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.
Table 1.
Prevalence of Prevotella intermedia and the presence of interpain A in subgingival plaque samples.
Figure 2.
Interpain A destroys hemolytic activity of human serum.
Sheep erythrocytes sensitized with antibodies (classical pathway, (A)) or rabbit erythrocytes (alternative pathway, (B)) were incubated with 1.25% or 10% NHS, respectively. Serum was supplemented with various concentrations of InpA and InpAC154A. After 1 h incubation, the degree of lysis was estimated by measurement of released hemoglobin (absorbance at 405 nm). Lysis obtained in the absence of interpain was set as 1. An average of three independent experiments is presented with bars indicating SD.
Figure 3.
Interpain A inhibits the classical pathway.
InpA and InpAC154A were incubated with 2% NHS (for C1q, C4b, C3b assays) and 10% NHS (for C9 assay) for 15 min and inactivated with an excess of E64 (20 µM) to prevent degradation of IgGs. Human IgGs were immobilized on microtiter plates and allowed to activate NHS pre-incubated with various concentrations of InpA and InpAC154A. After 20 min (C3b, C4b) and 45 min (C1q, C9) of incubation, the plates were washed, and deposited C1q (A), C4b (B), C3b (C), and C9 (D) were detected with specific polyclonal antibodies. Absorbance obtained in the absence of InpA was set as 1.0 unit. An average of three independent experiments is presented with bars indicating SD. Data points without error bars have minimal SD, which are not displayed by the graphing software (GraphPad Prism 4).
Figure 4.
Interpain A inhibits the lectin pathway.
Mannan was immobilized on microtiter plates and allowed to activate 4% NHS (for MBL, C4b, C3b assays) and 10% NHS (for C9 assay) that was pre-incubated for 15 min with various concentrations of InpA and InpAC154A. After 20 min (C3b, C4b) and 45 min (C9, MBL) of incubation, the plates were washed, and deposited MBL (A), C4b (B), C3b (C), and C9 (D) were detected with specific polyclonal antibodies. Absorbance obtained in the absence of interpain was set as 1. An average of three independent experiments is presented with bars indicating SD.
Figure 5.
Interpain A inhibits the alternative pathway.
Zymosan was immobilized on microtiter plates and allowed to activate 6% NHS (for C3b assay) and 10% NHS (for C9 assay) that was pre-incubated for 15 min with various concentrations of InpA and InpAC154A. After 20 min (C3b) and 45 min (C9) of incubation, the plates were washed, and deposited C3b (A) and C9 (B) were detected with specific polyclonal antibodies. Absorbance obtained in the absence of interpain was set as 1. (C) InpA was incubated with increasing concentrations of NHS, and the activity of InpA was determined using a synthetic substrate. The cysteine protease inhibitor E64 was used as a control. An average of three independent experiments is presented with bars indicating SD.
Figure 6.
Interpain A degrades preferentially α-chains of C3 and C4.
Purified C3 (A,C) and C4 (B,D) were incubated for 30 min with increasing concentrations of InpA or 1250 nM of InpAC154A and separated by SDS-PAGE. The gels were stained with silver (A,B), and protein band intensities corresponding to the α-chains and β-chains of C3 and C4 were analyzed by densitometry (C,D). The graphs show the % of native α- and β-chains remaining after incubation with InpA. An average of three independent experiments is presented with bars indicating SD. (E) In order to determine cleavage sites in C3 and C4, these proteins were digested and subjected to N-terminal sequencing. The N-terminal sequences of selected bands are listed on the right. (F) A schematic representation of C3 and C4 α-chains with indicated sites of cleavage by InpA.
Figure 7.
Kinetic parameters of InpA-mediated degradation of C3 and C4.
(A) Degradation of C3 and C4 incubated with one concentration of InpA for different time points. (B) Kinetic measurement of degradation of C3b by InpA. Various concentrations of InpA were injected over immobilized C3b, and the reduction in RU values indicates the extent of proteolytic cleavage of C3b. The inset shows a sensorgram obtained for a 3 µM InpA sample. The initial rates of proteolysis were determined for each InpA concentration from the slope in the sensorgram and plotted as a function of InpA concentration. (C) Michaelis-Menten plot of degradation of C3 by InpA. InpA was incubated with increasing concentrations of C3, and the amount of remaining α-chains (substrate) was determined using densitometry after separation on SDS-PAGE. (D) A similar analysis as in (C) but performed for C4.
Figure 8.
Deposition of C1q on plates and bacteria.
(A) Microtiter plates were blocked with BSA and incubated with 4% NHS containing various concentrations of InpA and InpAC154A for 45 min. Deposited C1q was detected with a specific antibody, and the absorbance obtained in the absence of InpA was set as 1. (B) P. nigrescens ATCC 25261 was incubated with 5% NHS and different concentrations of InpA and InpAC154A. Deposition of C1q was quantified using flow cytometry with specific FITC-labeled antibodies, and the absorbance obtained in the absence of InpA was set as 1. An average of three independent experiments is presented with bars indicating SD.
Figure 9.
Interpain A and gingipains act synergistically.
Mannan was immobilized on microtiter plates and allowed to activate 4% NHS containing 350 nM InpA and three gingipains, Kgp (44 nM), RgpB (55 nM), HRgpA (33 nM), alone or mixed together. After 20 min of incubation, the plates were washed, and deposited C3b was detected with specific antibodies. An average of three independent experiments is presented with bars indicating SD. Statistical significance of observed differences was estimated using Student's t-test; *** p<0.001.
Table 2.
Description of bacterial strains used in this study.