Listeriolysin O recruits UBA1 to ubiquitinate NLRP3 and suppress inflammasome defence

Abstract

The NLRP3 inflammasome is a central cytosolic defence pathway against infection, yet intracellular pathogens must restrain this response to preserve their replicative niche. Here we identify a ubiquitin-dependent immune-evasion mechanism in which L. monocytogenes uses listeriolysin O (LLO) to destabilise NLRP3. The N-terminal PEST-like sequence of LLO acted as an adaptor that recruited the host ubiquitin-activating enzyme UBA1 and promoted K48-linked polyubiquitination of NLRP3. This modification drove proteasomal NLRP3 degradation and limited caspase-1 activation, gasdermin D cleavage, IL-1{beta}/IL-18 release, and macrophage pyroptosis. Deleting the PEST region preserved basal haemolytic activity but impaired NLRP3 ubiquitination, thereby enhancing inflammasome activation during infection in macrophages and in vivo. Conversely, UBA1 overexpression suppressed NLRP3-mediated inflammatory responses and supported intracellular bacterial survival. UBA1 depletion restored NLRP3 protein levels, reduced bacterial burden and delayed lethal infection, albeit with increased inflammatory tissue injury. These findings define a ubiquitin-hijacking strategy that operates at the E1 step, whereby a bacterial toxin redirects host ubiquitin activation towards an innate immune sensor.