ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection (original) (raw)
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AJP: Lung Cellular and Molecular Physiology, 2014
Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY+), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoYK81M; ExoYK81M), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY+ i...
Scientific reports, 2017
Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cel...
2015
K. Adam Morrow, Cristhiaan D. Ochoa, Ron Balczon, Chun Zhou, Laura Cauthen, 6 Mikhail Alexeyev, Katherine M. Schmalzer, Dara W. Frank, and Troy Stevens 7 8 Departments of Physiology and Cell Biology, Biochemistry and Molecular Biology, and 9 Medicine, Center for Lung Biology, University of South Alabama, Mobile AL 36688; Physician10 Scientist Training Program, Department of Medicine and Division of Pulmonary and Critical 11 Care, University of Texas-Southwestern Medical Center, Dallas TX 75390; Department of 12 Medicine, Division of Hematology/Oncology, Department of Microbiology and Molecular 13 Genetics, Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee 14 WI 53226 15 16 17
The Role of ExoS in Dissemination of Pseudomonas aeruginosa during Pneumonia
PLOS Pathogens, 2015
Hospital-acquired pneumonia is associated with high rates of morbidity and mortality, and dissemination to the bloodstream is a recognized risk factor for particularly poor outcomes. Yet the mechanism by which bacteria in the lungs gain access to the bloodstream remains poorly understood. In this study, we used a mouse model of Pseudomonas aeruginosa pneumonia to examine this mechanism. P. aeruginosa uses a type III secretion system to deliver effector proteins such as ExoS directly into the cytosol of eukaryotic cells. ExoS, a bi-functional GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT), inhibits phagocytosis during pneumonia but has also been linked to a higher incidence of dissemination to the bloodstream. We used a novel imaging methodology to identify ExoS intoxicated cells during pneumonia and found that ExoS is injected into not only leukocytes but also epithelial cells. Phagocytic cells, primarily neutrophils, were targeted for injection with ExoS early during infection, but type I pneumocytes became increasingly injected at later time points. Interestingly, injection of these pneumocytes did not occur randomly but rather in discrete regions, which we designate ""fields of cell injection" (FOCI). These FOCI increased in size as the infection progressed and contained dead type I pneumocytes. Both of these phenotypes were attenuated in infections caused by bacteria secreting ADPRTdeficient ExoS, indicating that FOCI growth and type I pneumocyte death were dependent on the ADPRT activity of ExoS. During the course of infection, increased FOCI size was associated with enhanced disruption of the pulmonary-vascular barrier and increased bacterial dissemination into the blood, both of which were also dependent on the ADPRT activity of ExoS. We conclude that the ADPRT activity of ExoS acts upon type I pneumocytes to disrupt the pulmonary-vascular barrier during P. aeruginosa pneumonia, leading to bacterial dissemination.
Infection and Immunity, 2000
Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gen...
ExoU-induced procoagulant activity in Pseudomonas aeruginosa-infected airway cells
European Respiratory Journal, 2008
The present study addressed the question whether ExoU, a Pseudomonas aeruginosa toxin with phospholipase A2 (PLA2) activity, may induce airway epithelial cells to overexpress tissue factor (TF) and exhibit a procoagulant phenotype. Cells from the human bronchial epithelial BEAS-2B line were infected with an ExoU-producing P. aeruginosa strain, pre-treated or not with the cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP), or with two ExoU-deficient mutants. Control noninfected and infected cells were assessed for the expression of: 1) TF mRNA by RT-PCR; 2) cell-associated TF by enzyme immunoassay and flow cytometry; 3) procoagulant activity by a colorimetric assay; and 4) microparticle-associated TF by flow cytometry. An enzyme immunoassay was also used to assess cell-associated TF in lung extracts from mice infected intratracheally with ExoU-producing anddeficient bacteria. Cells infected with the wild-type bacteria had higher levels of TF mRNA, cell-associated TF expression, procoagulant activity and released microparticle-associated TF than cells infected with the mutants. Bacterial treatment with MAFP significantly reduced the expression of TF by infected cells. Lung samples from mice infected with the wild-type bacteria exhibited higher levels of cell-associated TF and procoagulant activity. The present results demonstrate that ExoU may contribute to the pathogenesis of lung injury by inducing a tissue factor-dependent procoagulant activity in airway epithelial cells.
Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy
American journal of physiology. Lung cellular and molecular physiology, 2015
Here, we tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors ExoY and ExoU induce release of a high molecular weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU, and the conditional expression of an activity-attenuated ExoU, induced time-dependent pulmonary microvascular endothelial cell (PMVEC) gap formation that was paralleled by loss of intracellular tau and concomitant appearance of high molecular weight extracellular tau. Transfer of the high molecular weight tau in filtered supernatant onto naïve endothelial cells resulted in the intracellular accumulation of tau clusters, which was accompanied by cell injury, inter-endothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant, but did not retrieve higher molecular ...
Toxins
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 cultu...
Shock, 2010
To address the question whether ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A 2 activity, can induce hemostatic abnormalities during the course of pneumosepsis, mice were instilled i.t. with the ExoUproducing PA103 P. aeruginosa or with a mutant obtained by deletion of the exoU gene. Control animals were instilled with sterile vehicle. To assess the role of ExoU in animal survival, mice were evaluated for 72 h. In all the other experiments, animals were studied at 24 h after infection. PA103-infected mice showed significantly higher mortality rate, lower blood leukocyte concentration, and higher platelet concentration and hematocrit than animals infected with the bacterial mutant, as well as evidences of increased vascular permeability and plasma leakage, which were confirmed by our finding of higher protein concentration in bronchoalveolar lavage fluids and by the Evans blue dye assay. Platelets from PA103infected mice demonstrated features of activation, assessed by the flow cytometric detection of higher percentage of Pselectin expression and of platelet-derived microparticles as well as by the enzyme immunoassay detection of increased thromboxane A 2 concentration in animal plasma. Histopathology of lung and kidney sections from PA103-infected mice exhibited evidences of thrombus formation that were not detected in sections of animals from the other groups. Our results demonstrate the ability of ExoU to induce vascular hyperpermeability, platelet activation, and thrombus formation during P. aeruginosa pneumosepsis, and we speculate that this ability may contribute to the reported poor outcome of patients with severe infection by ExoU-producing P. aeruginosa.