Pseudomonas aeruginosa is an opportunistic, Gram-negative pathogen and an important cause of hospital acquired infections, especially in immunocompromised patients. Highly virulent P. aeruginosa strains use a type III secretion system (T3SS) to inject exoenzyme effectors directly into the cytoplasm of a target host cell. P. aeruginosa strains that express the T3SS effector, ExoU, associate with adverse outcomes in critically ill patients with pneumonia, owing to the ability of ExoU to rapidly damage host cell membranes and subvert the innate immune response to infection. Herein, we review the structure, function, regulation, and virulence characteristics of the T3SS effector ExoU, a highly cytotoxic phospholipase A₂ enzyme.

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY⁺/ExoU⁻) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY⁻/ExoU⁺). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors.