FIGURE 7.

C4BP fusion proteins bind to NTHi but do not promote serum killing. (A–C) NTHi strains 3655 and KR271 and corresponding P5 mutant strains NTHi 3655ΔompP5 and KR271ΔompP5 were incubated with 10 μg/ml of fluorescently labeled C4BP (A), C4BP-IgM (B), or C4BP–IgG (C) for 30 min at 37°C, and binding was evaluated by flow cytometry. N. gonorrhoeae FA1090 strain was used as positive control for binding to C4BP. (D and E) Wild-type NTHi 3655 and KR271 were preincubated with or without C4BP–IgM (50 μg/ml) (D) or C4BP–IgG (20 or 50 μg/ml) (E) for 30 min at 37°C prior to addition of 3% and 5% NHS, respectively. Bacteria were counted at t = 0 and t = 30 min postaddition of serum, and survival percentage was calculated. Each bar represents the mean ± SEM of at least three independent experiments with technical duplicates. Statistical significance was calculated using one-way ANOVA. (A) F(3,14) = 8.955, p = 0.001; (B) F(3, 8) = 8.959, p = 0.006; (C) F(3,8) = 8.416, p = 0.007 with Tukey post hoc test.