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The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi*

Open AccessPublished:March 01, 2013DOI:https://doi.org/10.1074/jbc.M112.426049
      Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn2+ ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn2+-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

      Introduction

      Phospholipases are important enzymes that bacteria utilize to modulate the host environment into one well suited for the requirements of the pathogen at specific stages of infection. Various types of phospholipases hydrolyze unique phospholipid ester bonds. Phospholipases A (PLA)
      The abbreviations used are: PLA–D
      phospholipases A–D
      LPLA
      lysophospholipase A
      DG
      diacylglycerol
      PG
      phosphatidylglycerol
      PLP
      patatin-like protein
      p-NPPC
      para-nitrophenylphosphorylcholine
      PC
      phosphatidylcholine
      BCYE
      buffered charcoal yeast extract
      BYE
      buffered yeast extract
      DPPC
      dipalmitoylphosphatidylcholine
      DPPG
      dipalmitoylphosphatidylglycerol
      DPPE
      dipalmitoylphosphatidylethanolamine
      DPPS
      dipalmitoylphosphatidylserine
      PI
      phosphatidylinositol
      SM
      sphingomyelin
      CL
      cardiolipin.
      and B (PLB) release fatty acids from the phospholipid molecule, thereby generating lysophospholipids or glycerophosphorylcholine. The phosphodiesterases, phospholipase C (PLC) and phospholipase D (PLD), hydrolyze phosphodiester bonds to generate either 1,2-diacylglycerol (1,2-DG) and a phosphoryl alcohol or phosphatidic acid and an alcohol, respectively (
      • Titball R.W.
      Bacterial phospholipases C.
      ,
      • Songer J.G.
      Bacterial phospholipases and their role in virulence.
      ,
      • Schmiel D.H.
      • Miller V.L.
      Bacterial phospholipases and pathogenesis.
      ).
      Bacteria can employ these phospholipases to accomplish manifold tasks, including the induction of host disintegration and bacterial exit or to fine-tune the host milieu (
      • Titball R.W.
      Bacterial phospholipases C.
      ,
      • Songer J.G.
      Bacterial phospholipases and their role in virulence.
      ,
      • Schmiel D.H.
      • Miller V.L.
      Bacterial phospholipases and pathogenesis.
      ,
      • Sato H.
      • Frank D.W.
      ExoU is a potent intracellular phospholipase.
      ,
      • Sitkiewicz I.
      • Stockbauer K.E.
      • Musser J.M.
      Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis.
      ). For example, Listeria monocytogenes can destroy its host membrane via the secreted PLCs PlcA and PlcB to egress from the phagosome and spread intercellularly (
      • Marquis H.
      • Doshi V.
      • Portnoy D.A.
      The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells.
      ,
      • Smith G.A.
      • Marquis H.
      • Jones S.
      • Johnston N.C.
      • Portnoy D.A.
      • Goldfine H.
      The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread.
      ,
      • Camilli A.
      • Tilney L.G.
      • Portnoy D.A.
      Dual roles of plcA in Listeria monocytogenes pathogenesis.
      ). Another example is the type III-secreted PLA ExoU, a patatin-like protein (PLP) found in Pseudomonas aeruginosa that can act as a cytotoxin or modulate host cell signaling on a minute to minute basis. ExoU in particular also has been shown to trigger an arachidonic acid-dependent inflammatory cascade in vivo, activate several transcription factors, and promote the production of proinflammatory cytokines (
      • Sitkiewicz I.
      • Stockbauer K.E.
      • Musser J.M.
      Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis.
      ,
      • Saliba A.M.
      • Nascimento D.O.
      • Silva M.C.
      • Assis M.C.
      • Gayer C.R.
      • Raymond B.
      • Coelho M.G.
      • Marques E.A.
      • Touqui L.
      • Albano R.M.
      • Lopes U.G.
      • Paiva D.D.
      • Bozza P.T.
      • Plotkowski M.C.
      Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU.
      ). In the case of PLCs, both Clostridium perfringens α-toxin and nonhemolytic Bacillus cereus PC-PLC can trigger the arachidonic acid cascade and stimulate thromboxane or prostaglandin production to result in inflammation (
      • Fujii Y.
      • Sakurai J.
      Contraction of the rat isolated aorta caused by Clostridium perfringens α toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism.
      ,
      • Levine L.
      • Xiao D.M.
      • Little C.
      Increased arachidonic acid metabolites from cells in culture after treatment with the phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus.
      ). Furthermore, the Pseudomonas aeruginosa hemolytic PLC analogously activates the lipoxygenase pathway that contributes to increased vascular permeability (
      • Meyers D.J.
      • Berk R.S.
      Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent.
      ). Given the fact that protein kinase C, which is activated by the PLC reaction product 1,2-DG, influences several processes including cell proliferation, it is plausible that several bacterial PLCs, such as B. cereus PLC and C. perfringens α-toxin, analogously can influence diverse cellular processes (
      • Diaz-Laviada I.
      • Larrodera P.
      • Diaz-Meco M.T.
      • Cornet M.E.
      • Guddal P.H.
      • Johansen T.
      • Moscat J.
      Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by rassrc oncogenes.
      ,
      • Parkinson E.K.
      Phospholipase C mimics the differential effects of phorbol-12-myristate-13-acetate on the colony formation and cornification of cultured normal and transformed human keratinocytes.
      ).
      Like the latter two, Legionella pneumophila, a bacterium known to cause life-threatening pneumonia, also possesses phospholipase activity. Most of its identified enzymes are PLAs that belong to three families consisting of a total of at least 15 proteins, including the GDSL enzymes (three members), the PLPs (depending upon the strain, 10–11 members), and PlaB (
      • Lang C.
      • Flieger A.
      Characterisation of Legionella pneumophila phospholipases and their impact on host cells.
      ,
      • Banerji S.
      • Aurass P.
      • Flieger A.
      The manifold phospholipases A of Legionella pneumophila: identification, export, regulation, and their link to bacterial virulence.
      ). L. pneumophila employs a variety of protein secretion mechanisms important to the virulence of the pathogen and a multitude of exported proteins, including the PLPs VipD, VpdA, VpdB, VpdC, which are secreted by the type IV Dot/Icm secretion system, and the GDSL proteins PlaA and PlaC, both secreted by the type II Lsp secretion system (
      • Hubber A.
      • Roy C.R.
      Modulation of host cell function by Legionella pneumophila type IV effectors.
      ,
      • Cianciotto N.P.
      Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila.
