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Principal Investigator: JONATHON PETER AUDIA
Organization: UNIVERSITY OF SOUTH ALABAMA
Fiscal Year: 2024
Award: $231,000
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <65 and older><65 or older><65 years of age and older><65 years of age or more><65 years of age or older><65+ years><65+ years old><> 65 years><APF-1><ATP-Dependent Proteolysis Factor 1><Active Sites><Acute Lung Injury><Acute Pulmonary Injury><Aged 65 and Over><Airway failure><Antibiotic Resistance><Apoptosis-Related Cysteine Protease Caspase 1><Aspartate><Bacterial Toxins><Bypass><CASP-1><CASP1><CASP1 gene><COPD><Ca2+-Activated Protease><Calcium-Activated Neutral Protease><Calcium-Activated Neutral Proteinase><Calcium-Activated Protease><Calcium-Dependent Neutral Protease><Calcium-Dependent Neutral Proteinase><Calpain><Caspase><Caspase Gene><Caspase-1><Caspase-1 Gene><Causality><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Death><Cell Signaling><Cell model><Cell-Death Protease><Cells><Cellular Matrix><Cellular injury><Cellular model><Chronic Obstruction Pulmonary Disease><Chronic Obstructive Lung Disease><Chronic Obstructive Pulmonary Disease><Chronic lung disease><Combined Modality Therapy><Critical Illness><Critically Ill><Cysteine><Cysteine Endopeptidases><Cysteine Protease><Cysteine Proteinases><Cystic Fibrosis><Cytolysis><Cytoskeletal System><Cytoskeleton><Data><Desminase><Dysfunction><EC 3.1.1.4><ESKAPE><ESKAPE pathogens><Endothelial Cells><Endothelium><Epithelial Cells><Esteroproteases><Etiology><Functional disorder><Genes><HMG-20><Half-Cystine><Heart><High Mobility Protein 20><Human><ICE Protease><ICE-like protease><IL-1 beta Convertase><IL-1 beta-Converting Enzyme><IL-1BC><IL-1b Converting Enzyme><IL1B-Convertase><IL1BC><IL1BCE><Immune infiltrates><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><Infection><Inflammasome><Inflammation><Inflammatory><Intensive Care Units><Interleukin 1-B Converting Enzyme><Interleukin 1-Beta Convertase><Interleukin-1 Beta Converting Enzyme><Interleukin-1 Converting Enzyme><Intoxication><Intracellular Communication and Signaling><Knowledge><L-Aspartate><L-Cysteine><Lead><Lecithinase A2><Lipopolysaccharides><Lung><Lung Respiratory System><Lung damage><Lysis><Lytotoxicity><Macrophage><Mediating><Mediator><Membrane><Modeling><Modern Man><Morbidity><Morbidity - disease rate><Mucoviscidosis><Multimodal Therapy><Multimodal Treatment><Mφ><Nosocomial pneumonia><P aeruginosa><P aeruginosa associated pneumonia><P aeruginosa caused pneumonia><P aeruginosa induced pneumonia><P aeruginosa pneumonia><P aeruginosa-associated pneumonia><P aeruginosa-caused pneumonia><P aeruginosa-induced pneumonia><P. aeruginosa><P. aeruginosa associated pneumonia><P. aeruginosa caused pneumonia><P. aeruginosa induced pneumonia><P. aeruginosa infection><P. aeruginosa pneumonia><P. aeruginosa-associated pneumonia><P. aeruginosa-caused pneumonia><P. aeruginosa-induced pneumonia><PLA2><Papain-Like Cysteine Protease><Pathogenesis><Pathogenicity><Pathogenicity Factors><Pathway interactions><Patients><Pb element><Peptidases><Peptide Hydrolases><Permeability><Phospholipase A2><Phospholipases A><Physiology><Physiopathology><Pneumonia><Position><Positioning Attribute><Prevalence><Process><Protease Gene><Proteases><Proteinases><Proteolytic Enzymes><Pseudomonas aeruginosa><Pseudomonas aeruginosa associated pneumonia><Pseudomonas aeruginosa caused pneumonia><Pseudomonas aeruginosa induced pneumonia><Pseudomonas aeruginosa infection><Pseudomonas aeruginosa pneumonia><Pseudomonas pyocyanea><Publications><Publishing><Recovery><Resistance to antibiotics><Resistant to antibiotics><Respiratory Failure><Role><Scientific Publication><Sepsis><Severities><Signal Transduction><Signal Transduction Systems><Signaling><T3SS><Technology><Testing><Type III Secretion System><Type III Secretion System Pathway><Ubiquitin><Virulence><Virulence Factors><Virulent><Work><World Health Organization><above age 65><adverse consequence><adverse outcome><after age 65><age 65 and greater><age 65 and older><age 65 or older><age > 65><age of 65 years onward><aged 65 and greater><aged 65+><aged ≥65><antibiotic drug resistance><antibiotic resistant><biological signal transduction><blood infection><bloodstream infection><causation><cell culture><cell cultures><cell damage><cell injury><cell type><cellular damage><chronic obstructive pulmonary disorder><chronic pulmonary disease><combination therapy><combined modality treatment><combined treatment><cystein protease><cystein proteinase><cysteine endopeptidase><cytotoxicity><damage to cells><disease causation><exoenzyme><experiment><experimental research><experimental study><experiments><gene editing method><gene editing methodology><gene editing strategy><gene editing techniques><gene-editing approach><healthcare-associated pneumonia><heavy metal Pb><heavy metal lead><hospital acquired pneumonia><hospital associated pneumonia><human old age (65+)><immune cell infiltrate><immunosuppressed patient><inducible expression><inducible gene expression><infected with P. aeruginosa><infected with Pseudomonas aeruginosa><injury to cells><innovate><innovation><innovative><intracellular skeleton><lecithinase A><lung injury><membrane structure><mortality><multi-modal therapy><multi-modal treatment><necrocytosis><novel><old age><opportunistic pathogen><over 65 years><pathogen><pathophysiology><pathway><pharmacologic><phosphatidase><phosphatidolipase><phosphatidylcholine 2 acylhydrolase><pneumonia therapy><pneumonia treatment><pulmonary><pulmonary damage><pulmonary injury><pulmonary tissue damage><pulmonary tissue injury><response><social role><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><therapeutic agent development><therapeutic development><treat pneumonia><type 3 secretion system><≥65 years>