Activation and proteolytic activity of the Treponema pallidum metalloprotease, pallilysin.
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.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
Simon
S
Houston
Rebecca
R
Hof
Lisa
L
Honeyman
Julia
J
Hassler
Caroline E
CE
Cameron
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D020134:Catalytic Domain; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism*; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010587:Phagocytosis; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism; D014210:Treponema pallidum / Q000201:enzymology* / Q000472:pathogenicity; D015032:Zinc / Q000378:metabolism
D018829:Adhesins, Bacterial; D007797:Laminin; C049192:adhesin, Treponema pallidum; D005337:Fibrin; D005340:Fibrinogen; D045726:Metalloproteases; D013917:Thrombin; D015032:Zinc