      ,
      • Isberg R.R.
      • O'Connor T.J.
      • Heidtman M.
      The Legionella pneumophila replication vacuole: making a cosy niche inside host cells.
      ,
      • Franco I.S.
      • Shuman H.A.
      • Charpentier X.
      The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors.
      ,
      • Rossier O.
      • Starkenburg S.R.
      • Cianciotto N.P.
      Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia.
      ,
      • Vogel J.P.
      • Andrews H.L.
      • Wong S.K.
      • Isberg R.R.
      Conjugative transfer by the virulence system of Legionella pneumophila.
      ,
      • Segal G.
      • Purcell M.
      • Shuman H.A.
      Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.
      ,
      • VanRheenen S.M.
      • Luo Z.Q.
      • O'Connor T.
      • Isberg R.R.
      Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells.
      ,
      • Shohdy N.
      • Efe J.A.
      • Emr S.D.
      • Shuman H.A.
      Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.
      ,
      • Flieger A.
      • Neumeister B.
      • Cianciotto N.P.
      Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine.
      ,
      • Banerji S.
      • Bewersdorff M.
      • Hermes B.
      • Cianciotto N.P.
      • Flieger A.
      Characterization of the major secreted zinc metalloprotease-dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila.
      ,
      • Hales L.M.
      • Shuman H.A.
      The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii.
      ,
      • Zhu W.
      • Banga S.
      • Tan Y.
      • Zheng C.
      • Stephenson R.
      • Gately J.
      • Luo Z.Q.
      Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.
      ,
      • DebRoy S.
      • Dao J.
      • Söderberg M.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.
      ).
      In addition to PLA enzymes, secreted hydrolytic activity acting upon the water-soluble phosphodiester substrate para-nitrophenylphosphorylcholine (p-NPPC) was found in L. pneumophila, which may indicate the presence of PLC activity (
      • Fliegera A.
      • Gong S.
      • Faigle M.
      • Neumeister B.
      Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C.
      ). Although this activity is presumably of PLC origin, conversion of a phospholipid substrate into typical cleavage products such as 1,2-DG or phosphoryl alcohol has not yet been proven (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Flieger A.
      • Gong S.
      • Faigle M.
      • Deeg M.
      • Bartmann P.
      • Neumeister B.
      Novel phospholipase A activity secreted by Legionella species.
      ,
      • Baine W.B.
      A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate.
      ,
      • Baine W.B.
      Cytolytic and phospholipase C activity in Legionella species.
      ). The signal peptide-containing protein PlcA, responsible for up to ∼70% of the secreted L. pneumophila p-NPPC-hydrolase activity, was identified due to its homology to the phosphatidylcholine (PC)-specific PLC PlcC of Pseudomonas fluorescens (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Rossier O.
      • Cianciotto N.P.
      The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
      ,
      • Rossignol G.
      • Merieau A.
      • Guerillon J.
      • Veron W.
      • Lesouhaitier O.
      • Feuilloley M.G.
      • Orange N.
      Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
      ,
      • Preuss I.
      • Kaiser I.
      • Gehring U.
      Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.
      ). Furthermore, an L. pneumophila Lsp type II secretion mutant is about 80–90% defective in secreted p-NPPC hydrolase activity, with export of p-NPPC hydrolase activity partially dependent (up to 30%) upon the Tat pathway (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Rossier O.
      • Cianciotto N.P.
      The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
      ). Because of the observed difference (70 versus 30%), it is likely that PlcA is also exported via the sec system (
      • Rossier O.
      • Cianciotto N.P.
      The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
      ). The virulence factor peptidylprolyl cis-trans-isomerase Mip promotes p-NPPC hydrolase activity as well, albeit independently of PlcA (
      • Debroy S.
      • Aragon V.
      • Kurtz S.
      • Cianciotto N.P.
      Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants.
      ). These data suggest that additional Lsp- and/or Mip-dependent enzymes contributing to p-NPPC hydrolysis exist in L. pneumophila. A protein homologous to PlcA was recently identified and designated PlcB (
      • McCoy-Simandle K.
      • Stewart C.R.
      • Dao J.
      • DebRoy S.
      • Rossier O.
      • Bryce P.J.
      • Cianciotto N.P.
      Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia.
      ). Whether PlcB, which contains a putative signal peptide akin to PlcA, also contributes to secreted p-NPPC hydrolase activity is currently unknown. L. pneumophila plcA and plcB single and double knock-out mutants replicate intracellularly in a manner comparable with that of wild type bacteria in macrophage, epithelial cell, and/or amoeba infection models, indicating that these enzymes are not necessary for these in vitro virulence characteristics (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • McCoy-Simandle K.
      • Stewart C.R.
      • Dao J.
      • DebRoy S.
      • Rossier O.
      • Bryce P.J.
      • Cianciotto N.P.
      Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia.
      ). Moreover, L. pneumophila plcA mutants display no attenuation compared with wild type bacteria in in vivo mouse infections (
      • DebRoy S.
      • Dao J.
      • Söderberg M.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.
      ).
      To summarize, it is still unclear whether L. pneumophila possesses one or several secreted PLC activities that target phospholipids. We therefore screened the L. pneumophila genome sequence for potentially encoded PLC enzymes and found, in addition to PlcA and PlcB described previously (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • McCoy-Simandle K.
      • Stewart C.R.
      • Dao J.
      • DebRoy S.
      • Rossier O.
      • Bryce P.J.
      • Cianciotto N.P.
      Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia.
      ), one further homologous protein that we designated PlcC. In earlier studies, PlcC was found in another context and described as a cytotoxic type IVB-secreted L. pneumophila effector protein when expressed in yeast, and it was named CegC1 (
      • Zhu W.
      • Banga S.
      • Tan Y.
      • Zheng C.
      • Stephenson R.
      • Gately J.
      • Luo Z.Q.
      Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.
      ,
      • Altman E.
      • Segal G.
      The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates.
      ,
      • Heidtman M.
      • Chen E.J.
      • Moy M.Y.
      • Isberg R.R.
      Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways.
      ,
      • Huang L.
      • Boyd D.
      • Amyot W.M.
      • Hempstead A.D.
      • Luo Z.Q.
      • O'Connor T.J.
      • Chen C.
      • Machner M.
      • Montminy T.
      • Isberg R.R.
      The E Block motif is associated with Legionella pneumophila translocated substrates.
      ). These three L. pneumophila genes were interesting candidates for further analysis because all of them are transcriptionally induced during host cell infection (
      • Brüggemann H.
