Bifunctional role of the Treponema pallidum extracellular matrix binding adhesin Tp0751.

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.

Heterologous expression of the Treponema pallidum laminin-binding adhesin Tp0751 in the culturable spirochete Treponema phagedenis.

Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.