Differential targeting of human pyroptotic Caspase-5 and Caspase-4 by Shigella OspC2 and OspC3

Pyroptosis is an inflammatory cell death pathway that is a key defense mechanism of intestinal epithelial cells. To successfully establish an infection, intracytosolic Gram-negative pathogens must block this host response. Indeed, Shigella OspC3 suppresses epithelial pyroptosis by targeting and inactivating Caspase-4 (CASP4). Here, we demonstrate that OspC2, which shares 96% identity with OspC3, targets Caspase-5 (CASP5), a close paralog of CASP4. Through a combination of yeast two-hybrid, transfection, and bacterial infection assays, we show that the distinct pyroptotic caspase specificities of OspC2 and OspC3 are determined by a short α-helical region, designated the Pyroptotic Caspase Specificity (PCS) domain. This domain is located upstream from the Ankyrin Rich Repeat (ARR) region previously established to promote OspC3 binding to CASP4. Swapping PCS domains between OspC2 and OspC3 is sufficient to redirect their caspase targeting. Evidence for CASP5-driven pyroptosis in response to infection has not yet been established. However, CASP5 displays signatures of positive selection at residues predicted to interact with the PCS domain of OspC2. Notably, the introduction of orangutan-specific residues into human CASP5 disrupt its interaction with and modification by OspC2, demonstrating that CASP5 natural variation can cause key functional differences in this host-microbe molecular interaction. These findings highlight the evolutionary interplay between bacterial effectors and host proteins, support a likely role for CASP5 in responding to Gram-negative bacteria, and identify promising therapeutic targets for enhancing epithelial defense against bacterial pathogens.