Figure 2. P. pneumotropica binds to Factor H and regulates the alternative complement pathway.

(A) P. pneumotropica suspensions (10⁸ bacteria/tube) were incubated with 1 µg of human Factor H (FH) for 2 h or normal human serum (NHS) for 30 min. After washing, bound FH was detected by incubation with goat polyclonal antibody anti-FH (primary antibody) and then with anti-goat IgG-FITC conjugated (secondary antibody) (line histogram). As a negative control (left peak), untreated P. pneumotropica was incubated only with primary and secondary antibodies (dashed histogram). (B) Serum FH binding to P. pneumotropica was quantified by ELISA (*p<0.01). (C) Suspensions containing 2×10⁸ bacteria/tube were incubated for 1 h at 37°C with FH (1 µg), or 10% NHS and then washed three times with PBS. C3b and FI (0.5 µg of each) were added to the bacterial suspensions pre-treated with FH, 10% NHS or PBS. These suspensions were then incubated at 37°C for 1, 2 or 4 h. The functional cofactor activity was evaluated by Western blot, and the cleavage fragments of C3b were detected using goat polyclonal primary anti-human C3. FH bound to P. pneumotropica acts as a co-factor of Factor I (FI) for the C3b cleavage. C3b cleavage fragments are indicated by arrows. Negative controls were included to observe spontaneous C3b cleavage in the absence of FH and/or FI.