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

Abstract

Summary The mechanisms that facilitate dissemination of the highly invasive spirochete, 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.

Conservation of the Host-Interacting Proteins Tp0750 and Pallilysin among Treponemes and Restriction of Proteolytic Capacity to Treponema pallidum

“…DNA fragments encoding S 29 -E 491 of Tde0840 (Tp0751 ortholog) and G 23 -P 220 of Tde0841 (Tp0750 ortholog) were PCR amplified from T. denticola (strain ATCC 35405) genomic DNA using the primers listed in Table 1 (23)(24)(25). All constructs were confirmed as correct by DNA sequencing.…”

“…Recombinant Tp0750, wild-type pallilysin, inactive pallilysin (E 199 A), and the control protein Tp0327 were expressed and purified as previously described (24,25). Recombinant orthologs of Tp0750 (Tde0841 and DD750) and pallilysin (Tde0840 and DD751) were expressed and purified under identical conditions used for the corresponding T. pallidum proteins to ensure consistent levels of protein purity between each ortholog.…”

“…The highly invasive nature of T. pallidum is further highlighted by its ability to traverse the placental barrier to cause congenital syphilis and the blood-brain barrier to invade the central nervous system (9) and by the diverse and widespread clinical manifestations associated with secondary and tertiary syphilis (6). Numerous mechanisms have been suggested to contribute to the invasiveness and persistence of T. pallidum, including its corkscrew motility (4,11), chemotactic ability (6), antigenic variation capability (6,(12)(13)(14)(15)(16)(17), low outer membrane protein content (18,19), host component adhesion capabilities (20)(21)(22)(23)(24), and its ability to cause host damage by inducing host-mediated inflammatory and immune responses (6).…”

“…Pallilysin exists as a proprotease that, upon activation through cleavage of the prodomain, binds and degrades laminin (basement membrane lining blood vessels), fibrin, and fibrinogen (a component of the host's coagulation cascade that functions to entrap bacteria and prevent dissemination), while Tp0750 binds fibrinogen and is also capable of degrading fibrinogen and fibronectin, but not fibrin (21,(23)(24)(25). The genes encoding Tp0750 and pallilysin are cotranscribed and the two proteins coassociate to form a heterodimeric complex in vitro, suggesting shared function (24). The results from opsonophagocytosis assays are in support of pallilysin residing on the treponemal surface, suggesting pallilysin can directly mediate host-pathogen interactions (25).…”

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

“…This strain is pathogenic, and its pathogenicity can be tested in animal models (46)(47)(48). Most importantly, more than two-thirds of T. pallidum genes can be found in the genome of ATCC 35405 (4), including the majority of known T. pallidum virulence factors (10,16,44,(49)(50)(51)(52)(53). These advantages make this strain an ideal model a Parallel to the growth curve measurements shown in Fig.…”