Abstract 1014 Pseudomonas aeruginosa Exoenzyme Y inhibits Exoenzyme S-induced Caspase-3/7 activation in Pulmonary Microvascular Endothelial Cells

Pseudomonas aeruginosa (P. aer.) is the most common cause of ventilator associated pneumonia in intensive care unit patients. P. aer. utilizes a type III secretion system that injects exoenzymes U, S, T, and/or Y into the host cell cytosol. ExoY is present in approximately 90% of clinically isolated P. aer. strains along with ExoS and T. In pulmonary microvascular endothelial cells (PMVECs ) that line capillary walls, intoxication with either ExoS or ExoY results in alveolar flooding and impaired gas exchange. ExoS causes PMVEC death, whereas ExoY causes cell rounding, without PMVEC death. Previous studies show that in lung epithelial cells , when ExoY and ExoS are expressed together, ExoY inhibits ExoS-induced cytotoxicity. Other studies have shown that ExoY infection of PMVECs does not induce the activation of executioner caspases 3 and/or 7 that mediate apoptotic cell death. Despite these findings, our preliminary data indicates that ExoY does not inhibit ExoS mediated cell death in PMVECs. We therefore sought to determine whether ExoY inhibits ExoS-induced intracellular caspase-3/7 activation in PMVECs , preventing apoptotic mechanisms of cell death. PMVECs were infected with isogenic P. aer. strains expressing various combinations of exoenzymes S, T, and Y. PMVECs were infected at a multiplicity of infection of 20:1 in a 12-well dish for 5.5 hours. At 2.5 hours post-infection, fluorescently labeled caspase-3/7 indicator (FLICA) was added to each well. FLICA is membrane permeable, and irreversibly binds to intracellular caspase-3/7 activation. Post-infection, cells were washed with a buffer to remove any unbound FLICA. PMVECs were assessed for the amount of intracellular active caspase-3/7 by flow cytometry. Infection with S- and ST-expressing strains resulted in statistically significant intracellular caspase-3/7 activation compared to the uninfected control. Conversely, infection with SY- and STY-expressing strains significantly decreased intracellular caspase-3/7 activation compared to S and ST. These data indicate that ExoY inhibits ExoS-mediated caspase-3/7 activation in PMVECs, regardless of the presence or absence of ExoT. Paradoxically, our previous studies indicate that this decrease in apoptotic caspase activation is not accompanied with a similar decrease in cell death. Indeed, ExoY does not inhibit ExoS-mediated cell death in PMVECs. This suggests that in the presence of both ExoS and ExoY, ExoY may shift ExoS-induced apoptosis to necrotic pathways. Further investigation is required to determine the mechanism by which ExoY inhibits apoptotic caspase activation. This research was funded by South Alabama's Summer Undergraduate Research Fellowship (SURF), our South Alabama ASBMB Student Chapter Award, and the ASBMB undergraduate research award.