Contribution of the platelet activating factor signaling pathway to cerebral microcirculatory dysfunction during experimental sepsis by ExoU producing Pseudomonas aeruginosa.

Intravital microscopy was used to assess the involvement of ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, in dysfunction of cerebral microcirculation during experimental pneumosepsis. Cortical vessels from mice intratracheally infected with low density of the ExoU-producing PA103 P. aeruginosa strain exhibited increased leukocyte rolling and adhesion to venule endothelium, decreased capillar density and impaired arteriolar response to vasoactive acetylcholine. These phenomena were mediated by the platelet activating factor receptor (PAFR) pathway because they were reversed in mice treated with a PAFR antagonist prior to infection. Brains from PA103-infected animals exhibited a perivascular inflammatory infiltration that was not detected in animals infected with an exoU deficient mutant or in mice treated with the PAFR antagonist and infected with the wild type bacteria. No effect on brain capillary density was detected in mice infected with the PAO1 P. aeruginosa strain, which do not produce ExoU. Finally, after PA103 infection, mice with a targeted deletion of the PAFR gene exhibited higher brain capillary density and lower leukocyte adhesion to venule endothelium, as well as lower increase of systemic inflammatory cytokines, when compared to wild-type mice. Altogether, our results establish a role for PAFR in mediating ExoU-induced cerebral microvascular failure in a murine model of sepsis.

ExoU-induced redox imbalance and oxidative stress in airway epithelial cells during Pseudomonas aeruginosa pneumosepsis.

ExoU is a potent proinflammatory toxin produced by Pseudomonas aeruginosa, a major agent of severe lung infection and sepsis. Because inflammation is usually associated with oxidative stress, we investigated the effect of ExoU on free radical production and antioxidant defense mechanisms during the course of P. aeruginosa infection. In an experimental model of acute pneumonia, ExoU accounted for increased lipid peroxidation in mice lungs as soon as 3 h after intratracheal instillation of PA103 P. aeruginosa strain. The contribution of airway cells to the generation of a redox imbalance was assessed by in vitro tests carried out with A549 airway epithelial cells. Cultures infected with the ExoU-producing PA103 P. aeruginosa strain produced significantly increased concentrations of lipid hydroperoxides, 8-isoprostane, reactive oxygen intermediates, peroxynitrite and nitric oxide (NO), when compared to cells infected with exoU-deficient mutants. Overproduction of NO by PA103-infected cells likely resulted from overexpression of both inducible and endothelial NO synthase isoforms. PA103 infection was also associated with a significantly increased activity of superoxide dismutase (SOD) and decreased levels of reduced glutathione (GSH), a major antioxidant compound. Our findings unveil another potential mechanism of tissue damage during infection by ExoU-producing P. aeruginosa strains.

Central role of PAFR signalling in ExoU-induced NF-κB activation.

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR-/- or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion.

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

ExoU activates NF-κB and increases IL-8/KC secretion during Pseudomonas aeruginosa infection.

ExoU, a Pseudomonas aeruginosa cytotoxin injected into host cytosol by type III secretion system, exhibits a potent proinflammatory activity that leads to a marked recruitment of neutrophils to infected tissues. To evaluate the mechanisms that account for neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We demonstrate that ExoU increases IL-8 mRNA and protein levels in P. aeruginosa-infected epithelial and endothelial cell lines. Also, ExoU induces the nuclear translocation of p65/p50 NF-κB transactivator heterodimer as well as NF-κB-dependent transcriptional activity. ChIP assays clearly revealed that ExoU promotes p65 binding to NF-κB site in IL-8 promoter and the treatment of cultures with the NF-κB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. These results were corroborated in a murine model of pneumonia that revealed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage when mice were treated with Bay 11-7082 before infection with an ExoU-producing strain. In conclusion, our data demonstrate that ExoU activates NF-κB, stimulating IL-8 expression and secretion during P. aeruginosa infection, and unveils a new mechanism triggered by this important virulence factor to interfere in host signaling pathways.

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis.

BACKGROUND: ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1). METHODS: Mice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection. RESULTS: In PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively. CONCLUSION: ExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection.

BACKGROUND: Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays. METHODS: B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase. RESULTS: ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs. CONCLUSION: B. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.

ExoU modulates soluble and membrane-bound ICAM-1 in Pseudomonas aeruginosa-infected endothelial cells.

ExoU, a Pseudomonas aeruginosa cytotoxin injected via the type III secretion system into host cells, possesses eicosanoid-mediated proinflammatory properties due to its phospholipase A(2) (PLA(2)) activity. This report addressed the question whether ExoU may modulate the expression of adhesion molecules in host cells, therefore contributing to the recruitment of leukocyte into infected tissues. ExoU was shown to down-regulate membrane-bound ICAM-1 (mICAM-1) and up-regulate the release of soluble ICAM-1 (sICAM-1) from P. aeruginosa-infected endothelial cells. The modulation of ICAM-1 depended on the direct effect of the ExoU PLA(2) activity and involved the cyclooxygenase (COX) pathway. No differences in mICAM-1 and sICAM-1 mRNA levels were observed when cultures were infected with the ExoU-producing PA103 strain or the mutant PA103DeltaexoU, suggesting that ExoU may proteolytically cleave mICAM-1, producing sICAM-1 in a COX-dependent pathway.

