Filamentous-actin as a determinant of ExoY-induced purine and pyrimidine cyclic nucleotide monophosphate signaling in pulmonary microvascular endothelium

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa type III secretion system effector that possesses promiscuous nucleotidyl cyclase activity. Host cell filamentous-actin (F-actin) is the ExoY cofactor. F-actin binding to ExoY promotes enzymatic activity, leading to time-dependent production of both purine (cAMP and cGMP) and pyrimidine (cUMP and cCMP) cyclic nucleotide monophosphates that are responsible for microtubule reorganization, endothelial cell retraction, and increased alveolar-capillary permeability. However, mechanisms underlying ExoY substrate selection are poorly understood. Here, we tested the hypothesis that F-actin structure is a determinant of which cyclic nucleotide monophosphates are produced and accumulate within endothelium. Infection with P. aeruginosa (PA103 exoUexoT::Tc pUCP exoY; ExoY + ) resulted in ExoY intoxication of pulmonary microvascular endothelial cells. ExoY predominantly generated cGMP in association with modest VASP 157 and VASP 239 phosphorylation and microtubule reorganization. Cytochalasin D dose-dependently reduced the cortical F-actin rim and stress fibers without impacting microtubule organization. However, cytochalasin D in combination with ExoY + infection revealed cUMP, and to a lesser extent cCMP, accumulation that corresponded with prominent VASP 239 phosphorylation and the presence of an extensive microtubule architecture. These data therefore reveal an essential role for F-actin configuration on the generation and accumulation of pyrimidine cyclic nucleotide monophosphates. Whereas ExoY is a guanylyl cyclase in pulmonary microvascular endothelial cells upon the initial infection, F-actin remodeling reveals ExoY pyrimidine cyclase activity. These studies illustrate a functional coupling between F-actin and microtubules that allows for ExoY to breakdown microtubules during infection. This research was funded by HL66299 (T.S.), HL140182 (M.T.L), HL148069 (T.S.), and HL167997 (T.S.). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.