Pseudomonas aeruginosa exoenzyme Y activity inhibits exoenzyme S-induced caspase activation in pulmonary microvascular endothelial cells.

Pseudomonas aeruginosa is the most common cause of ventilator-associated pneumonia in intensive care unit (ICU) patients. Virulent P. aeruginosa strains utilize a type III secretion system that injects exoenzymes S, T, U, and/or Y (ExoS, T, U, and/or Y, respectively) into the host cell cytosol. Most clinically isolated P. aeruginosa strains encode ExoY, often in combination with ExoU and ExoT, or ExoS and ExoT. The effects of each individual exoenzyme on host cell signaling have been extensively studied, but how these signaling mechanisms are altered during co-intoxication is poorly understood. In this study, we sought to determine whether co-intoxication of ExoY with ExoS and/or ExoT modulated ExoS-induced caspase activation. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa isogenic PAK strains expressing differing combinations of exoenzymes S, T, and Y. ExoS alone, but not ExoT or ExoY, induced significant caspase activation and cell death in PMVECs. Importantly, ExoY significantly decreased ExoS-induced caspase activation, but not cell death. To determine whether ExoY enzymatic activity was necessary to inhibit ExoS-induced caspase activation, ExoS-expressing P. aeruginosa strains were transformed with plasmids containing catalytically active or inactive ExoY. Enzymatically active ExoY inhibited ExoS-mediated caspase activation, whereas enzymatically inactive ExoY did not. The presence of ExoS did not significantly diminish ExoY-mediated cGMP production. Thus, we conclude that ExoY enzymatic activity inhibits ExoS-induced caspase activation in PMVECs.NEW & NOTEWORTHY P. aeruginosa strain PAK utilizes a type III secretion system to inject exoenzymes S, T, and Y directly into the host cell cytosol. In pulmonary microvascular endothelial cells, exoenzyme S (ExoS) alone, but not exoenzymes T or Y (ExoT or ExoY) alone, induces apoptotic caspase activation and cell death. When exoenzymes are coinjected, ExoY, but not ExoT, inhibits ExoS-induced caspase activation, but not cell death. ExoY must be catalytically active to prevent ExoS-induced caspase activation.