FIGURE 5.

NTHi devoid of P5 binds significantly less human serum C4BP, resulting in more C3d deposition. NTHi strains 3655, KR271, and their corresponding P5 mutant strains NTHi 3655ΔompP5 and KR271ΔompP5 in addition to the laboratory strain E. coli BL21 were incubated with human serum (NHS: 3% and 5% for NTHi 3655 and KR271, respectively, and 0.2% for E.coli BL21) for 30 min at 37°C followed by flow cytometry analyses. (A and B) C4BP binding (A) and C3d complement deposition (B) on the bacterial surface was detected by specific Abs. Each bar represents the mean MFI ratio ± SEM of four and six independent experiments, respectively. MFI ratio was calculated as follows: MFI of cells incubated with primary and FITC-conjugated secondary Ab/MFI of cells incubated with secondary Ab only. Statistical significance was calculated using one-way ANOVA. (A) F(4, 15) = 6.54, p = 0.003; (B) F(4, 24) = 11.67, p < 0.001 with Tukey post hoc test. (C) Representative flow cytometry profiles from experiments included in (A) and (B). Background (light gray) represents bacteria incubated with FITC-conjugated secondary Abs only.