      • Hagman A.
      • Jules M.
      • Sismeiro O.
      • Dillies M.A.
      • Gouyette C.
      • Kunst F.
      • Steinert M.
      • Heuner K.
      • Coppée J.Y.
      • Buchrieser C.
      Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila.
      ,
      • Weissenmayer B.A.
      • Prendergast J.G.
      • Lohan A.J.
      • Loftus B.J.
      Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.
      ,
      • Faucher S.P.
      • Mueller C.A.
      • Shuman H.A.
      Legionella Pneumophila transcriptome during intracellular multiplication in human macrophages.
      ). Here we describe our observation that all three proteins exhibit PLC activity and demonstrate the importance of zinc ions for activity. Conserved amino acid residues in all of the L. pneumophila enzymes and PlcC of P. fluorescens were targeted for mutagenesis, with the finding that 15 residues are essential for PLC activity of PlcC. These residues also were completely conserved in other homologs of fungal proteins that have not yet been characterized. This set of properties, namely (a) lack of significant homology to enzymes of other PLC families, (b) considerable homology between the respective enzymes and conservation of specific essential amino acids embedded within conserved protein motifs, and (c) the necessity of Zn2+ for enzymatic activity, distinguishes these enzymes from other defined PLC groups. Therefore, we propose that the enzymes belong to a novel PLC family present in Legionella, some Pseudomonas spp., and fungi.

      DISCUSSION

      Here, we present evidence that L. pneumophila does indeed possess three PLC enzymes that release the signaling molecule 1,2-DG from phospholipids. Earlier descriptions of L. pneumophila PLC activity had left open the possibility that other enzymes, in addition to PLC, may cause the observed release of a water-soluble, tritium-labeled reaction product from phosphatidylcholine, such as PLD (release of choline) or PLA/LPLA (release of glycerophosphorylcholine) (
      • Baine W.B.
      A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate.
      ,
      • Baine W.B.
      Cytolytic and phospholipase C activity in Legionella species.
      ). Indeed, the PLA/LPLA reaction product, glycerophosphorylcholine (but not phosphorylcholine) was found when phosphatidylcholine was incubated with L. pneumophila culture supernatants and may be responsible for some of the previous PLC descriptions (
      • Flieger A.
      • Gong S.
      • Faigle M.
      • Deeg M.
      • Bartmann P.
      • Neumeister B.
      Novel phospholipase A activity secreted by Legionella species.
      ). On the other hand, Aragon et al. (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ) were unable to assay for PLC by monitoring 1,2-DG release due to the presence of multiple PLA, LPLA, and lipases potentially deacylating the PLC reaction product. We also hypothesized that the prominently secreted and cell-associated L. pneumophila PLA/LPLA prevented the detection of PLC reaction products. This is consistent with the observation that cell-associated PLA/LPLA PlaB knock-out mutants, when assayed for cell-associated phospholipase activity, released not only the PLA reaction products lysophosphatidylcholine and free fatty acids but also 1,2-DG. 1,2-DG was not detected, however, in wild type incubations (
      • Flieger A.
      • Rydzewski K.
      • Banerji S.
      • Broich M.
      • Heuner K.
      Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity.
      ).
      S. Banerji and A. Flieger, unpublished observation.
      We attribute the detection of 1,2-DG in our study in part to the addition of Zn2+, which not only boosted L. pneumophila PLC activity but also seemed to inhibit bacterial PLA/LPLA activities.
      P. Aurass, M. Schlegel, and A. Flieger, unpublished observation.
      That Zn2+ may inhibit PLA activity has already been described for several snake venom PLA2 (
      • Mezna M.
      • Ahmad T.
      • Chettibi S.
      • Drainas D.
      • Lawrence A.J.
      Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms.
      ,
      • Wells M.A.
      Spectral perturbations of Crotalus adamanteus phospholipase A 2 induced by divalent cation binding.
      ). Moreover, the specific reaction conditions (e.g. choosing appropriate dilutions and reaction lengths) and the use of DPPG as a substrate further optimized PLC activity, thereby allowing detectable 1,2-DG development.
      The L. pneumophila PLC enzymes described here display significant protein homology to enzymes in other Legionella species and P. fluorescens (FIGURE 2, FIGURE 3, FIGURE 4 and Table 1) (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Rossignol G.
      • Merieau A.
      • Guerillon J.
      • Veron W.
      • Lesouhaitier O.
      • Feuilloley M.G.
      • Orange N.
      Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
      ,
      • McCoy-Simandle K.
      • Stewart C.R.
      • Dao J.
      • DebRoy S.
      • Rossier O.
      • Bryce P.J.
      • Cianciotto N.P.
      Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia.
      ). Further bacterial protein homologs in addition to those found in Legionella spp. and some Pseudomonas spp. (no homolog in P. aeruginosa) were not found but were present, interestingly, in fungi, including G. zeae, C. militaris, and T. rubrum. None of these fungal proteins have been characterized yet, although they may prove to be interesting candidates for virulence factor analyses because of their potential PLC activity. The related bacterial and fungal PLC-like enzymes do not share significant homology to the well characterized family of Zn2+-dependent (broad spectrum) PC-preferring PLC (PC-PLC) enzymes of several Gram-positive bacteria, such as B. cereus, L. monocytogenes, and C. perfringens (Fig. 4) (
      • Titball R.W.
      Bacterial phospholipases C.
      ,
      • Rossignol G.
      • Merieau A.
      • Guerillon J.
      • Veron W.
      • Lesouhaitier O.
      • Feuilloley M.G.
      • Orange N.
      Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
      ,
      • Geoffroy C.
      • Raveneau J.
      • Beretti J.L.
      • Lecroisey A.
      • Vazquez-Boland J.A.
      • Alouf J.E.
      • Berche P.
      Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.
      ,
      • Hough E.
      • Hansen L.K.
      • Birknes B.
      • Jynge K.
      • Hansen S.
      • Hordvik A.
      • Little C.
      • Dodson E.
      • Derewenda Z.
      High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus.
      ,
      • Krug E.L.
      • Kent C.
      Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme.
      ,
      • Raynaud C.
      • Guilhot C.
      • Rauzier J.
      • Bordat Y.
      • Pelicic V.
      • Manganelli R.
      • Smith I.
      • Gicquel B.
      • Jackson M.
      Phospholipases C are involved in the virulence of Mycobacterium tuberculosis.
