Dual NLRC4 and non-canonical inflammasome signaling drives human GSDMD-mediated killing of Shigella flexneri independently of bacterial cardiolipin

ABSTRACT Macrophages internalize and kill bacteria and thus are crucial for clearing bacterial infections. Although macrophage killing of some intracellular bacteria requires inflammasomes, the specific mechanisms of inflammasome-dependent killing are incompletely understood. Here we show that, upon infection with an intracellular pathogen Shigella flexneri , human macrophages activate a robust NLRC4-caspase-1 inflammasome response that restricts intracellular bacterial replication independently of pyroptosis. Gasdermin D (GSDMD) cleavage is required for bactericidal activity, revealing a GSDMD-dependent mechanism of bacterial killing independent of host cell death. We find that GSDMD-mediated killing of S. flexneri does not require bacterial cardiolipin, identifying a cardiolipin-independent mode of bacterial targeting. Priming macrophages with interferon (IFN)-γ enhances killing of intracellular S. flexneri by promoting involvement of caspase-4, which cooperates with caspase-1 to potentiate GSDMD function. These results identify a dual engagement of canonical and non-canonical inflammasomes that leads to macrophage killing S. flexneri while preserving host cell integrity. Furthermore, these findings uncover a previously unrecognized cardiolipin-independent mechanism of GSDMD-mediated bacterial killing with broad implications for immune cell control of cytosolic bacterial pathogens.