Corynebacterium diphtheriaecauses keratinocyte-intrinsic ribotoxic stress and NLRP1 inflammasome activation in a model of cutaneous diphtheria

ABSTRACT NLRP1 is an innate immune sensor protein that activates inflammasome-driven pyroptotic cell death. Recent work demonstrates that human NLRP1 has evolved to sense viral infections. Whether and how human NLRP1 responds to other infectious agents is unclear. Here, and in a companion manuscript, we report that human NLRP1, as an integral component of the ribotoxic stress response (RSR), is activated by bacterial exotoxins that target human ribosome elongation factors EEF1 and EEF2, including Diphtheria Toxin (DT) from Corynebacterium diphtheria e, exotoxin A from Pseudomonas aeruginosa and sidI from Legionella pneumophila . In human keratinocytes, DT activates RSR kinases ZAKα, p38 and JNKs, upregulates a set of signature RSR transcripts and triggers rapid NLRP1-dependent pyroptosis. Mechanistically, these processes require 1) DtxR-mediated de-repression of DT production in the bacteria, as well as 2) diphthamide synthesis and 3) ZAKα/p38-driven NLRP1 phosphorylation in the host. In 3D human skin cultures, Corynebacterium diphtheria e infection disrupts barrier function and induces IL-1 driven inflammation. Pharmacologic inhibition of p38 and ZAKα suppresses keratinocyte pyroptosis and rescues barrier integrity of Corynebacterium diphtheria e-treated organotypic skin. In summary, these findings implicate RSR and the NLRP1 inflammasome in antibacterial innate immunity and might explain certain aspects of diphtheria pathogenesis. KEY POINTS EEF1/EEF2-targeting bacterial exotoxins activate the human NLRP1 inflammasome. DT+ve toxigenic Corynebacterium diphtheriae induces ZAKα-driven RSR and NLRP1-driven pyroptosis in human keratinocytes. Identification of transcripts that are induced by multiple RSR agents across multiple cell types. p38 and ZAKα inhibition rescues epidermal integrity by limiting pyroptosis in 3D skin mode of cutaneous diphtheria.