Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors.

Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

Plant phospholipases: an overview.

Plant phospholipases can be grouped into four major types, phospholipase D, phospholipase C, phospholipase A1 (PLA(1)), and phospholipase A2 (PLA(2)), that hydrolyze glycerophospholipids at different ester bonds. Within each type, there are different families or subfamilies of enzymes that can differ in substrate specificity, cofactor requirement, and/or reaction conditions. These differences provide insights into determining the cellular function of specific phospholipases in plants, and they can be explored for different industrial applications.

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge.

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.

Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity.

In eukaryotes phospholipase D (PLD) is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom.

Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12(1)1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, ß = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively.

Signal-activated phospholipase regulation of leukocyte chemotaxis.

Signal-activated phospholipases are a recent focus of the rapidly growing field of lipid signaling. The extent of their impact on the pathways regulating diverse cell functions is beginning to be appreciated. A critical step in inflammation is the attraction of leukocytes to injured or diseased tissue. Chemotaxis of leukocytes, a requisite process for monocyte and neutrophil extravasation from the blood into tissues, is a critical step for initiating and maintaining inflammation in both acute and chronic settings. Recent studies have identified new important and required roles for two signal-activated phospholipases A2 (PLA2) in regulating chemotaxis. The two intracellular phospholipases, cPLA2alpha (Group IVA) and iPLA2beta (Group VIA), act in parallel to provide distinct lipid mediators at different intracellular sites that are both required for leukocytes to migrate toward the chemokine monocyte chemoattractant protein-1. This review will summarize the separate roles of these phospholipases as well as what is currently known about the influence of two other classes of intracellular signal-activated phospholipases, phospholipase C and phospholipase D, in regulating chemotaxis in eukaryotic cells, but particularly in human monocytes. The contributions of these phospholipases to chemotaxis both in vitro and in vivo will be highlighted.

Phospholipase D1 is specifically required for regulated secretion of von Willebrand factor from endothelial cells.

Endothelial cells regulate thrombosis, hemostasis, and inflammatory responses by supplying the vasculature with several factors that include procoagulant von Willebrand factor (VWF) and fibrinolytic tissue-type plasminogen activator (tPA). Both proteins can be secreted in a Ca(2+)-regulated manner after endothelial activation but exhibit opposing physiologic effects. In search for factors that could modulate endothelial responses by selectively affecting the secretion of procoagulant or anticoagulant proteins, we identify here phospholipase D1 (PLD1) as a specific regulator of VWF secretion. PLD1 is translocated to the plasma membrane upon stimulation of endothelial secretion, and this process correlates with the generation of phosphatidic acid (PA) in the plasma membrane. Histamine-evoked secretion of VWF, but not tPA, is inhibited by blocking PLD-mediated production of PA, and this effect can be attributed to PLD1 and not PLD2. Thus, different mechanisms appear to control the agonist-induced secretion of VWF and tPA, with only the former requiring PLD1.

DNase II is a member of the phospholipase D superfamily.

MOTIVATION: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown. RESULTS: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence-function analyses of this important nuclease.

Retinoic acid specifically activates an oleate-dependent phospholipase D in the nuclei of LA-N-1 neuroblastoma cells.

Earlier studies showed that treatment of LA-N-1 cells with TPA, a tumoral promoter, leads to the stimulation of a G protein-regulated phospholipase D (PLD) in the nuclei. Now we demonstrate that retinoic acid, a cellular differentiation inducing agent, activates a nuclear oleate-dependent PLD in LA-N-1 cells. Treatment of the nuclei with retinoic acid induces the breakdown of phosphatidylcholine (PtdCho). Our results indicate that PLD is regulated differentially depending on the nature of the stimulatory agent. These results strongly suggest the existence of two nuclear PLD isoforms in LA-N-1 nuclei that hydrolyze PtdCho.

A novel phospholipase D of Arabidopsis that is activated by oleic acid and associated with the plasma membrane.

Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli. PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta. Oleate stimulated PLDdelta's binding to phosphatidylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.

The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily.

The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

Substrate selectivities and lipid modulation of plant phospholipase D alpha, -beta, and -gamma.

