P. aeruginosa ExoY Disrupts Microtubules and Induces Endothelial Cell Gap Formation

Abstract

Microtubule architecture controls endothelial cell barrier function, as acute microtubule disruption leads to inter‐endothelial gap formation. The P. aeruginosa toxin, ExoY, is injected through a type III secretion system (TTSS) into the cytosol of endothelial cells, where it associates with microtubules, and produces a cytosolic cAMP pool that disrupts the endothelial barrier. However, it is unclear whether cytosolic cAMP reorganizes microtubules as a necessary prerequisite. To test this idea, confluent pulmonary microvascular endothelial cell monolayers were infected with two strains of P. aeruginosa that possess a: (1) functional TTSS, and only injects ExoY (ExoY+), and (2) functional TTSS, but injects a catalytically inactive ExoY (ExoYK81M) due to a single point mutation at the ATP binding site. After 4 hours of incubation, cells were stained for microtubule organization. Although microtubules were disrupted and inter‐endothelial gaps were formed in ExoY+‐treated cells, neither microtubules nor inter‐endothelial gaps were formed in ExoYK81M or vehicle control‐treated cells. However, stabilizing microtubules prior to infection prevented ExoY+‐induced microtubule disruption and inter‐endothelial gap formation. Collectively, these data suggest that ExoY generates a cytosolic cAMP pool that reorganizes microtubules and causes endothelial barrier disruption. [HL‐60024 & HL‐66299]