FIG. 3.

Binding of FH to YadA. (A) Binding of purified FH to YadA shown by affinity blotting. Triton X-114 membrane protein extracts from E. coli expressing YadA (Tx-YadA) or from E. coli carrying the empty vector (Tx-Ctrl) were subjected to 8% SDS-PAGE and transferred onto nitrocellulose membrane. The part of the membrane containing the proteins with molecular masses greater than 160 kDa was incubated with purified FH. Binding of FH was detected by immunoblotting with preadsorbed goat anti-human FH and HRP-conjugated rabbit anti-goat antibodies. Binding of FH to the YadA band is indicated by the arrow. (B) Divergent effects of gelatin and collagen on FH binding to YadA. Binding of purified FH to YadA-expressing wild-type Y. enterocolitica bacteria was tested in VBS (gelatin free), GVB (contains 0.1% gelatin), or VBS supplemented with type I collagen (0.01%). After extensive washings, bound FH was identified by immunoblotting with nonadsorbed polyclonal antiserum against human FH. The use of nonadsorbed serum allowed the simultaneous detection of the YadA band (indicated by the arrow), which served as a loading control.