ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection

Abstract

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa 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 A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa 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 P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-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 P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-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 P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.

Amyloid-β and caspase-1 are indicators of sepsis and organ injury

“…Our previous studies using cell culture and animal models have described paradoxical functions of caspase-1 activation during infection. In cultured lung endothelial cells and macrophages, pathogen-induced caspase-1 activation can be pro-inflammatory and incite damage [ 37 , 38 ]. Conversely, caspase-1 can also work to protect pulmonary barrier function during infection [ 39 ].…”

Role of Host and Bacterial Lipids in Pseudomonas aeruginosa Respiratory Infections

“…Although ExoU injection causes reactive oxygen species (ROS) imbalance ( 116 ) and is cytotoxic for airway epithelial cells ( 102 , 117 ), the remaining non-infected cells activate several transcriptional regulators, such as AP1 and NF-kB, modulating the host response ( 111 , 112 , 118 , 119 ). Furthermore, the cytotoxic activity of ExoU also promotes endothelial cell damage, which is associated with ROS generation, membrane lipid peroxidation, and caspase-1 activation ( 107 , 120 ). The ability to break down cellular barriers to bacterial dissemination, such as epithelium and endothelium, helps explain why ExoU is a predictor of invasive infections and has been associated with severe pneumonia followed by bacteremia and sepsis.…”