      ). One important exception, however, is a short stretch of essential amino acids harboring the signature F(A/T)XH(Y/F)(Y/L)XDXF(A/S)XGH, where the histidines and the aspartate of the motif are also conserved in those Gram-positive bacterial PLCs (Fig. 4). This specific region includes residues involved in co-factor binding (
      • Titball R.W.
      Bacterial phospholipases C.
      ,
      • Geoffroy C.
      • Raveneau J.
      • Beretti J.L.
      • Lecroisey A.
      • Vazquez-Boland J.A.
      • Alouf J.E.
      • Berche P.
      Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.
      ,
      • Hough E.
      • Hansen L.K.
      • Birknes B.
      • Jynge K.
      • Hansen S.
      • Hordvik A.
      • Little C.
      • Dodson E.
      • Derewenda Z.
      High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus.
      ,
      • Naylor C.E.
      • Eaton J.T.
      • Howells A.
      • Justin N.
      • Moss D.S.
      • Titball R.W.
      • Basak A.K.
      Structure of the key toxin in gas gangrene.
      ,
      • Hansen S.
      • Hansen L.K.
      • Hough E.
      The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution. The nature of the metal ion in site 2.
      ,
      • Vazquez-Boland J.A.
      • Kocks C.
      • Dramsi S.
      • Ohayon H.
      • Geoffroy C.
      • Mengaud J.
      • Cossart P.
      Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread.
      ). Furthermore, the PLCs described here possess no significant protein homology to the acidic phosphatase/PLC family of several Gram-negative bacteria that do not require additional metal ions for activity and that can effectively hydrolyze p-NPPC (e.g. P. aeruginosa PlcH and PlcN, Francisella tularensis AcpA, M. tuberculosis PlcA, PlcB, PlcC, and PlcD, and P. fluorescens CGDEase) (
      • Titball R.W.
      Bacterial phospholipases C.
      ,
      • Rossignol G.
      • Merieau A.
      • Guerillon J.
      • Veron W.
      • Lesouhaitier O.
      • Feuilloley M.G.
      • Orange N.
      Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
      ,
      • Costas M.J.
      • Pinto R.M.
      • Cordero P.M.
      • Cabezas A.
      • Alves-Pereira I.
      • Cameselle J.C.
      • Ribeiro J.M.
      CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions.
      ,
      • Stonehouse M.J.
      • Cota-Gomez A.
      • Parker S.K.
      • Martin W.E.
      • Hankin J.A.
      • Murphy R.C.
      • Chen W.
      • Lim K.B.
      • Hackett M.
      • Vasil A.I.
      • Vasil M.L.
      A novel class of microbial phosphocholine-specific phospholipases C.
      ,
      • Felts R.L.
      • Reilly T.J.
      • Tanner J.J.
      Structure of Francisella tularensis AcpA. Prototype of a unique superfamily of acid phosphatases and phospholipases C.
      ,
      • Reilly T.J.
      • Baron G.S.
      • Nano F.E.
      • Kuhlenschmidt M.S.
      Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.
      ). The lack of protein homology therefore supports the notion that these enzymes may belong to a novel family of PLC enzymes, as already suggested by Preuss et al. and Rossignol et al. (
      • Rossignol G.
      • Merieau A.
      • Guerillon J.
      • Veron W.
      • Lesouhaitier O.
      • Feuilloley M.G.
      • Orange N.
      Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
      ,
      • Preuss I.
      • Kaiser I.
      • Gehring U.
      Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.
      ). This new family of PLCs possesses the following distinguishing characteristics: (a) it includes proteins from Gram-negative bacteria as well as fungi, (b) its members share defined blocks of amino acid homology, and (c) its members seem to require Zn2+.
      Export into the bacterial supernatant, surface presentation, or injection into a eukaryotic cell is a common feature of host-targeting phospholipases. Clearly, bacteria utilize a variety of enzymes to cleave phospholipids, and the transport systems used are likewise multifaceted. For example, the pathogen P. aeruginosa employs different modes of phospholipase application, such as Sec- or Tat-dependent and subsequent Xcp type II-dependent secretion of PlcB, hemolytic PlcH, and non-hemolytic PlcN; the type III-dependent injection of the PLA cytotoxin ExoU (
      • Phillips R.M.
      • Six D.A.
      • Dennis E.A.
      • Ghosh P.
      In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors.
      ,
      • Sato H.
      • Frank D.W.
      • Hillard C.J.
      • Feix J.B.
      • Pankhaniya R.R.
      • Moriyama K.
      • Finck-Barbançon V.
      • Buchaklian A.
      • Lei M.
      • Long R.M.
      • Wiener-Kronish J.
      • Sawa T.
      The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.
      ,
      • Finck-Barbançon V.
      • Goranson J.
      • Zhu L.
      • Sawa T.
      • Wiener-Kronish J.P.
      • Fleiszig S.M.
      • Wu C.
      • Mende-Mueller L.
      • Frank D.W.
      ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.
      ,
      • Hauser A.R.
      • Kang P.J.
      • Engel J.N.
      PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.
      ); or type V autotransport for a second patatin-like PLA PlpD (
      • Finck-Barbançon V.
      • Goranson J.
      • Zhu L.
      • Sawa T.
      • Wiener-Kronish J.P.
      • Fleiszig S.M.
      • Wu C.
      • Mende-Mueller L.
      • Frank D.W.
      ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.
      ,
      • Hauser A.R.
      • Kang P.J.
      • Engel J.N.
      PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.
      ,
      • Voulhoux R.
      • Ball G.
      • Ize B.
      • Vasil M.L.
      • Lazdunski A.
      • Wu L.F.
      • Filloux A.
      Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.
      ,
      • Ochsner U.A.
      • Snyder A.
      • Vasil A.I.
      • Vasil M.L.
      Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis.
      ,
      • Barker A.P.
      • Vasil A.I.
      • Filloux A.
      • Ball G.
      • Wilderman P.J.
      • Vasil M.L.
      A novel extracellular phospholipase C of Pseudomonas aeruginosa is required for phospholipid chemotaxis.
      ,
      • Salacha R.
      • Kovaci F.
      • Brochier-Armanet C.
      • Wilhelm S.
      • Tommassen J.
      • Filloux A.
      • Voulhoux R.
      • Bleves S.
      The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.
      ). This suggests that each specific function correlates with a corresponding unique mode of enzyme transport, which seems to be important in the case of Legionella. L. pneumophila PlcA and PlcB harbor predicted signal peptides and therefore are candidates for type II secretion after inner membrane crossing via Sec- or Tat-dependent processes (Table 2) (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Rossier O.
      • Cianciotto N.P.
