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.
Highlights
Pseudomonas aeruginosa is an opportunistic, Gram-negative pathogen and a common cause of respiratory infections in mechanically ventilated patients, often leading to acute respiratory distress syndrome (ARDS) and sepsis [1–4]
The overarching goal of this study was to examine the effects of the P. aeruginosa T3SS effector, ExoU, on the barrier and inflammatory functions of pulmonary microvascular endothelial cell (PMVEC) using a cell culture
To determine the effects of ExoU on PMVECs, cultured cells were inoculated with the various strains and inter-endothelial gap formation and cell lysis were assessed by microscopy and lactate dehydrogenase (LDH) release, respectively
Summary
Pseudomonas aeruginosa is an opportunistic, Gram-negative pathogen and a common cause of respiratory infections in mechanically ventilated patients, often leading to acute respiratory distress syndrome (ARDS) and sepsis [1–4]. P. aeruginosa virulence is primarily driven by the type III secretion system (T3SS) and its cognate exoenzyme effectors, which are associated with poor disease outcomes in patients with pneumonia, ARDS, and/or sepsis [3,4,9]. There are four wellcharacterized T3SS effectors, each of which is defined by the eukaryotic cofactor(s) required to activate enzymatic function upon injection into a host cell [10–13]. Each of the four exoenzyme effectors differ in the mechanism and/or extent of damaging effects elicited upon injection into a host cell. These differences define the relative contribution of each exoenzyme effector to P. aeruginosa virulence
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