Figure 1.
P. intermedia strains bind purified 125I-FI and FI from HI-NHS.
A) P. intermedia ATCC 25611, OMZ 248, OMZ 324, MH6, S. aureus strains ATCC 25923 and Newman (FI binding positive control) as well as E. coli DH5α (FI binding negative control) were incubated with 125I-FI (250 kcpm). Bound and free FI were separated by centrifugation through sucrose, the radioactivity associated with pellets and supernatants was measured in a gamma counter and the percentage of radioactivity bound to the pellet was calculated. Samples containing 125I-labeled FI that was incubated with buffer alone served as a negative control. Averages of three independent experiments performed in duplicates are shown; error bars show SD. One-way ANOVA followed by Tukey's post-hoc test was used for statistical evaluation of the data as compared to the negative control (*** p<0.001; ** p<0.01, n.s., not significant). B) HI-NHS (1%) was mixed with P. intermedia ATCC 25611, OMZ 248, OMZ 324, MH6 or S. aureus strains ATCC 25923 and Newman, as well as E. coli DH5α and samples were incubated for 1 h at RT followed by centrifugation. Aliquots of the supernatants were added to the wells of a microtiter plate coated with the polyclonal anti-FI antibody PK9205. Captured FI was detected with a polyclonal goat anti-FI antibody. Binding of FI to bacteria resulted in a decreased FI concentration in the serum. This value was then used to calculate bound FI. The graph shows averages of three independent experiments performed in duplicates and error bars indicate SD. One-way ANOVA followed by Tukey's post-hoc test was applied for statistical evaluation of the data as compared to values calculated for E. coli DH5α (** p<0.01; * p<0.05, n.s., not significant).
Figure 2.
Competition of 125I-FI binding by P. intermedia with unlabelled FI and sensitivity of FI binding to ionic strength.
A) Bacteria were mixed with unlabelled FI (final concentrations 0–5000 nM/0–0.44 µg/µl) and incubated with 250 kcpm 125I-FI for 1 h at RT and bound protein was measured as described in Fig. 1. B) Increasing concentrations of NaCl (150–950 mM) were added together with 250 kcpm 125I-FI to bacteria. Averages of three independent experiments performed in duplicates are shown with error bars indicating SD and FI acquisition expressed as protein bound relative to protein bound without addition of competitor. One-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis (*** p<0.001).
Figure 3.
Degradation of 125I-C4b by FI bound to P. intermedia.
A) P. intermedia ATCC 25611 was incubated at 56°C for 30 min and subsequently 5 µg, 10 µg, and 15 µg FI (final concentration 125 ng/µl (1.42 µM), 250 ng/µl (2.84 µM) and 375 ng/µl (4.26 µM)) were added to the bacterial suspension and samples incubated for 1 h at RT. Thereafter, bacteria were washed and mixed with E-64, C4met, C4BP and 125I-C4b. Samples were incubated for 4 h at 37°C, centrifuged to remove bacteria followed by separation of proteins on a 10–15% SDS-PAGE gradient gel under reducing conditions. Gels were dried and 125I-C4b was detected using a phosphorimager. Controls were samples only containing purified proteins without bacteria; either FI and C4BP (positive control) or only FI without cofactor (negative control) were added to degradation reactions. B) Data were quantified using the Image Gauge Software. Averages of four independent experiments are shown, error bars denoting the SD. The ratio of C4d/α-chain was calculated and data from samples containing FI bound to bacteria compared to the control containing bacteria without FI using one-way ANOVA followed by Tukey's post-hoc test applied to samples containing bacteria (*** p<0.001; ** p<0.01; n.s., not significant). C) Effect of elevated temperature on FI binding by P. intermedia. P. intermedia ATCC 25611 was incubated at 56°C or RT for 30 min and a binding assay was performed with 250 kcpm 125I-labeled FI to exclude false positive binding by bacteria treated at elevated temperature. Data were compared to values measured for samples incubated at RT using one-way ANOVA followed by Tukey's post-hoc test (** p<0.01).
Figure 4.
P. intermedia isolates bind purified FI cofactors C4BP and FH.
The indicated P. intermedia strains, as well as A) M. catarrhalis RH 4, P. gingivalis W50 and W83 (C4BP binding positive controls), M. catarrhalis ΔuspA1/2, and E. coli DH5α (C4BP binding negative controls) or B) S. pyogenes CCUG 25571 (FH binding positive control), S. aureus ATCC 25923, and E. coli DH5α (FH binding negative controls) were mixed with (A) 500 kcpm 125I-C4BP or (B) 125I-FH and incubated for 1 h at RT. Proteins bound to bacteria were detected as described in Fig. 1. Samples containing 125I-labeled proteins that were incubated with buffer alone served as negative controls. Bars represent averages of three independent experiments performed in duplicates and SD are indicated as error bars. One-way ANOVA and Tukey's post-hoc test was used for statistical analysis as compared to negative controls (*** p<0.001; * p<0.05; n.s., not significant).
Figure 5.
Specificity and sensitivity of C4BP and FH binding by P. intermedia ATCC 25611.
A) Binding of 250 kcpm 125I-C4BP was competed by using 30 µg (final concentration 1.32 µM) unlabeled C4BP, C) 125I-FH binding by P. intermedia ATCC 25611 was competed by 7.5 µg (final concentration 1.25 µM) unlabeled FH. The decrease of 125I-C4BP and 125I-FH binding was evaluated comparing the results of samples with and without radiolabeled protein and bacteria. Samples without bacteria served as negative controls. The sensitivity of the interaction to ionic strength was tested adding increasing concentrations of NaCl to binding reactions with B) 125I-C4BP and D) 125I-FH. Three independent experiments were performed in duplicates; averages and SD were calculated and binding expressed as protein bound relative to protein bound without addition of competitor. One-way ANOVA followed by Tukey's post-hoc test was applied for statistical evaluation of the data (** p<0.01; *** p<0.001).
Figure 6.
C4BP and FH bound to P. intermedia ATCC 25611 retain their cofactor activity.
P. intermedia ATCC 25611 was incubated for 30 min at 56°C and subsequently incubated with C4BP (5–15 µg) (final concentration 125 ng/µl (0.22 µM), 250 ng/µl (0.44 µM) and 375 ng/µl (0.66 µM)) (A) and B)) or FH (1–5 µg) (final concentration 0.025 µg/µl (0.16 µM), 0.05 µg/µl (0.32 µM), 0.125 µg/µl (0.8 µM)) (D) and E)) for 1 h at RT. Samples without C4BP and FH were used as a reference. Bacteria were washed and transferred to fresh reaction tubes. A) and B) Bacteria were mixed with E-64, C4met, FI and trace amounts of 125I-C4b. Samples were incubated for 1.5 h at 37°C, centrifuged and proteins separated on a 10–15% SDS-PAGE gradient gel under reducing conditions. Samples without bacteria, which contained C4BP but no FI served as negative controls. Samples supplemented with C4BP and FI were positive controls in this experimental setup. B) and E) Protein bands were quantified using the Image Gauge Software. C) and F) P. intermedia ATCC 25611 was incubated at 56°C for 30 min and binding assays were performed as described earlier with 125I-labeled protein. D) and E) Bacterial suspensions were mixed with E-64, C3met, FI and trace amounts of 125I-C3b. Samples were incubated for 1.5 h at 37°C and separated as described above. A negative control without bacteria was included, in which FI was omitted. A positive control without bacteria supplemented with FH and FI was prepared. All experiments were performed in triplicates, the mean values and SD were calculated. One-way ANOVA and Tukey's post-hoc test were used for statistical analysis on bacteria containing samples. Degradation mediated by cofactors bound to P. intermedia was evaluated by comparing the ratios of C4d or α′43 degradation products to α-chains of samples where protein was omitted during preincubation (*** p≤0.001; ** p<0.01; * p<0.05; n.s., not significant).