      The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
      ). It has been shown previously that PlcA secretion contributes about 50–70% to secreted p-NPPC hydrolase activity, whereas Tat- and Lsp-dependent secretion contribute 30 and 80–90%, respectively, to secreted p-NPPC hydrolase activity (
      • Aragon V.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila genes that encode lipase and phospholipase C activities.
      ,
      • Rossier O.
      • Cianciotto N.P.
      The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
      ). Type II-secreted enzymes such as zinc metalloproteinase ProA have been found in the Legionella phagosome (
      • DebRoy S.
      • Dao J.
      • Söderberg M.
      • Rossier O.
      • Cianciotto N.P.
      Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.
      ,
      • Rechnitzer C.
      • Williams A.
      • Wright J.B.
      • Dowsett A.B.
      • Milman N.
      • Fitzgeorge R.B.
      Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea pig and human alveolar macrophages.
      ,
      • Liles M.R.
      • Edelstein P.H.
      • Cianciotto N.P.
      The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.
      ,
      • Aragon V.
      • Kurtz S.
      • Flieger A.
      • Neumeister B.
      • Cianciotto N.P.
      Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.
      ,
      • Rossier O.
      • Cianciotto N.P.
      Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila.
      ), so it is conceivable that PlcA and PlcB also may have a function in lipid hydrolysis within the phagosome. This may be important for phagosome remodeling, for release of signal transducers allowing intracellular replication, or even for the destruction of the membrane inclusion upon commencement of bacterial replication. It also may allow bacteria to manipulate host-signaling pathways for the purpose of directly injecting a PLC enzyme into the host cell cytosol, such as type IVB-secreted PlcC/CegC1. There are examples of bacterial phospholipases that are directly injected into the host cell, but this has been described only for PLA thus far, such as P. aeruginosa ExoU, which is injected into the host cell via type III secretion (
      • Finck-Barbançon V.
      • Goranson J.
      • Zhu L.
      • Sawa T.
      • Wiener-Kronish J.P.
      • Fleiszig S.M.
      • Wu C.
      • Mende-Mueller L.
      • Frank D.W.
      ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.
      ,
      • Hauser A.R.
      • Kang P.J.
      • Engel J.N.
      PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.
      ,
      • Finck-Barbançon V.
      • Yahr T.L.
      • Frank D.W.
      Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin.
      ). Now that the PLC activity of the type IVB-secreted effector PlcC/CegC1 and the importance in virulence of the three PLC have been established, a variety of mechanisms of host cell modulation via the known effects of PKC or arachidonic acid cascade activation become possible (
      • Fujii Y.
      • Sakurai J.
      Contraction of the rat isolated aorta caused by Clostridium perfringens α toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism.
      ,
      • Levine L.
      • Xiao D.M.
      • Little C.
      Increased arachidonic acid metabolites from cells in culture after treatment with the phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus.
      ,
      • Meyers D.J.
      • Berk R.S.
      Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent.
      ,
      • Diaz-Laviada I.
      • Larrodera P.
      • Diaz-Meco M.T.
      • Cornet M.E.
      • Guddal P.H.
      • Johansen T.
      • Moscat J.
      Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by rassrc oncogenes.
      ,
      • Parkinson E.K.
      Phospholipase C mimics the differential effects of phorbol-12-myristate-13-acetate on the colony formation and cornification of cultured normal and transformed human keratinocytes.
      ,
      • de Felipe K.S.
      • Glover R.T.
      • Charpentier X.
      • Anderson O.R.
      • Reyes M.
      • Pericone C.D.
      • Shuman H.A.
      Legionella eukaryotic-like type IV substrates interfere with organelle trafficking.
      ). However, although all three PLC enzymes have been found up-regulated during host cell infection (
      • Brüggemann H.
      • Hagman A.
      • Jules M.
      • Sismeiro O.
      • Dillies M.A.
      • Gouyette C.
      • Kunst F.
      • Steinert M.
      • Heuner K.
      • Coppée J.Y.
      • Buchrieser C.
      Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila.
      ,
      • Weissenmayer B.A.
      • Prendergast J.G.
      • Lohan A.J.
      • Loftus B.J.
      Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.
      ,
      • Faucher S.P.
      • Mueller C.A.
      • Shuman H.A.
      Legionella Pneumophila transcriptome during intracellular multiplication in human macrophages.
      ), we did not observe an essential impact of the three PLC on infection and intracellular replication using macrophage and amoeba infection models. It remains to be elucidated in the future whether they play a role in in vivo infection models in addition to G. mellonella, as has been described for the four PLC of M. tuberculosis (
      • Raynaud C.
      • Guilhot C.
      • Rauzier J.
      • Bordat Y.
      • Pelicic V.
      • Manganelli R.
      • Smith I.
      • Gicquel B.
      • Jackson M.
      Phospholipases C are involved in the virulence of Mycobacterium tuberculosis.
      ).
      Why bacteria such as P. aeruginosa or M. tuberculosis express a multitude of PLC enzymes remains an unanswered question. Our work adds L. pneumophila, with its three PLC, to this list of PLC-expressing bacteria. Interestingly, these three PLC homologs were conserved in all L. pneumophila genomes, although only the type IVB-secreted PlcC was conserved in all other (currently accessible) Legionella genomes (Table 1). L. pneumophila therefore seems to harbor a variety of these enzymes, which conceivably could have related functions, although differences in secretion type and substrate preferences suggest distinct functions. This raises the question of whether type IVB effectors are more versatile than type II effectors in allowing L. pneumophila to adapt to diverse hosts and environments. The answer to this latter question may begin to shed light upon the means by which L. pneumophila ultimately causes the clinical manifestations of Legionnaires' disease.

      Acknowledgments

      We thank Susanne Karste and Simone Dumschat for excellent technical assistance.

      REFERENCES

        • Titball R.W.
        Bacterial phospholipases C.
        Microbiol. Mol. Biol. Rev. 1993; 57: 347-366
        • Songer J.G.
        Bacterial phospholipases and their role in virulence.
        Trends Microbiol. 1997; 5: 156-161
        • Schmiel D.H.
        • Miller V.L.
        Bacterial phospholipases and pathogenesis.
        Microbes Infect. 1999; 1: 1103-1112
        • Sato H.
        • Frank D.W.
        ExoU is a potent intracellular phospholipase.
        Mol. Microbiol. 2004; 53: 1279-1290
        • Sitkiewicz I.
        • Stockbauer K.E.
        • Musser J.M.
        Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis.
