Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.     

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 A₂ (PLA₂) 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 PLA₂ 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 PA103ΔexoU, suggesting that ExoU may proteolytically cleave mICAM-1, producing sICAM-1 in a COX-dependent pathway.     

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.     

The Pseudomonas aeruginosa type III secreted toxin ExoT is necessary and sufficient to induce apoptosis in epithelial cells

Type III secreted (T3SS) effectors are important virulence factors in acute infections caused by Pseudomonas aeruginosa . PA103, a well-studied human lung isolate, encodes and secretes two effectors, ExoU and ExoT. ExoU is a potent cytotoxin that causes necrotic cell death. In addition, PA103 can induce cell death in macrophages in an ExoU-independent but T3SS-dependent manner. We now demonstrate that ExoT is both necessary and sufficient to cause apoptosis in HeLa cells and that it activates the mitochondrial/cytochrome c -dependent apoptotic pathway. We further show that ExoT induction of cell death is primarily dependent on its ADP ribosyltransferase domain activity. Our data also indicate that the T3SS apparatus can cause necrotic cell death, which is effectively blocked by ExoT, suggesting that P. aeruginosa may have evolved strategies to prevent T3SS-induced necrosis.     

Proapoptotic effects of P. aeruginosa involve inhibition of surfactant phosphatidylcholine synthesis

Pseudomonas aeruginosa causes sepsis-induced acute lung injury, a disorder associated with deficiency of surfactant phosphatidylcholine (PtdCho). P. aeruginosa (PA103) utilizes a type III secretion system (TTSS) to induce programmed cell death. Herein, we observed that PA103 reduced alveolar PtdCho levels, resulting in impaired lung biophysical activity, an effect partly attributed to caspase-dependent cleavage of the key PtdCho biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Expression of recombinant CCTalpha variants harboring point mutations at putative caspase cleavage sites in murine lung epithelia resulted in partial proteolytic resistance of CCTalpha to PA103. Further, caspase-directed CCTalpha degradation, decreased PtdCho levels, and cell death in murine lung epithelia were lessened after exposure of cells to bacterial strains lacking the TTSS gene product, exotoxin U (ExoU), but not ExoT. These observations suggest that during the proapoptotic program driven by P. aeruginosa, deleterious effects on phospholipid metabolism are mediated by a TTSS in concert with caspase activation, resulting in proteolysis of a key surfactant biosynthetic enzyme.     

Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.     

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.     

Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis

Phospholipases are ubiquitous and diverse enzymes that induce changes in membrane composition, activate the inflammatory cascade and alter cell signaling pathways. Recent evidence suggests that certain bacterial pathogens have acquired genes encoding secreted phospholipase A2 enzymes through lateral gene transfer events. The two best-studied members of this class of enzyme are ExoU and SlaA, which are produced by Pseudomonas aeruginosa and group A Streptococcus, respectively. These enzymes modulate the host inflammatory response, increase the severity of disease and otherwise alter host-pathogen interactions. We propose that a key function of ExoU and SlaA is to increase the fitness of the subclones expressing these enzymes, thereby increasing the population size of the PLA2-positive strains and enhancing the likelihood of encountering an at-risk host.     

Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system. Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain. We investigated the phospholipase A (PLA) activity of ExoU. ExoU, but not non-catalytic ExoU-S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca(2+)-independent PLA(2) activity. When activated ExoU was mixed with secretory type PLA(2), more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity. A significant increase in lysoPLA activity was also observed. Glycerol enhanced this activity and inhibitors of iPLA(2) suppressed ExoU's lysoPLA activity. Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser(142) with an unknown eukaryotic cell factor(s) for its activation.     

Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU

Pseudomonas aeruginosa is an opportunistic pathogen that uses a type III secretion system and four effector proteins to avoid innate immune responses. ExoS, ExoT, ExoY, and ExoU all possess enzymatic activities that disrupt host cellular physiology and prevent bacterial clearance by host defense mechanisms. The specificity of these toxins for eukaryotic cells depends on the presence of substrate targets and eukaryotic cofactors responsible for effector activation. We used a combined biochemical and proteomic approach to identify Cu(2+), Zn(2+)-superoxide dismutase (SOD1) as a cofactor that activates the phospholipase activity of ExoU. Recombinant ExoU (rExoU) was activated in a dose-dependent manner by either bovine liver SOD1 or the yeast ortholog, Sod1p, but not by either Fe or Mn-containing SODs from E. coli or small molecule SOD mimetics. Inhibitor studies indicated that SOD enzymatic activity was not required for the activation of rExoU. The physical interaction between rExoU and SOD was demonstrated by capture techniques using either of the two proteins immobilized onto the solid phase. Identification of SOD as a cofactor allowed us to develop a new assay using a fluorescent substrate to measure the phospholipase activity of rExoU. The ability of SOD to act as a cytoplasmic cofactor stimulating ExoU phospholipase activity has significant implications for the biological activity of the toxin. Further elucidation of the structural mechanism of ExoU activation by this eukaryotic cofactor may provide a rational approach to the design of inhibitors that can diminish tissue damage during infection by ExoU-producing strains of P. aeruginosa.     

Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii

Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.     

In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors

A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown. Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoU-expressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA2) and Ca2+ -independent (iPLA2) phospholipase A2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A2 inhibitors may represent a novel mode of treatment for acute P. aeruginosa infections. We find that 300-600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion. We also show that ExoU interacts in vitro with an inhibitor of cPLA2 and iPLA2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA2 and iPLA2 enzymes. Mutation of either the serine or the aspartate renders ExoU non-cytotoxic. Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.     

Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 is a multihost pathogen that can infect Arabidopsis. We found that PA14 pathogenesis in Arabidopsis involves the following steps: attachment to the leaf surface, congregation of bacteria at and invasion through stomata or wounds, colonization of intercellular spaces, and concomitant disruption of plant cell wall and membrane structures, basipetal movement along the vascular parenchyma, and maceration and rotting of the petiole and central bud. Distinctive features of P. aeruginosa pathogenesis are that the surface of mesophyll cell walls adopt an unusual convoluted or undulated appearance, that PA14 cells orient themselves perpendicularly to the outer surface of mesophyll cell walls, and that PA14 cells make circular perforations, approximately equal to the diameter of P. aeruginosa, in mesophyll cell walls. Taken together, our data show that P. aeruginosa strain PA14 is a facultative pathogen of Arabidopsis that is capable of causing local and systemic infection, which can result in the death of the infected plant.     

ExoU is a potent intracellular phospholipase

The combination of a large genome encoding metabolic versatility and conserved secreted virulence determinants makes Pseudomonas aeruginosa a model pathogen that can be used to study host-parasite interactions in many eukaryotic hosts. One of the virulence regulons that likely plays a role in the ability of P. aeruginosa to avoid innate immune clearance in mammals is a type III secretion system (TTSS). Upon cellular contact, the P. aeruginosa TTSS is capable of delivering a combination of at least four different effector proteins, exoenzyme S (ExoS), ExoT, ExoU, and ExoY. Two of the four translocated proteins, ExoS and ExoU, are cytotoxic to cells during infection and transfection. The mechanism of cytotoxicity of ExoS is unclear. ExoU, however, has recently been characterized as a member of the phospholipase A family of enzymes, possessing at least phospholipase A2 activity. Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryotic-specific modification or cofactor for its activity in vitro. The biologic effects of minimal expression of ExoU in yeast can be visualized by membrane damage to different organelles and fragmentation of the vacuole. In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. ExoU likely represents a unique enzyme and is the first identified phopholipase virulence factor that is translocated into the cytosol by TTSS.     

Inhibition of phospholipase A2 reduces neurite outgrowth and neuronal viability

Phospholipase A2 (PLA(2)) has been implicated in neurodevelopmental processes and in the early development of the nervous system. We investigated the effects of the inhibition of calcium-dependent and calcium-independent subtypes of cytosolic PLA2 (cPLA2 and iPLA2) on the development and viability of primary cultures of cortical and hippocampal neurons. PLA2 in these cultures was continuously inhibited with methylarachidonyl-fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and iPLA2, or with bromoenol lactone (BEL), an irreversible selective iPLA2 inhibitor. The effect of PLA2 inhibitors on the development of neuronal cultures was ascertained by total cell count and morphological characterisation. Neuronal viability was quantified with MTT assays. Inhibition of PLA2 resulted in reduction of neuritogenesis and neuronal viability, disrupting neuronal homeostasis and leading to neuronal death. We conclude that the functional integrity of both calcium-dependent and calcium-independent cytosolic PLA2 is necessary for the in vitro development of cortical and hippocampal neurons.     

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.     

Eukaryotic localization, activation and ubiquitinylation of a bacterial type III secreted toxin

Type III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.     

Bordetella type III secretion induces caspase 1-independent necrosis

The Bordetella bronchiseptica type III (TIII) secretion system induces cytotoxicity in infected macrophages and epithelial cells. In this report we characterize the cell death phenotype and compare it to the TIII-dependent cytotoxicity induced by Yersinia enterocolitica and Shigella flexneri. Bordetella bronchiseptica strain RB58 was able to induce cell death in J774A.1 macrophages with the same efficiency as Shigella and Yersinia, but only B. bronchiseptica was able to kill epithelial cells in a TIII-dependent manner. Primary macrophages from caspase 1-/- mice were susceptible to RB58-mediated killing, suggesting that unlike Shigella and Salmonella, caspase 1 does not mediate cell death. RB58-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD, and Western blot analyses of RB58-infected HeLa cells indicated that neither caspase 3 nor 7 was cleaved and PARP remained in its full-length active form. Morphologically the RB58-infected HeLa cells resembled necrotic rather than apoptotic cells, exhibiting cytoplasmic swelling and extensive membrane blebbing in the absence of nuclear changes. The addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking non-specific ion fluxes across the plasma membrane, blocked RB58-induced cytotoxicity. Addition of cyclosporin A which prevents the opening of the mitochondrial permeability pore, had no effect on RB58-infected cells. We conclude that the B. bronchiseptica TIII secretion system induces a mode of cell death consistent with necrosis that is distinct from that of Yersinia and Shigella.