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

Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by Pseudomonas 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 promote 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 ZAKα- 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 ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2.Published versionThis project was supported by the ATIP-Avenir program (to E. Meunier), Fondation pour la Recherche Médicale “Amorçage Jeunes Equipes” (AJE20151034460 to E. Meunier), the Agence Nationale de la Recherche (ANR Psicopak to E. Meunier), the Agence nationale de recherche sur le sida et les hépatites-Maladies infectieuses émergentes (to E. Meunier), the European Research Council (StG INFLAME 804249 to E. Meunier), the European Society of Clinical Microbiology and Infectious Diseases (to R. Planès), Invivogen-Conventions industrielles de formation par la recherche PhD grant (to M. Pinilla), Vaincre La Mucoviscidose, and Region Occitanie (Groupement de Recherche pour des Applications INnovantes avec les Entreprises) grants to C. Cougoule

Druggable redox pathways against M. abscessus in cystic fibrosis patient-derived airway organoids

Abstract Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose antioxidants as a potential host-directed strategy to improve Mabs infection control