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The multifunctional role of the pallilysin-associated Treponema pallidum protein, Tp0750, in promoting fibrinolysis and extracellular matrix component degradation.

ABSTRACT: The mechanisms that facilitate dissemination of the highly invasive spirochaete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein-protein interaction domain commonly observed in extracellular matrix (ECM)-binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose-dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection-limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP-like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T.?pallidum dissemination.

SUBMITTER: Houston S

PROVIDER: S-EPMC3954913 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Project description:Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹⁹⁸A], HAXXH [E¹⁹⁹A], and HEXXA [H²⁰²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.

Project description:Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete Borrelia burgdorferi to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein's lipocalin fold.

Project description: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.

Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

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The multifunctional role of the pallilysin-associated Treponema pallidum protein, Tp0750, in promoting fibrinolysis and extracellular matrix component degradation.

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