ExoU-induced vascular hyperpermeability and platelet activation in the course of experimental Pseudomonas aeruginosa pneumosepsis.

To address the question whether ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, can induce hemostatic abnormalities during the course of pneumosepsis, mice were instilled i.t. with the ExoU-producing 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 PA103-infected mice demonstrated features of activation, assessed by the flow cytometric detection of higher percentage of P-selectin expression and of platelet-derived microparticles as well as by the enzyme immunoassay detection of increased thromboxane A2 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.

Lack of MyD88 protects the immunodeficient host against fatal lung inflammation triggered by the opportunistic bacteria Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen of major concern for cystic fibrosis patients as well as immunocompromised cancer patients and transplant recipients. The mechanisms by which B. cenocepacia triggers a rapid health deterioration of the susceptible host have yet to be characterized. TLR and their key signaling intermediate MyD88 play a central role in the detection of microbial molecular patterns and in the initiation of an effective immune response. We performed a study to better understand the role of TLR-MyD88 signaling in B. cenocepacia-induced pathogenesis in the immunocompromised host, using an experimental murine model. The time-course of several dynamic parameters, including animal survival, bacterial load, and secretion of critical inflammatory mediators, was compared in infected and immunosuppressed wild-type and MyD88(-/-) mice. Notably, when compared with wild-type mice, infected MyD88(-/-) animals displayed significantly reduced levels of inflammatory mediators (including KC, TNF-alpha, IL-6, MIP-2, and G-CSF) in blood and lung airspaces. Moreover, despite a higher transient bacterial load in the lungs, immunosuppressed mice deficient in MyD88 had an unexpected survival advantage. Finally, we showed that this B. cenocepacia-induced life-threatening infection of wild-type mice involved the proinflammatory cytokine TNF-alpha and could be prevented by corticosteroids. Altogether, our findings demonstrate that a MyD88-dependent pathway can critically contribute to a detrimental host inflammatory response that leads to fatal pneumonia.

TLR 5, but neither TLR2 nor TLR4, is involved in lung epithelial cell response to Burkholderia cenocepacia.

Burkholderia cenocepacia is known to induce a harmful inflammatory response in the airways of cystic fibrosis patients. Toll-like receptors (TLRs) play key roles in sensing microbial-associated molecular patterns and initiating host innate immunity, but their role in the inflammatory response elicited by B. cenocepacia has not been precisely examined. In this study, we evaluated the contribution of TLR2, TLR4 and TLR5 to the signaling pathways triggered by B. cenocepacia in human bronchial epithelial cells. By quantitative reverse transcriptase (RT)-PCR, we demonstrated that the expression of both TLR2 and TLR4 was significantly upregulated by B. cenocepacia infection, whereas TLR5 expression remained unchanged. Using a dominant-negative approach and airway epithelial cells isolated from MyD88(-/-) mice, we found that B. cenocepacia activated a signaling complex that required the adapter molecule MyD88. Moreover, using epithelial cells from TLR2(-/-), TLR4(-/-) or TLR2/4(-/-) mice or cells overexpressing a functional form of TLR5, we established that TLR5, but neither TLR2 nor TLR4, critically regulated B. cenocepacia-induced lung epithelial inflammatory response.

Lipid body mobilization in the ExoU-induced release of inflammatory mediators by airway epithelial cells.

This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE(2) that was reduced by the bacterial treatment with MAFP, a cPLA(2) inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE(2). Also, in PA103-infected cells, PGE(2) was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE(2) and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.

Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells.

To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells.

Burkholderia cenocepacia, B. multivorans, B. ambifaria and B. vietnamiensis isolates from cystic fibrosis patients have different profiles of exoenzyme production.

Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC-infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.

Up-regulation of Fas expression by Pseudomonas aeruginosa-infected endothelial cells depends on modulation of iNOS and enhanced production of NO induced by bacterial type III secreted proteins.

