Differential targeting of human pyroptotic caspase-5 and caspase-4 by <i>Shigella</i> 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, a <i>Shigella</i> effector OspC3, injected into cells by its type III secretion system, 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 disrupts 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.IMPORTANCE<i>Shigella</i> species are human-specific gram-negative pathogens that establish a replicative niche in intestinal epithelial cells by blocking pyroptosis, a key inflammatory cell death pathway. We reveal that two closely related <i>Shigella</i> type III secreted effectors, OspC2 and OspC3, specifically inactivate the human caspase paralogs CASP5 and CASP4, respectively. This specificity is determined by their newly identified pyroptotic caspase specificity (PCS) domains. In addition, we find that positively selected residues in CASP5 alter the OspC2/CASP5 interaction, underscoring the impacts of ongoing evolutionary arms races between bacterial effectors and host immune proteins. By elucidating the molecular basis of caspase targeting and adaptation, this work provides new insight into the diversification of host defense mechanisms and identifies potential therapeutic targets for enhancing epithelial resistance to bacterial infection.