Figure 1.

The FhaB prodomain affects the conformation of the MCD. (A) At top is an illustration of mature FHA with an N-teriminal hexahistadine tag and a TEV protease cleavage site upstream of the MCD. Below are schematics of the B. pertussis FhaB proteins as initially synthesized. From left, the domains illustrated are the signal peptide (purple), the TPS domain (orange), the β-helical shaft (light blue), the MCD (green), the PNT (dark red), and the remainder of the prodomain (red). The hexahistadine tag is located between the signal peptide and the TPS domain. The TEV protease cleavage site is located between the β-helix and the MCD. The approximate site of SphB1-dependent cleavage is noted. The proline-rich region (PRR) of the prodomain is also noted. Residue numbering is based on the B. pertussis FhaB protein. (B) The strains were tested to determine if the hexahistadine tag and TEV protease site altered FHA mediated adherence to A549 human lung epithelial cells. BPSMAQ adhered in a manner indistinguishable from wildtype B. pertussis, while BPSMAQT-N adhered as inefficiently as an fhaB deletion strain. (C) Anti-MCD immunoblot of purified MCD polypeptides. Semi-native gel analysis indicated altered mobility of the MCD derived from the prodomain-truncated (T-N) FhaB compared to the MCD derived from full-length (AQ) FhaB. When denatured by either heat or chemical denaturation, the MCDs of both proteins migrate identically. (D) Western blot of MCD polypeptides after proteinase-K treatment. Purified MCDs were tested for sensitivity to proteinase-K. Samples were incubated with increasing concentrations of proteinase-K, separated by SDS-PAGE, and detected with anti-MCD antibody. Additionally, samples were denatured/renatured prior to analysis of proteinase- K sensitivity. Densitometery values proportionate to the undigested sample of each Western blot are listed below the blots.