To obtain a better understanding of the mechanisms involved in the up-regulation of the Fas apoptotic signaling cascade induced by P. aeruginosa type III secretion system (TTSS), human umbilical vein endothelial cells (HUVEC) were infected with P. aeruginosa PAO-1 or its TTSS-negative mutant PAO-1::exsA. PAO-1 was significantly more cytotoxic than the mutant and features of apoptosis (DNA fragmentation and annexin V reactivity) were more prominent in cultures infected with the wild-type bacteria. PAO-1 induced the up-regulation of Fas and the release of soluble FasL (sFasL) from infected cells but cell treatment with antagonist anti-Fas did not completely abrogate apoptosis suggesting that, besides the activated Fas-FasL pathway, other mechanisms are likely to be associated with the induction of apoptosis. LNMMA, a potent inhibitor of NO synthesis, completely inhibited apoptosis in both PAO-1 and PAO-1::exsA infected cultures. Moreover, PAO-1 was shown to up-regulate both the expression of iNOS and NO production by HUVEC. Treatment of cells with LNMMA completely inhibited cell expression of mFas. Based on these results we speculate that P. aeruginosa TTSS not only accounts for HUVEC higher expression of Fas and release of sFasL but also leads to overproduction of NO and to a NO-dependent up-regulation of the Fas-FasL proteins.

Implications of oxidative stress in the cytotoxicity of Pseudomonas aeruginosa ExoU.

ExoU PLA2-like activity has been shown to account for membrane lysis and acute death of infected cells. Translocation of effector proteins by the type III secretion systems depends on close contact between microbial and host cells. Our finding that both the ExoU-producing PA103 Pseudomonas aeruginosa and its mutant obtained by deletion of exoU adhered poorly to endothelial cells (EC) led to the hypothesis that, in some cells, the amount of injected toxin may not be enough to induce cell lysis but cells would suffer from a long-term effect of ExoU intoxication. To address this question, cells were exposed to both bacteria for 1 h and then treated with gentamicin-containing medium, to eliminate infecting microorganisms. After 24 h, the percentage of viable EC in PA103-infected cultures was significantly lower than in cultures exposed to the mutant, as determined by the MTT assay. Cell death was not likely to depend on the ExoU lytic activity since cell labeling with propidium iodide was similar in cultures infected with both bacterial strains. Bacterial cytotoxicity was significantly reduced by MAFP, a specific inhibitor of cPLA2 and iPLA2. Since the PLA2 activity on membrane phospholipids generates free fatty acid, including arachidonic acid (AA), we next compared the bacterial ability to release AA from infected EC. PA103 was shown to induce a potent AA release that was inhibited by MAFP. AA oxidation by oxygenases generates eicosanoids, known to induce both cell death and proliferation. However neither inhibitors of cyclooxygenases (ibuprofen) nor lipoxygenases (NDGA) reduced the ExoU toxicity. Since non-enzymatic oxidation of AA generates reactive radicals, we next investigated the PA103 ability to induce oxidative stress in infected cells. FACS analysis of cell labeling with the C-11 fluor probe and with anti-4-hydroxynonel antibody revealed a significant peroxidation of cell membrane lipids. These results, together with our finding that PA103-infected EC death was significantly attenuated by alpha-tocopherol, led to the conclusion that AA-induced oxidative stress may be another mechanism of cell damage in the course of infection by ExoU-producing P. aeruginosa.

Nod1 participates in the innate immune response to Pseudomonas aeruginosa.

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.

Pseudomonas aeruginosa-induced production of free radicals by IFNgamma plus TNFalpha-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?

We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNgamma plus TNFalpha to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed. In this study we investigated the fate of IFNgamma plus TNFalpha-activated HUVEC that have harboured IC bacteria for up to 24 h. At 10 h, the endothelial cell killing of P. aeruginosa isolates exceeded 90%. IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC. P. aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH). Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability. Treatment of cells with the antioxidant alpha-lipoic acid significantly increased the survival of infected cells. Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells. Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.

Dynamic interaction between airway epithelial cells and Staphylococcus aureus.

Staphylococcus aureus is a major cause of pulmonary infection, particularly in cystic fibrosis (CF) patients. However, few aspects of the interplay between S. aureus and host airway epithelial cells have been investigated thus far. We investigated by videomicroscopy the time- and bacterial concentration-dependent (10(4), 10(6), and 10(8) CFU/ml) effect of S. aureus on adherence, internalization, and the associated damage of the airway epithelial cells. The balance between the secretion by S. aureus of the alpha-toxin virulence factor and by the airway cells of the antibacterial secretory leukoproteinase inhibitor (SLPI) was also analyzed. After 1 h of interaction, whatever the initial bacterial concentration, a low percentage of S. aureus (<8%) adhered to airway cells, and no airway epithelial cell damage was observed. In contrast, after 24 h of incubation, more bacteria adhered to airway epithelial cells, internalized bacteria were observed, and a bacterial concentration-dependent effect on airway cell damage was observed. At 24 h, most airway cells incubated with bacteria at 10(8) CFU/ml exhibited a necrotic phenotype. The necrosis was preceded by a transient apoptotic process. In parallel, we observed a time- and bacterial concentration-dependent decrease in SLPI and increase in alpha-toxin expression. These results suggest that airway cells can defend against S. aureus in the early stages of infection. However, in later phases, there is a marked imbalance between the bactericidal capacity of host cells and bacterial virulence. These findings reinforce the potential importance of S. aureus in the pathogenicity of airway infections, including those observed early in CF patients.