Thursday, April 3, 2025
4/3/2025
The Pseudomonas aeruginosa virulence factor ExoU activates pyroptosis - PROJECT SUMMARY/ABSTRACT Our recent publications described pathogenic effects of the Pseudomonas aeruginosa type III secretion system effector ExoU on caspase-1 regulated inflammation. Our new preliminary data indicate that ExoU bypasses the inflammasome to induce a non-canonical form of caspase-1 activation in lung endothelial cells. However, the mechanisms underlying ExoU-directed caspase-1 activation are unknown. The consequences of ExoU-directed caspase-1 activation on a form of cell death known as pyroptosis are also undefined. ExoU is a phospholipase A2 (PLA2) that directly interacts with the host cell plasmalemmal membrane to induce lysis. We have discovered that ExoU also activates an indirect lysis pathway involving the gasdermin D (GSDMD) executioner of pyroptosis. Together, the data raise the intriguing prospect that ExoU-induced cell damage involves a combination of direct (ExoU PLA2 activity) and indirect (GSDMD-mediated) lysis pathways. The ExoU-induced indirect lysis pathway represents a novel virulence mechanism that contributes to P. aeruginosa pathogenesis. Based on our published and preliminary data, two complementary Specific Aims will test the Hypothesis that ExoU elicits non-canonical caspase-1 activation and processing of GSDMD to incite lung endothelial cell pyroptosis during P. aeruginosa infection. Aim 1 will elucidate mechanisms underlying ExoU-induced caspase-1 activation. Proposed experiments will determine whether: 1) ExoU PLA2 increases cytosolic Ca++ to stimulate calpain protease activation and 2) ExoU- mediated calpain activation liberates caspase-1 from the cytoskeleton to induce auto-activation. Aim 2 will examine the role of ExoU in GSDMD activation by: 1) rigorously validating the role of GSDMD in the ExoU indirect lysis pathway and 2) determining the role of ExoU PLA2 activity in GSDMD activation. The studies proposed herein are highly significant. P. aeruginosa is the most frequent Gram-negative, opportunistic pathogen causing pneumonia in patients with chronic lung disease (e.g., chronic obstructive pulmonary disease and cystic fibrosis), older age, and/or immunocompromised status. P. aeruginosa is also prevalent in critically ill patients with respiratory failure in the intensive care unit. Importantly, ExoU-expressing strains associate with the highest levels of patient morbidity and mortality. Thus, combined therapies targeting both ExoU and caspase-1 represent a pharmacological strategy to treat the most severe cases of P. aeruginosa induced pneumonia, acute lung injury, and sepsis. Moreover, the discovery that ExoU induces non-canonical caspase-1 and GSDMD activation, which drives lung cell death and dysfunction during P. aeruginosa infection is conceptually innovative. Our proposed studies will use technically innovative gene editing and inducible expression technologies to demonstrate cause-and-effect relationships between ExoU PLA2 activity, caspase- 1, and GSDMD towards testing our hypothesis.
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