Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin

Abstract

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 [H 198 A], HAXXH [E 199 A], and HEXXA [H 202 A]) 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 .

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

“…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). These findings implicate T. pallidum-mediated host component degradation as an additional pathogenic mechanism promoting treponemal invasion and dissemination during infection.…”

“…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.…”

“…Therefore, a fluorescence-based degradation assay was performed to compare the host protein-degrading capabilities of pallilysin, Tde0840, DD751, and inactive pallilysin (E 199 A) using our previously described methodology (23) with the following modifications. Recombinant proteins were diluted in protease reaction buffer (20 mM HEPES [pH 7.0], 25 mM CaCl 2 ) and preincubated at 37°C for 24 h. This preincubation step was performed since it has been demonstrated that attainment of full pallilysin proteolytic activity is dependent upon autocatalytic cleavage of an N-terminal prodomain, a process that can take up to 24 h in vitro (25). The physiological pH of the buffer used for these studies (pH 7.0) was selected to be representative of the pH of the local environment encountered by the treponemes within the host.…”

“…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).…”