Fig. 5. BfCDCL proteolytic activation and protection by an immunity protein.

a The proteolytic cleavage sites identified by the in vitro assays were tested for activity against the sensitive P. dorei 9_1_42FAA strain. The growth of P. dorei 9_1_42FAA in liquid cultures is shown when treated with unactivated wildtype BfCDCLs (as in Fig. 4) and the BfCDCL^L and BfCDCL^S proteolytic cleavage site mutants, R70A and R62A, respectively when paired with its wildtype cognate CDCL^L or CDCL^S. Note that the HBS control is the same in a, as they were performed simultaneously. b P. dorei CL02 and its DpnA and DpnB gene knockouts¹⁷ were treated with the unactivated wildtype BfCDCLs. c Mixtures were prepared of unactivated BfCDCLs, CF liposomes and BcdI, which was added at 1:10, 3:10, 5:10, and 10:10 molar ratios to the BfCDCL^S. Fragipain (FP) (3.1 μg) was injected at 120 seconds to activate the BfCDCLs and pore formation was followed by the release of CF. In the last panel of (c) the BcdI was injected at a 3:10 molar ratio to BfCDCL^S at 540 sec. The HBS control is the same for all these experiments, as they were performed simultaneously. d The BcdI gene was deleted from in B. fragilis YCH46 and added in trans to the sensitive P. dorei strains CL09T03C04 and 9_1_42FAA. Their sensitivity to unactivated BfCDCL pair was tested in the liquid culture assay (as in Fig. 4b). The B. fragilis YCH46 was also treated with fragipain-activated BfCDCLs. The growth curves in a, b represent the standard error of the mean of the OD₆₀₀ for each time point derived from three individual growth assays. The growth assays in a, b were performed on a Stratus Kinetic plate reader (Cerillo), whereas those in (d) were performed on a Biotek Epoch 2 plate reader. The CF release assays in c are representative of 2 assays.