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 [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.
Highlights
The expression of host-interacting proteases has been shown to contribute to the pathogenesis of bacteria of medical interest by promoting host colonization and immune evasion, acquisition of nutrients, tissue invasion and dissemination of infection
Student’s two-tailed t test indicated both forms of wild-type pallilysin exhibited statistically significant levels of fibrinolysis compared to the levels exhibited by water and HEXXA (* indicates p,0.0001). (C) Fibrin clots were incubated with TYGVS growth medium in the presence of thrombin (2 mg/ml), T. phagedenis transformed with the shuttle plasmid pKMR in the presence of thrombin (2 mg/ml), or T. phagedenis transformed with tp0751/pKMR in both the presence (2 mg/ml) and absence of thrombin
Student’s two-tailed t test indicated that T. phagedenis transformed with tp0751/pKMR exhibited statistically significant levels of fibrinolysis compared to the levels exhibited by T. phagedenis transformed with pKMR alone (* indicates p,0.005)
Summary
The expression of host-interacting proteases has been shown to contribute to the pathogenesis of bacteria of medical interest by promoting host colonization and immune evasion, acquisition of nutrients, tissue invasion and dissemination of infection. Several pathogenic bacteria, including Streptococcus pneumoniae [1], Yersinia pestis [2], Vibrio cholerae [3], and Clostridium perfringens [4], express bacterial proteases and toxins which play a central role in the infection process through facilitation of bacterial dissemination and tissue invasion by proteolytic degradation of host proteins. Host component degradation is common to many disseminating pathogens, such a mechanism has yet to be confirmed within the highly invasive causative agent of syphilis, Treponema pallidum subsp. The highly invasive nature of the pathogen is further emphasized by the diverse clinical manifestations that can occur in untreated syphilis infections, including skin rashes, meningitis, ocular disease, and cardiovascular and neurological complications, and by the fact that T. pallidum can cause bone destruction in congenital and tertiary stage syphilis [15]. T. pallidum is one of only a few pathogens that can traverse the placental and blood-brain barriers
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