        Trends Microbiol. 2007; 15: 63-69
        • Marquis H.
        • Doshi V.
        • Portnoy D.A.
        The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells.
        Infect. Immun. 1995; 63: 4531-4534
        • Smith G.A.
        • Marquis H.
        • Jones S.
        • Johnston N.C.
        • Portnoy D.A.
        • Goldfine H.
        The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread.
        Infect. Immun. 1995; 63: 4231-4237
        • Camilli A.
        • Tilney L.G.
        • Portnoy D.A.
        Dual roles of plcA in Listeria monocytogenes pathogenesis.
        Mol. Microbiol. 1993; 8: 143-157
        • Saliba A.M.
        • Nascimento D.O.
        • Silva M.C.
        • Assis M.C.
        • Gayer C.R.
        • Raymond B.
        • Coelho M.G.
        • Marques E.A.
        • Touqui L.
        • Albano R.M.
        • Lopes U.G.
        • Paiva D.D.
        • Bozza P.T.
        • Plotkowski M.C.
        Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU.
        Cell. Microbiol. 2005; 7: 1811-1822
        • Fujii Y.
        • Sakurai J.
        Contraction of the rat isolated aorta caused by Clostridium perfringens α toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism.
        Br. J. Pharmacol. 1989; 97: 119-124
        • Levine L.
        • Xiao D.M.
        • Little C.
        Increased arachidonic acid metabolites from cells in culture after treatment with the phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus.
        Prostaglandins. 1987; 34: 633-642
        • Meyers D.J.
        • Berk R.S.
        Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent.
        Infect. Immun. 1990; 58: 659-666
        • Diaz-Laviada I.
        • Larrodera P.
        • Diaz-Meco M.T.
        • Cornet M.E.
        • Guddal P.H.
        • Johansen T.
        • Moscat J.
        Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by rassrc oncogenes.
        EMBO J. 1990; 9: 3907-3912
        • Parkinson E.K.
        Phospholipase C mimics the differential effects of phorbol-12-myristate-13-acetate on the colony formation and cornification of cultured normal and transformed human keratinocytes.
        Carcinogenesis. 1987; 8: 857-860
        • Lang C.
        • Flieger A.
        Characterisation of Legionella pneumophila phospholipases and their impact on host cells.
        Eur. J. Cell Biol. 2011; 90: 903-912
        • Banerji S.
        • Aurass P.
        • Flieger A.
        The manifold phospholipases A of Legionella pneumophila: identification, export, regulation, and their link to bacterial virulence.
        Int. J. Med. Microbiol. 2008; 298: 169-181
        • Hubber A.
        • Roy C.R.
        Modulation of host cell function by Legionella pneumophila type IV effectors.
        Annu. Rev. Cell Dev. Biol. 2010; 26: 261-283
        • Cianciotto N.P.
        Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila.
        Future Microbiol. 2009; 4: 797-805
        • Isberg R.R.
        • O'Connor T.J.
        • Heidtman M.
        The Legionella pneumophila replication vacuole: making a cosy niche inside host cells.
        Nat. Rev. Microbiol. 2009; 7: 13-24
        • Franco I.S.
        • Shuman H.A.
        • Charpentier X.
        The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors.
        Cell. Microbiol. 2009; 11: 1435-1443
        • Rossier O.
        • Starkenburg S.R.
        • Cianciotto N.P.
        Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia.
        Infect. Immun. 2004; 72: 310-321
        • Vogel J.P.
        • Andrews H.L.
        • Wong S.K.
        • Isberg R.R.
        Conjugative transfer by the virulence system of Legionella pneumophila.
        Science. 1998; 279: 873-876
        • Segal G.
        • Purcell M.
        • Shuman H.A.
        Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1669-1674
        • VanRheenen S.M.
        • Luo Z.Q.
        • O'Connor T.
        • Isberg R.R.
        Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells.
        Infect. Immun. 2006; 74: 3597-3606
        • Shohdy N.
        • Efe J.A.
        • Emr S.D.
        • Shuman H.A.
        Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4866-4871
        • Flieger A.
        • Neumeister B.
        • Cianciotto N.P.
        Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine.
        Infect. Immun. 2002; 70: 6094-6106
        • Banerji S.
        • Bewersdorff M.
        • Hermes B.
        • Cianciotto N.P.
        • Flieger A.
        Characterization of the major secreted zinc metalloprotease-dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila.
        Infect. Immun. 2005; 73: 2899-2909
        • Hales L.M.
        • Shuman H.A.
        The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii.
        J. Bacteriol. 1999; 181: 4879-4889
        • Zhu W.
        • Banga S.
        • Tan Y.
        • Zheng C.
        • Stephenson R.
        • Gately J.
        • Luo Z.Q.
        Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.
        PloS One. 2011; 6: e17638
        • DebRoy S.
        • Dao J.
        • Söderberg M.
        • Rossier O.
        • Cianciotto N.P.
        Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19146-19151
        • Fliegera A.
        • Gong S.
        • Faigle M.
        • Neumeister B.
        Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C.
        Enzyme Microb. Technol. 2000; 26: 451-458
        • Aragon V.
        • Rossier O.
        • Cianciotto N.P.
        Legionella pneumophila genes that encode lipase and phospholipase C activities.
        Microbiology. 2002; 148: 2223-2231
        • Flieger A.
        • Gong S.
        • Faigle M.
        • Deeg M.
        • Bartmann P.
        • Neumeister B.
        Novel phospholipase A activity secreted by Legionella species.
        J. Bacteriol. 2000; 182: 1321-1327
        • Baine W.B.
        A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate.
        J. Gen. Microbiol. 1988; 134: 489-498
        • Baine W.B.
        Cytolytic and phospholipase C activity in Legionella species.
        J. Gen. Microbiol. 1985; 131: 1383-1391
        • Rossier O.
        • Cianciotto N.P.
        The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.
        Infect. Immun. 2005; 73: 2020-2032
        • Rossignol G.
        • Merieau A.
        • Guerillon J.
        • Veron W.
        • Lesouhaitier O.
        • Feuilloley M.G.
        • Orange N.
        Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.
        BMC Microbiol. 2008; 8: 189
        • Preuss I.
        • Kaiser I.
        • Gehring U.
        Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.
        Eur. J. Biochem. 2001; 268: 5081-5091
        • Debroy S.
        • Aragon V.
        • Kurtz S.
        • Cianciotto N.P.
        Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants.
        Infect. Immun. 2006; 74: 5152-5160
        • McCoy-Simandle K.
