Interpain A, a Cysteine Proteinase from Prevotella intermedia, Inhibits Complement by Degrading Complement Factor C3
Figure 1
Interpain A destroys bactericidal activity of NHS.
(A) Western blotting analysis of InpA expression. Five-day old broth cultures of nine P. intermedia strains were adjusted to OD600 of 2, and the culture supernatants were separated by SDS-PAGE under reducing conditions and proteins transferred onto PVDF membranes. InpA was visualized with polyclonal antibodies. One lane shows purified InpA. (B) Western blotting analysis of gingival crevicular fluid (20 µL/lane) from patients with chronic periodontitis. Load of P. intermedia was determined with qPCR (<100 bacteria/sample “−”, 100–1,000 bacteria/sample “+”, 10,000–50 000 bacteria/sample “++”, >50,000 bacteria/sample “+++”). The leftmost lane contains 1 µg of a purified inactive recombinant mutant, InpAC154A. (C) E. coli DH5α were incubated with 2% NHS pretreated with increasing concentrations of InpA and InpAC154A, and the surviving bacteria were enumerated after overnight culture on LB agar plates. As a control, heat-inactivated NHS (ΔNHS) was used, and the survival of bacteria in this condition was set to 100%. (D) P. intermedia and E. coli were incubated with 20% NHS and 40% NHS for 1.5 h in anaerobic conditions, respectively, with and without supplementation with 1 mM E64 protease inhibitor, and the surviving bacteria were enumerated after culture onto TSB and LB plates, respectively. In (C) and (D) an average of three independent experiments is presented with bars indicating standard deviation (SD). Statistical significance of observed differences was estimated using Student's t-test; n.s. not significant, *p<0.05.