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.

Syphilis is a prominent disease in low- and middle-income countries, and a re-emerging public health threat in high-income countries. Syphilis elimination will require development of an effective vaccine that has thus far remained elusive. Here we assess the vaccine potential of Tp0751, a vascular adhesin from the causative agent of syphilis, Treponema pallidum subsp. pallidum. Tp0751-immunized animals exhibit a significantly reduced bacterial organ burden upon T. pallidum challenge compared with unimmunized animals. Introduction of lymph nodes from Tp0751-immunized, T. pallidum-challenged animals to naive animals fails to induce infection, confirming sterile protection. These findings provide evidence that Tp0751 is a promising syphilis vaccine candidate.

Syphilis is a sexually transmitted disease caused by Treponema pallidum subsp. pallidum; it can be effectively treated with penicillin yet remains prevalent worldwide, due in part to the shortcomings of current diagnostic tests. Here we report the production of soluble recombinant versions of three novel diagnostic candidate proteins, Tp0326, Tp0453, and a Tp0453-Tp0326 chimera. The sensitivities of these recombinant proteins were assessed by screening characterized serum samples from primary, secondary, and latent stages of infection (n ‫؍‬ 169). The specificities were assessed by screening false positives identified with the standard diagnostic testing algorithm (n ‫؍‬ 21), samples from patients with potentially cross-reactive infections (Leptospira spp., Borrelia burgdorferi, Helicobacter pylori, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, or cytomegalovirus) (n ‫؍‬ 38), and samples from uninfected individuals (n ‫؍‬ 11). The sensitivities of Tp0326, Tp0453, and the Tp0453-Tp0326 chimera were found to be 86%, 98%, and 98%, respectively, and the specificities were 99%, 100%, and 99%. In a direct comparison, the Captia syphilis (T. pallidum)-G enzyme immunoassay (Trinity Biotech) was used to screen the same serum samples and was found to have a sensitivity of 98% and a specificity of 90%. In particular, Tp0453 and the chimera exhibited superior accuracy in classifying analytical false-positive samples (100%, compared to 43% for the Captia assay). These findings identify Tp0453 and the Tp0453-Tp0326 chimera as novel syphilis-specific diagnostic candidates that surpass the performance of a currently available diagnostic enzyme immunoassay test for syphilis and that allow accurate detection of all stages of infection.

Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope.

Treponema pallidumis a highly invasive spirochete that disseminates to organ sites distal to the site of primary infection and is able to cross both the blood-brain and placental barriers during the course of infection. The corkscrew motility used byT. pallidumundoubtedly contributes to its invasive nature. However, this signature motility is shared with other spirochetes and thus the factors responsible for the widespread dissemination capability that is unique toT. pallidumremain unknown. We have identified the treponemal-specific, surface-localised protein pallilysin as a dual functioning adhesin/metalloprotease that exhibits specific attachment to, and degradation of, multiple extracellular matrix components. Pallilysin is produced as an inactive proprotease that can be activated via either autocatalytic cleavage or host-originating thrombin cleavage. Purified recombinant pallilysin, as well as a non-invasive model treponeme heterologously expressing pallilysin on its surface, exhibit specific degradation of fibrin clots. Pallilysin immunisation alters the course ofT. pallidumdissemination following challenge within the rabbit model of syphilis infection, with immunised rabbits exhibiting a reduced bacterial burden within organs distal to the site of challenge compared to unimmunized control rabbits. Further, rabbit infectivity tests (RIT) showed that rabbits receiving lymph nodes from challenged, unimmunized rabbits seroconverted and developed orchitis by 30 days post-transfer, while 66% of RIT rabbits receiving lymph nodes from challenged, pallilysin-immunised rabbits remained seronegative and had no signs of orchitis at the termination of the experiment (day 185 post-transfer). Collectively these studies identify pallilysin as aT. pallidum-specific metalloprotease that (1) exploits the host coagulation cascade to facilitate protease activation, (2) plays a central role in treponemal dissemination and (3) shows promise as a novel syphilis vaccine candidate.