Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

Abstract

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.

Syphilis: using modern approaches to understand an old disease

“…TP0136, a protein identified by reactivity with primary human syphilitic sera (9), also binds to human fibronectin (10). TP0751 can bind to laminin, which has the highest concentration in the basement membrane (11)(12)(13), and to fibrinogen, a blood-clotting protein that functions to contain bacteria (13). TP0751 can also degrade laminin and fibrinogen using its zinc-dependent protease domain, which may be a means by which T. pallidum disseminates to surrounding tissues and the bloodstream (13).…”

Rho GTPases as pathogen targets: Focus on curable sexually transmitted infections

“…Tp0136 is another bacterial adhesin that binds to fibronectin and laminin, 2 host extracellular matrix glycoproteins. 109 In addition, Tp0750 has a dual function, as it binds specifically to a variety of laminin isoforms, 110 and it is able to degrade host molecules, suggesting that it plays a role in the proteolytic mechanisms involved in tissue damage, destruction and dissemination of T. pallidum during infection 111 ( Fig. 3).…”

New Proteins for a New Perspective on Syphilis Diagnosis

“…Bacteria were harvested through centrifugation, resuspended in 20 mM Tris (pH 7.5), 500 mM NaCl, 20 mM imidazole (Tp0326 and the Tp0453-Tp0326 chimera) or 25 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole (Tp0453), and lysed either through sonication (Tp0326 and the Tp0453-Tp0326 chimera) or with a French press (Tp0453). The resulting lysates were centrifuged for 45 min at 20,000 ϫ g at 4°C, and the supernatants containing the soluble recombinant proteins were removed and subjected to immobilized metal ion affinity chromatography and size exclusion chromatography as described previously (23).…”