FIG 7.

CspA orthologs simultaneously bound to CFH and plasminogen. Hexahistidine-tagged recombinant proteins or BSA (5 μg/ml each) was immobilized onto microtiter plates and incubated with 10 μg/ml purified plasminogen (PLG) (A), 10 μg/ml purified CFH (B), or 100 μl 3% NHS (C). Simultaneous binding of plasminogen and CFH to CspA LW2 (D), CspA PKo (E), or CspA A14S (F) was assayed by ELISA. CFH and increasing amounts of plasminogen were added to the immobilized CspA orthologs (molar ratios are indicated). Protein-protein complexes formed in the assays were detected using either a polyclonal goat anti-plasminogen antibody or a polyclonal goat anti-CFH antibody. Absorbance of each test was measured at 490 nm. Experiments were performed in triplicate. Data represent means and SEM from three separate experiments, each performed at least in triplicate. Raw data were analyzed by one-way ANOVA with a post hoc Bonferroni correction. ***, P < 0.001; **, P < 0.01; *, P < 0.05.