The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA₂ and lyso-PLA₂ activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA₁ activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.     

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin,ExoU

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.     

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.     

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P₂. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.     

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.     

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.     

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.     

Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.     

Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P2-Associated,Cytoskeletal Collapse and Late Phase Membrane Blebbing

Pseudomonas aeruginosa is an opportunistic pathogen that is associated with hospital-acquired infections, ventilator-associated pneumonia, and morbidity of immunocompromised individuals. A subpopulation of P. aeruginosa encodes a protein, ExoU, which exhibits acute cytotoxicity. Toxicity is directly related to the phospholipase A₂ activity of the protein after injection into the host cytoplasm via a type III secretion system. ExoU enzymatic activity requires eukaryotic cofactors, ubiquitin or ubiquitin-modified proteins. When administered extracellularly, ExoU is unable to intoxicate epithelial cells in culture, even in the presence of the cofactor. Injection or transfection of ExoU is necessary to observe the acute cytotoxic response. Biochemical approaches indicate that ExoU possesses high affinity to a multifunctional phosphoinositide, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P₂ and that it is capable of utilizing this phospholipid as a substrate. In eukaryotic cells, PI(4,5)P₂ is mainly located in the cytoplasmic side of the plasma membrane and anchors adaptor proteins that are involved in cytoskeletal structures, focal adhesions, and plasma membranes. Time-lapse fluorescent microscopy analyses of infected live cells demonstrate that ExoU intoxication correlates with intracellular damage in the early phases of infection, such as disruption of focal adhesions, cytoskeletal collapse, actin depolymerization, and cell rounding. At later time points, a membrane blebbing phenotype was prominent prior to the loss of the plasma membrane integrity and barrier function. Membrane blebbing appears to accelerate membrane rupture and the release of intracellular markers. Our data suggest that in eukaryotic host cells, intracellular ExoU targets and hydrolyzes PI(4,5)P₂ on the plasma membrane, causing a subsequent disruption of cellular structures and membrane integrity.     

Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A(2) (PLA(2))zeta and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.     

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.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta

The entomopathogenic bacterium, Xenorhabdus nematophila, induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts. A hypothesis has been raised that the bacteria inhibit eicosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts. Here, we show direct evidence that X. nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eicosanoid-biosynthesis pathway. Inhibition of PLA2 was dependent on both incubation time with X. nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes. While living bacteria inhibited PLA2 activity, heat-killed X. nematophila rather increased PLA2 activity. X. nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium. The PLA2 inhibitory activity of the organic extract was lost after heat treatment. These results clearly indicate that X. nematophila inhibits PLA2 activity, and thereby inhibits eicosanoid biosynthesis which leads to immunodepression of the infected hosts.     

Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers

A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2.     

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A₂ activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P₂, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P₂ for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P₂ and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P₂. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P₂ binding domains.     

Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca²⁺-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.     

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.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

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.     

A [Lys⁴⁹]phospholipase A₂ from Protobothrops flavoviridis venom induces caspase-independent apoptotic cell death accompanied by rapid plasma-membrane rupture in human leukemia cells

Protobothrops flavoviridis venom contains plural phospholipase A(2) (PLA(2)) isozymes. A [Lys(49)]PLA(2) called BPII induced cell death in human leukemia cells. PLA2, an [Asp(49)]PLA(2) that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA(2) lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.