FIG 2.

OprF promotes binding to human C3b. (A, B) Mid-log-phase bacteria were opsonized with 20% human serum for 5 or 20 min, and the reactions were stopped. Bacteria were fixed and stained with anti-human C3 antibody (A) or anti-human C5 antibody (B) as outlined in Materials and Methods. (C) OprF-expressing E. coli and vector control strains were incubated with C5-depleted human serum, washed, fixed, and stained with anti-C3 antibody. (D) To evaluate the specificity of the interaction of OprF and C3b, other porin mutants, ΔoprD, ΔoprQ, ΔoprI, and ΔoprJ mutants, along with wild-type P. aeruginosa, were opsonized with human serum and stained with anti-C3 antibody. C3b binding efficiency was analyzed by flow cytometry using Flow Jo software. Means ± standard errors of the means (SEM) are given (n = 3). Nonopsonized bacteria were used in each experiment to set the gate for analysis. Data from 30,000 gated events were collected, and mean fluorescence intensity (MFI) was calculated for each sample. **, P ≤ 0.005; ***, P ≤ 0.0005.