        • Stewart C.R.
        • Dao J.
        • DebRoy S.
        • Rossier O.
        • Bryce P.J.
        • Cianciotto N.P.
        Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia.
        Infect. Immun. 2011; 79: 1984-1997
        • Altman E.
        • Segal G.
        The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates.
        J. Bacteriol. 2008; 190: 1985-1996
        • Heidtman M.
        • Chen E.J.
        • Moy M.Y.
        • Isberg R.R.
        Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways.
        Cell. Microbiol. 2009; 11: 230-248
        • Huang L.
        • Boyd D.
        • Amyot W.M.
        • Hempstead A.D.
        • Luo Z.Q.
        • O'Connor T.J.
        • Chen C.
        • Machner M.
        • Montminy T.
        • Isberg R.R.
        The E Block motif is associated with Legionella pneumophila translocated substrates.
        Cell. Microbiol. 2011; 13: 227-245
        • Brüggemann H.
        • Hagman A.
        • Jules M.
        • Sismeiro O.
        • Dillies M.A.
        • Gouyette C.
        • Kunst F.
        • Steinert M.
        • Heuner K.
        • Coppée J.Y.
        • Buchrieser C.
        Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila.
        Cell. Microbiol. 2006; 8: 1228-1240
        • Weissenmayer B.A.
        • Prendergast J.G.
        • Lohan A.J.
        • Loftus B.J.
        Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.
        PloS One. 2011; 6: e17570
        • Faucher S.P.
        • Mueller C.A.
        • Shuman H.A.
        Legionella Pneumophila transcriptome during intracellular multiplication in human macrophages.
        Front. Microbiol. 2011; 2: 60
        • Sadosky A.B.
        • Wiater L.A.
        • Shuman H.A.
        Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.
        Infect. Immun. 1993; 61: 5361-5373
        • Jepras R.I.
        • Fitzgeorge R.B.
        • Baskerville A.
        A comparison of virulence of two strains of Legionella pneumophila based on experimental aerosol infection of guinea pigs.
        J. Hyg. 1985; 95: 29-38
        • Aurass P.
        • Pless B.
        • Rydzewski K.
        • Holland G.
        • Bannert N.
        • Flieger A.
        bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.
        Appl. Environ. Microbiol. 2009; 75: 4506-4515
        • Edelstein P.H.
        Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens.
        J. Clin. Microbiol. 1981; 14: 298-303
        • Wiater L.A.
        • Sadosky A.B.
        • Shuman H.A.
        Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth phase-regulated pigmentation gene.
        Mol. Microbiol. 1994; 11: 641-653
        • Merzbacher M.
        • Detsch C.
        • Hillen W.
        • Stülke J.
        Mycoplasma pneumoniae HPr kinase/phosphorylase.
        Eur. J. Biochem. 2004; 271: 367-374
        • Bligh E.G.
        • Dyer W.J.
        A rapid method of total lipid extraction and purification.
        Can. J. Biochem. Physiol. 1959; 37: 911-917
        • Plekhanov A.Y.
        Rapid staining of lipids on thin-layer chromatograms with amido black 10B and other water-soluble stains.
        Anal. Biochem. 1999; 271: 186-187
        • Broich M.
        • Rydzewski K.
        • McNealy T.L.
        • Marre R.
        • Flieger A.
        The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32.
        J. Bacteriol. 2006; 188: 1218-1226
        • Moffat J.F.
        • Tompkins L.S.
        A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii.
        Infect. Immun. 1992; 60: 296-301
        • Cianciotto N.P.
        • Fields B.S.
        Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages.
        Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 5188-5191
        • Harding C.R.
        • Schroeder G.N.
        • Reynolds S.
        • Kosta A.
        • Collins J.W.
        • Mousnier A.
        • Frankel G.
        Legionella pneumophila pathogenesis in the Galleria mellonella infection model.
        Infect. Immun. 2012; 80: 2780-2790
        • Benfield A.P.
        • Goodey N.M.
        • Phillips L.T.
        • Martin S.F.
        Structural studies examining the substrate specificity profiles of PC-PLC(Bc) protein variants.
        Arch. Biochem. Biophys. 2007; 460: 41-47
        • Sakurai J.
        • Nagahama M.
        • Oda M.
        Clostridium perfringens α-toxin: characterization and mode of action.
        J. Biochem. 2004; 136: 569-574
        • Geoffroy C.
        • Raveneau J.
        • Beretti J.L.
        • Lecroisey A.
        • Vazquez-Boland J.A.
        • Alouf J.E.
        • Berche P.
        Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.
        Infect. Immun. 1991; 59: 2382-2388
        • Hough E.
        • Hansen L.K.
        • Birknes B.
        • Jynge K.
        • Hansen S.
        • Hordvik A.
        • Little C.
        • Dodson E.
        • Derewenda Z.
        High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus.
        Nature. 1989; 338: 357-360
        • Krug E.L.
        • Kent C.
        Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme.
        Arch. Biochem. Biophys. 1984; 231: 400-410
        • Zavaleta-Pastor M.
        • Sohlenkamp C.
        • Gao J.L.
        • Guan Z.
        • Zaheer R.
        • Finan T.M.
        • Raetz C.R.
        • López-Lara I.M.
        • Geiger O.
        Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 302-307
        • Srinivas M.
        • Rajakumari S.
        • Narayana Y.
        • Joshi B.
        • Katoch V.M.
        • Rajasekharan R.
        • Balaji K.N.
        Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis.
        J. Biosci. 2008; 33: 221-230
        • Lang C.
        • Rastew E.
        • Hermes B.
        • Siegbrecht E.
        • Ahrends R.
        • Banerji S.
        • Flieger A.
        Zinc metalloproteinase ProA directly activates Legionella pneumophila PlaC glycerophospholipid:cholesterol acyltransferase.
        J. Biol. Chem. 2012; 287: 23464-23478
        • Moser J.
        • Gerstel B.
        • Meyer J.E.
        • Chakraborty T.
        • Wehland J.
        • Heinz D.W.
        Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes.
        J. Mol. Biol. 1997; 273: 269-282
        • Kubiak R.J.
        • Yue X.
        • Hondal R.J.
        • Mihai C.
        • Tsai M.D.
        • Bruzik K.S.
        Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond.
        Biochemistry. 2001; 40: 5422-5432
        • Naylor C.E.
        • Eaton J.T.
        • Howells A.
        • Justin N.
        • Moss D.S.
        • Titball R.W.
        • Basak A.K.
        Structure of the key toxin in gas gangrene.
