FIG 1.
Expression and proteolytic activities of C. rodentium CroP and CroPMut. (A) Presence of CroP and CroPMut at the OM of C. rodentium. The OM fractions of C. rodentium wild-type, ΔcroP, ΔcroP(pYCcroP), and ΔcroP(pYCcroPMut) strains were resolved on an SDS–11% PAGE gel. Western blotting was done using a polyclonal rabbit antibody raised against purified CroP. (B) Proteolytic cleavage of the FRET substrate by CroP and CroPMut. The various C. rodentium strains were incubated with the FRET substrate for 60 min. The results are expressed as means ± the standard errors of the mean (SEM) of triplicate samples and are expressed relative to the activity of the C. rodentium ΔcroP strain. Statistical significance was assessed using a one-way analysis of variance, followed by Dunnett's post hoc comparison test (***, P < 0.001). (C) Proteolytic cleavage of CRAMP by CroP and CroPMut. CRAMP (0.1 μM) was incubated with the various C. rodentium strains for 15 min. After incubation, bacteria were pelleted by centrifugation, and supernatant aliquots were resolved by Tris-Tricine SDS-PAGE on 10 to 20% polyacrylamide gels and visualized by Coomassie blue staining. The data shown are representative of three independent experiments. Ctl., control; WT, wild type.