Illuminating the structural mechanism of non-canonical inflammasomes

forthcoming

Inflammasomes are essential components of the innate immune system that form cytosolic multi-protein complexes restricting pathogen growth and coordinating inflammatory signalling. Among these, non-canonical inflammasomes are unique lipid-protein assemblies where inflammatory caspases directly sense and oligomerise on bacterial lipopolysaccharide (LPS), triggering a response that ultimately induces lytic cell death. Despite the critical role of non-canonical inflammasomes, the transient and heterogeneous nature of their assembly on the complex Gram-negative bacterial membrane has left the structural mechanisms underlying their specificity, assembly, and activation largely undefined. In this project, I will leverage technical advancements uniquely tailored to study dynamic membrane-associated complexes to zoom into the structural mechanisms governing non-canonical inflammasome assembly and activation across increasing levels of biological complexity. By innovating tools and workflows for time-resolved and correlative cryoEM imaging I will capture sequential snapshots of non-canonical inflammasomes assembly on bacterial membranes, both in vitro and in infected cells. I will elucidate how the inflammasome initiation complex assembles on the bacterial membrane, how it cooperatively employs GTP hydrolysis to deform and expose bacterial LPS and how this platform directs caspases to exposed membrane sites where they form the non-canonical inflammasome. Understanding the fundamental principles that fuel the non-canonical inflammasome engine has broad implications for innate immune signalling, as excessive inflammasome activity provokes severe conditions such as sepsis and blood-brain barrier breakdown, highlighting the relevance of this research for guiding inflammation-modulating strategies. The approach and methodology developed will also broadly benefit the study of structure and dynamics of other supramolecular assemblies on complex membrane environments.