        Nat. Struct. Biol. 1998; 5: 738-746
        • Hansen S.
        • Hansen L.K.
        • Hough E.
        The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution. The nature of the metal ion in site 2.
        J. Mol. Biol. 1993; 231: 870-876
        • Vazquez-Boland J.A.
        • Kocks C.
        • Dramsi S.
        • Ohayon H.
        • Geoffroy C.
        • Mengaud J.
        • Cossart P.
        Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread.
        Infect. Immun. 1992; 60: 219-230
        • Kirby J.E.
        • Vogel J.P.
        • Andrews H.L.
        • Isberg R.R.
        Evidence for pore-forming ability by Legionella pneumophila.
        Mol. Microbiol. 1998; 27: 323-336
        • Roy C.R.
        • Berger K.H.
        • Isberg R.R.
        Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake.
        Mol. Microbiol. 1998; 28: 663-674
        • Flieger A.
        • Rydzewski K.
        • Banerji S.
        • Broich M.
        • Heuner K.
        Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity.
        Infect. Immun. 2004; 72: 2648-2658
        • Mezna M.
        • Ahmad T.
        • Chettibi S.
        • Drainas D.
        • Lawrence A.J.
        Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms.
        Biochem. J. 1994; 301: 503-508
        • Wells M.A.
        Spectral perturbations of Crotalus adamanteus phospholipase A 2 induced by divalent cation binding.
        Biochemistry. 1973; 12: 1080-1085
        • Raynaud C.
        • Guilhot C.
        • Rauzier J.
        • Bordat Y.
        • Pelicic V.
        • Manganelli R.
        • Smith I.
        • Gicquel B.
        • Jackson M.
        Phospholipases C are involved in the virulence of Mycobacterium tuberculosis.
        Mol. Microbiol. 2002; 45: 203-217
        • Costas M.J.
        • Pinto R.M.
        • Cordero P.M.
        • Cabezas A.
        • Alves-Pereira I.
        • Cameselle J.C.
        • Ribeiro J.M.
        CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions.
        Mol. Microbiol. 2010; 78: 1556-1576
        • Stonehouse M.J.
        • Cota-Gomez A.
        • Parker S.K.
        • Martin W.E.
        • Hankin J.A.
        • Murphy R.C.
        • Chen W.
        • Lim K.B.
        • Hackett M.
        • Vasil A.I.
        • Vasil M.L.
        A novel class of microbial phosphocholine-specific phospholipases C.
        Mol. Microbiol. 2002; 46: 661-676
        • Felts R.L.
        • Reilly T.J.
        • Tanner J.J.
        Structure of Francisella tularensis AcpA. Prototype of a unique superfamily of acid phosphatases and phospholipases C.
        J. Biol. Chem. 2006; 281: 30289-30298
        • Reilly T.J.
        • Baron G.S.
        • Nano F.E.
        • Kuhlenschmidt M.S.
        Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.
        J. Biol. Chem. 1996; 271: 10973-10983
        • Phillips R.M.
        • Six D.A.
        • Dennis E.A.
        • Ghosh P.
        In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors.
        J. Biol. Chem. 2003; 278: 41326-41332
        • Sato H.
        • Frank D.W.
        • Hillard C.J.
        • Feix J.B.
        • Pankhaniya R.R.
        • Moriyama K.
        • Finck-Barbançon V.
        • Buchaklian A.
        • Lei M.
        • Long R.M.
        • Wiener-Kronish J.
        • Sawa T.
        The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.
        EMBO J. 2003; 22: 2959-2969
        • Finck-Barbançon V.
        • Goranson J.
        • Zhu L.
        • Sawa T.
        • Wiener-Kronish J.P.
        • Fleiszig S.M.
        • Wu C.
        • Mende-Mueller L.
        • Frank D.W.
        ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.
        Mol. Microbiol. 1997; 25: 547-557
        • Hauser A.R.
        • Kang P.J.
        • Engel J.N.
        PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.
        Mol. Microbiol. 1998; 27: 807-818
        • Voulhoux R.
        • Ball G.
        • Ize B.
        • Vasil M.L.
        • Lazdunski A.
        • Wu L.F.
        • Filloux A.
        Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.
        EMBO J. 2001; 20: 6735-6741
        • Ochsner U.A.
        • Snyder A.
        • Vasil A.I.
        • Vasil M.L.
        Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8312-8317
        • Barker A.P.
        • Vasil A.I.
        • Filloux A.
        • Ball G.
        • Wilderman P.J.
        • Vasil M.L.
        A novel extracellular phospholipase C of Pseudomonas aeruginosa is required for phospholipid chemotaxis.
        Mol. Microbiol. 2004; 53: 1089-1098
        • Salacha R.
        • Kovaci F.
        • Brochier-Armanet C.
        • Wilhelm S.
        • Tommassen J.
        • Filloux A.
        • Voulhoux R.
        • Bleves S.
        The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.
        Environ. Microbiol. 2010; 12: 1498-1512
        • Rechnitzer C.
        • Williams A.
        • Wright J.B.
        • Dowsett A.B.
        • Milman N.
        • Fitzgeorge R.B.
        Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea pig and human alveolar macrophages.
        J. Gen. Microbiol. 1992; 138: 1671-1677
        • Liles M.R.
        • Edelstein P.H.
        • Cianciotto N.P.
        The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.
        Mol. Microbiol. 1999; 31: 959-970
        • Aragon V.
        • Kurtz S.
        • Flieger A.
        • Neumeister B.
        • Cianciotto N.P.
        Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.
        Infect. Immun. 2000; 68: 1855-1863
        • Rossier O.
        • Cianciotto N.P.
        Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila.
        Infect. Immun. 2001; 69: 2092-2098
        • Finck-Barbançon V.
        • Yahr T.L.
        • Frank D.W.
        Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin.
        J. Bacteriol. 1998; 180: 6224-6231
        • de Felipe K.S.
        • Glover R.T.
        • Charpentier X.
        • Anderson O.R.
        • Reyes M.
        • Pericone C.D.
        • Shuman H.A.
        Legionella eukaryotic-like type IV substrates interfere with organelle trafficking.
        PLoS Pathog. 2008; 4: e1000117
        • Hovel-Miner G.
        • Pampou S.
        • Faucher S.P.
        • Clarke M.
        • Morozova I.
        • Morozov P.
        • Russo J.J.
        • Shuman H.A.
        • Kalachikov S.
        SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.
        J. Bacteriol. 2009; 191: 2461-2473