ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Infection
The Gram-negative, opportunistic pathogen utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A (PLA) that hydrolyses host cell membranes, and strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from -infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during infection that may inhibit endothelial barrier and inflammatory functions.
Hardy, Kierra S.; Tuckey, Amanda N.; Renema, Phoibe; Patel, Mita; Al-Mehdi, Abu-Bakr; Spadafora, Domenico; Schlumpf, Cody A.; Barrington, Robert A.; Alexeyev, Mikhail F.; Stevens, Troy; Pittet, Jean-Francois; Wagener, Brant M.; Simmons, Jon D.; Alvarez, Diego F.; and Audia, Jonathon P., "ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Infection" (2022). Department of Microbiology and Immunology Faculty and Staff Publications and Presentations. 13.