentioning

confidence: 99%

Biological Basis for Syphilis

2006

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SUMMARY Syphilis is a chronic sexually transmitted disease caused by Treponema pallidum subsp. pallidum. Clinical manifestations separate the disease into stages; late stages of disease are now uncommon compared to the preantibiotic era. T. pallidum has an unusually small genome and lacks genes that encode many metabolic functions and classical virulence factors. The organism is extremely sensitive to environmental conditions and has not been continuously cultivated in vitro. Nonetheless, T. pallidum is highly infectious and survives for decades in the untreated host. Early syphilis lesions result from the host's immune response to the treponemes. Bacterial clearance and resolution of early lesions results from a delayed hypersensitivity response, although some organisms escape to cause persistent infection. One factor contributing to T. pallidum's chronicity is the paucity of integral outer membrane proteins, rendering intact organisms virtually invisible to the immune system. Antigenic variation of TprK, a putative surface-exposed protein, is likely to contribute to immune evasion. T. pallidum remains exquisitely sensitive to penicillin, but macrolide resistance has recently been identified in a number of geographic regions. The development of a syphilis vaccine, thus far elusive, would have a significant positive impact on global health.

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“…Although it is now 13 years since the publication of the Nichols strain complete genome sequence (24) and over 30 years since initial binding studies demonstrated that T. pallidum is capable of adhering to mammalian cells (3,21,30) and components of the extracellular matrix (ECM) (23,70), data regarding the molecular basis of these important pathogenic interactions have only recently begun to emerge. Previously, our laboratory identified the T. pallidum adhesin Tp0751 and demonstrated that this adhesin binds specifically to a variety of laminin isoforms, which are abundant glycoprotein components of the basement membrane that underlie endothelial cell layers, in a dose-dependent manner (8,9).…”

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Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

et al. 2011

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Treponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidum adhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidum with host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitro degradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coli expression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidum protease capable of degrading host components and thus provides novel insight into the mechanism of T. pallidum dissemination.

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Molecular characterization of receptor binding proteins and immunogens of virulent Treponema pallidum.

Cited by 123 publications

References 29 publications

A heat-labile protein of Chlamydia trachomatis binds to HeLa cells and inhibits the adherence of chlamydiae.

A heat-labile protein of Chlamydia trachomatis binds to HeLa cells and inhibits the adherence of chlamydiae.

Biological Basis for Syphilis

Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

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