Three classes of phospholipase D (PLD), designated PLD alpha, -beta, and -gamma, have been cloned from plants, but their substrate selectivities have not been established. Using active PLDs expressed from their cDNAs in Escherichia coli, we compared the hydrolytic activities of these three PLDs toward various phospholipids and the influence of substrate composition on their substrate selectivities. When single-class phospholipid vesicles of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2), N-acylphosphatidylethanolamine (NAPE), and cardiolipin (CL) were examined, PLD alpha hydrolyzed PC, PE, and PG but PLD beta and -gamma showed no activity toward any of these lipids. When PIP2 was included in mixed vesicles with the phospholipids above, PLD alpha showed the same PC-, PE-, and PG-hydrolyzing ability, whereas PLD beta and -gamma were able to hydrolyze both PE and PS. When both PE and PIP2 were included in substrate vesicles, PLD beta and PLD gamma hydrolyzed PC, PG, and NAPE, showing that both PE and PIP2 are required for PC, PG, and NAPE hydrolysis by PLD beta and -gamma. The PE activation of PLD beta and -gamma required lipid vesicles made of mostly PE, suggesting that PE may affect the substrate presentation rather than serve as a cofactor of these PLDs. Under equivalent reaction conditions, PLD beta displayed a similar preference for PC and NAPE, whereas PLD gamma preferred NAPE to PC by nearly three times. None of the three PLDs used PI, CL, or PIP2 as substrates. These results have identified PS- and NAPE-hydrolyzing PLDs and have indicated an important role for lipid composition in regulating the substrate selectivity of PLD beta and -gamma.

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells.

Phospholipase D (PLD) in lymphocytic mouse leukemic L1210 cells has been found to be activated by oleate both in vitro and in intact cells. The PLD activity was measured by phosphatidylethanol produced from radiolabeled phosphatidylcholine or myristic acid in the presence of ethanol. This oleate-activated PLD was further characterized in intact cells and compared with that in HL60 cells. Unlike PLD in HL60 cells, the PLD in L1210 cells was activated by unsaturated fatty acids, stimulated by melittin, insensitive to guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PIP2), independent of phorbol 12-myristate 13-acetate (PMA) and staurosporine, and inhibited by pervanadate. These observations indicate that the PLD present in L1210 cells is distinct from that in HL60 cells. Key PLD properties of L1210 cells such as insensitivity to GTP gamma S, ARF, PIP2, or PMA were in good agreement with currently known in vitro properties of the oleate-activated PLD found in mammalian sources. Therefore, the L1210 cells could be used as an intact-cell source for an oleate-activated PLD.

A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues.

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.

v-Src activates a unique phospholipase D activity that can be distinguished from the phospholipase D activity activated by phorbol esters.

Phospholipase D (PLD) activity, as measured by the transphosphatidylation of cellular phospholipids, is elevated in BALB/c 3T3 cells transformed by v-Src. Phorbol esters that activate protein kinase C (PKC) also increase PLC activity in BALB/c 3T3 cells. v-Src-induced PLD activity could be distinguished from phorbol ester-induced PLD activity by differential radiolabelling of phospholipids, which are the substrates of PLD. Both v-Src- and phorbol ester-induced PLD activity could be detected when phospholipids were prelabelled with either radiolabelled myristate or palmitate; however, only phorbol ester-induced PLD activity could be detected when either arachidonate or 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (alkyl-lysoPC) was used to prelabel the phospholipids. The increased PLD activity in v-Src-transformed cells was not detected when the cells were prelabelled with either arachidonic acid or alkyl-lysoPC, which contains an ether linkage at sn-1 of the glycerol backbone. As both arachidonic acid and alkyl-lysoPC are incorporated into phosphatidylcholine (PC), the substrate for v-Src-induced PLD activity, these data suggest that the PLD activated by v-Src can distinguish PCs lacking arachidonic acid and ether linkages. Consistent with v-Src activating a PLD activity that is distinct from that activated by phorbol esters that activate PKC directly, neither depleting cells of PKC nor treatment with the protein kinase inhibitor, staurosporine, had any effect on v-Src-induced PLD activity, whereas both PKC depletion and staurosporine inhibited phorbol ester-induced PLD activity. Taken together, these data suggest that v-Src activates a PKC-independent PLD activity that is specific for a subpopulation of PC and distinct from the PLD activity induced by PKC activity induced by phorbol esters. The diacylglycerol produced from PC by the action of the v-Src-induced PLD may therefore be responsible for the activation of PKC by v-Src.