EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption upon Pseudomonas infection

The intracellular inflammasome complex have been implicated in the maladaptive tissue damage and inflammation observed in chronic Pseudomonas aeruginosa infection. Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by P. aeruginosa, specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects Exotoxin A (EXOA), a ribotoxin released by P. aeruginosa Type 2 Secretion System (T2SS) during chronic infection. Mechanistically, EXOA-driven Eukaryotic Elongation Factor 2 (EEF2) ribosylation and covalent inactivation promotes ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, Diphtheria Toxin and Cholix Toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAK- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, Cystic Fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAK inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAK as a critical sensor of virulence-inactivated EEF2. KEY POINTSO_LIP. aeruginosa induces NLRP1-dependent pyroptosis in human corneal and nasal epithelial cells C_LIO_LIP. aeruginosa Exotoxin A (EXOA) and other EEF2-inactivating bacterial exotoxins activate the human NLRP1 inflammasome C_LIO_LIEEF2 inactivation promotes ribotoxic stress response and ZAK kinase-dependent NLRP1 inflammasome activation. C_LIO_LIBronchial epithelial cells from Cystic Fibrosis patients show extreme sensitivity to ribotoxic stress-dependent NLRP1 inflammasome activation in response to Exotoxin A C_LIO_LIP38 and ZAK inhibition protects Cystic Fibrosis epithelial cell from EXOA-induced pyroptosis C_LI