PROJECT SUMMARY/ABSTRACT
The alveolar-capillary membrane facilitates efficient gas exchange while maintaining a restrictive permeability
barrier. Pseudomonas aeruginosa infection disrupts the alveolar-capillary barrier leading to exudative edema
and impaired oxygenation. P. aeruginosa utilizes a type III secretion system and its effectors to disrupt barrier
integrity. In particular, the exoenzyme Y is introduced into lung endothelium, where it acquires nucleotidylyl
cyclase activity and produces cGMP, cAMP, and cUMP. These cyclic nucleotide monophosphates activate
protein kinase A resulting in endothelial tau phosphorylation, tau dissociation from microtubules, and microtubule
breakdown, which collectively hinders repair following infection. Phosphorylated tau is released from
endothelium as cytotoxic variants that contribute to lung injury. The signaling mechanisms used by exoenzyme
Y to produce cytotoxic tau is incompletely understood, yet cUMP is produced at especially high concentrations
and the cUMP signal parallels the generation of cytotoxic tau. Elevations in cUMP are sufficient to promote the
production of cytotoxic tau variants. Our preliminary data demonstrate that the exoenzyme Y-induced cUMP
signal also decreases endothelial nicotinamide adenine dinucleotide (NAD+) and increases nicotinamide, the
product of NAD+ hydrolase activity, which may impair recovery following infection. Lung endothelium expresses
sterile alpha and TIR motif containing 1 (SARM1), the only TIR (Toll/Interleukin-1 Receptor) domain protein in
mammalian cells that possesses NAD+ hydrolase activity. Recent studies revealed a SARM1 bacterial
homologue is directly activated by cUMP as an essential innate immune mechanism. While our studies illustrate
an important role for cUMP in the endothelial cell response to infection, how exoenzyme Y generates the cUMP
that leads to tau phosphorylation and production of cytotoxic tau variants, and how cUMP lowers NAD+ while
hindering endothelial cell repair remains poorly understood. To address this knowledge gap in a rigorous way,
this project tests the hypothesis that the P. aeruginosa exoenzyme Y generates cUMP, which contributes to the
tau phosphorylation, microtubule breakdown, and SARM1-dependent NAD+ hydrolase activity that causes lung
injury and hinders repair.