Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice Océane Dufies 1, Anne Doye 1, Johan Courjon 1,2, Cédric Torre 1, Gregory Michel 1 ,Celine Loubatier 1, Arnaud Jacquel 1, Paul Chaintreuil 1, Alissa Majoor 1, Rodolphe R. Guinamard 1, Alexandre Gallerand 1, Pedro H. V. Saavedra 3, Els Verhoeyen 1,4, Amaury Rey 4, Sandrine Marchetti 1, Raymond Ruimy 1,2, Dorota Czerucka 5,6, Mohamed Lamkanfi 3, Bénédicte F. Py 4, Patrick Munro 1, Orane Visvikis 1 and Laurent Boyer 1,6,* 1 Université Côte d’Azur, Inserm, C3M, Nice, France. 2 Université Côte d’Azur, CHU Nice, Nice, France. 3 Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium. 4 CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS UMR5308, ENS de Lyon, Lyon, France. 5 Centre Scientifique de Monaco, Monaco, Monaco. 6 LIA ROPSE, Laboratoire International Associé Université Côte d’Azur, Centre Scientifique de Monaco, Nice, France. *Corresponding author It is crucial for the innate immune system to detect microbes and more precisely to sense pathogens. Many pathogens target the host Rho GTPases to invade the host and hijack the immune response. Among them, the CNF1 toxin form UPEC constitutively activates the host Rho GTPases. We have previously shown that the CNF1 activity is detected and triggers an immune response depending on Caspase-1 and IL-1ß, suggesting the involvement of an inflammasome (Boyer et al., Immunity, 2011; Diabate et al., PLoS Pathogens, 2015). In our study, we aimed to identify which inflammasome is responsible for the detection of CNF1 and to decipher the signaling pathway leading to inflammasome activation (Dufies et al., Nature Microbiology, 2021). We found that the NLRP3 inflammasome detects the Rac2 constitutive activation by CNF1. This signaling pathway involves the Rac2 effector Pak1, which phosphorylates NLRP3 on Thr659 allowing NLRP3-Nek7 interaction and inflammasome activation. Importantly, we found that CNF1-induced IL-1ß secretion occurs without cell death and is GSDMD-independent. Furthermore, inhibition of the Pak–NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Our results reveal the importance of Pak1 and NLRP3 in controlling the bacterial burden during bacteraemia in mice. References Boyer, L., Magoc, L., Dejardin, S., Cappillino, M., Paquette, N., Hinault, C., Charriere, G.M., Ip, W.K.E., Fracchia, S., Hennessy, E., et al. (2011). Pathogen-derived effectors trigger protective immunity via activation of the Rac2 enzyme and the IMD or Rip kinase signaling pathway. Immunity 35, 536–549. Diabate, M., Munro, P., Garcia, E., Jacquel, A., Michel, G., Obba, S., Goncalves, D., Luci, C., Marchetti, S., Demon, D., et al. (2015). Escherichia coli α-hemolysin counteracts the anti-virulence innate immune response triggered by the Rho GTPase activating toxin CNF1 during bacteremia. PLoS Pathog. 11, e1004732. Dufies, O., Doye, A., Courjon, J., Torre, C., Michel, G., Loubatier, C., Jacquel, A., Chaintreuil, P., Majoor, A., Guinamard, R.R., et al. (2021). Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice. Nat. Microbiol. 6, 401–412.