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.