Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.026021
ISSN1083-351X
AutoresJennifer K. Bender, Kerstin Rydzewski, Markus Broich, Eva Schunder, Klaus Heuner, Antje Flieger,
Tópico(s)Legionella and Acanthamoeba research
ResumoLegionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity. Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity. Bacterial phospholipases are involved in many disease-promoting processes ranging from production of bioactive molecules influencing cellular signal transduction pathways to formation of membrane pores and depletion of essential components from lipid layers (1Bender J. Flieger A. Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. Springer-Verlag GmBH, Heidelberg, Germany2009Google Scholar, 2Istivan T.S. Coloe P.J. Microbiology. 2006; 152: 1263-1274Crossref PubMed Scopus (106) Google Scholar, 3Schmiel D.H. Miller V.L. Microbes Infect. 1999; 1: 1103-1112Crossref PubMed Scopus (138) Google Scholar). They often induce massive membrane destruction, as shown for Pseudomonas aeruginosa type III secreted cytotoxin ExoU, a phospholipase A (PLA) 2The abbreviations used are: PLAphospholipaseLPLAlysophospholipaseDPPGdipalmitoylphosphatidylglycerolDPPCdipalmitoylphosphatidylcholineMPLPG1-monopalmitoylphosphatidylglycerolMPLPC1-monopalmitoyphosphatidylcholinePCphosphatidylcholine1-MPG1-monopalmitoylglycerol. 2The abbreviations used are: PLAphospholipaseLPLAlysophospholipaseDPPGdipalmitoylphosphatidylglycerolDPPCdipalmitoylphosphatidylcholineMPLPG1-monopalmitoylphosphatidylglycerolMPLPC1-monopalmitoyphosphatidylcholinePCphosphatidylcholine1-MPG1-monopalmitoylglycerol. /lysophospholipase A (LPLA), or Clostridium perfringens α- toxin, a phospholipase C (4Jepson M. Titball R. Microbes Infect. 2000; 2: 1277-1284Crossref PubMed Scopus (16) Google Scholar, 5Phillips R.M. Six D.A. Dennis E.A. Ghosh P. J. Biol. Chem. 2003; 278: 41326-41332Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 6Sato 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. EMBO J. 2003; 22: 2959-2969Crossref PubMed Scopus (268) Google Scholar, 7Sato H. Frank D.W. Mol. Microbiol. 2004; 53: 1279-1290Crossref PubMed Scopus (225) Google Scholar, 8Titball R.W. Naylor C.E. Basak A.K. Anaerobe. 1999; 5: 51-64Crossref PubMed Scopus (145) Google Scholar). We recently identified PlaB, the major cell-associated PLA/LPLA of Legionella pneumophila, to be hemolytic and thereby a membrane-destroying enzyme (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). Accordingly, activity of PlaB fits into the picture of Legionnaires' disease, the severe pneumonia caused by L. pneumophila bacteria, showing loss of a functional epithelial cell layer in the lung as well as degradation of lung macrophages in infected individuals (10Winn Jr., W.C. Myerowitz R.L. Hum. Pathol. 1981; 12: 401-422Crossref PubMed Scopus (147) Google Scholar, 11Blackmon J.A. Chandler F.W. Cherry W.B. England 3rd, A.C. Feeley J.C. Hicklin M.D. McKinney R.M. Wilkinson H.W. Am. J. Pathol. 1981; 103: 429-465PubMed Google Scholar). Now it is indeed clear that PlaB contributes to pathogenesis in a guinea pig infection model (1Bender J. Flieger A. Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. Springer-Verlag GmBH, Heidelberg, Germany2009Google Scholar), 3E. Schunder, P. Adam, F. Higa, K. A. Remer, U. Lorenz, J. Bender, A. Flieger, M. Steinert, J. Hacker, and K. Heuner, submitted for publication. 3E. Schunder, P. Adam, F. Higa, K. A. Remer, U. Lorenz, J. Bender, A. Flieger, M. Steinert, J. Hacker, and K. Heuner, submitted for publication. although it is dispensable for intracellular replication in macrophage and amoeba host cells (1Bender J. Flieger A. Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. Springer-Verlag GmBH, Heidelberg, Germany2009Google Scholar, 9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). 3E. Schunder, P. Adam, F. Higa, K. A. Remer, U. Lorenz, J. Bender, A. Flieger, M. Steinert, J. Hacker, and K. Heuner, submitted for publication. This clearly underlines the importance of PlaB as a virulence factor of L. pneumophila; however, its mechanism of action has not yet been studied in detail. phospholipase lysophospholipase dipalmitoylphosphatidylglycerol dipalmitoylphosphatidylcholine 1-monopalmitoylphosphatidylglycerol 1-monopalmitoyphosphatidylcholine phosphatidylcholine 1-monopalmitoylglycerol. phospholipase lysophospholipase dipalmitoylphosphatidylglycerol dipalmitoylphosphatidylcholine 1-monopalmitoylphosphatidylglycerol 1-monopalmitoyphosphatidylcholine phosphatidylcholine 1-monopalmitoylglycerol. PlaB was initially detected by screening an L. pneumophila genomic library in Escherichia coli, thus yielding a clone with remarkable hemolytic activity on human red blood agar (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). Subsequently, plaB insertion mutagenesis revealed that the gene encodes the most prominent cell-associated PLA/LPLA activity, being ∼100-fold more active than secreted phospholipolytic activities of L. pneumophila. Interestingly, PlaB has no significant protein homology to any established lipase/phospholipase at all. Further, considering the putative domains responsible for catalytic activity, it does not align significantly to any defined class of lipolytic enzymes. As proposed by Arpigny and Jaeger (12Arpigny J.L. Jaeger K.E. Biochem. J. 1999; 343: 177-183Crossref PubMed Scopus (1021) Google Scholar), lipases can be divided into eight families characterized by conserved amino acid motifs. Members of those families in general exhibit a conserved pentapeptide of GXSXG surrounding the catalytically important serine residue except of proteins belonging to GDSL hydrolases, which harbor the catalytic active serine within a differing GDSL motif (13Upton C. Buckley J.T. Trends Biochem. Sci. 1995; 20: 178-179Abstract Full Text PDF PubMed Scopus (254) Google Scholar). Further, lipases or phospholipases usually constitute a catalytic triad of serine, aspartate, and histidine; however, combinations of Ser-Glu-His or catalytic dyads have been reported as well (12Arpigny J.L. Jaeger K.E. Biochem. J. 1999; 343: 177-183Crossref PubMed Scopus (1021) Google Scholar, 14Rydel T.J. Williams J.M. Krieger E. Moshiri F. Stallings W.C. Brown S.M. Pershing J.C. Purcell J.P. Alibhai M.F. Biochemistry. 2003; 42: 6696-6708Crossref PubMed Scopus (213) Google Scholar, 15Vernet T. Ziomek E. Recktenwald A. Schrag J.D. de Montigny C. Tessier D.C. Thomas D.Y. Cygler M. J. Biol. Chem. 1993; 268: 26212-26219Abstract Full Text PDF PubMed Google Scholar). The latter one is present within a recently discovered group of bacterial lipolytic enzymes, the patatin-like proteins (16Banerji S. Flieger A. Microbiology. 2004; 150: 522-525Crossref PubMed Scopus (92) Google Scholar, 17Banerji S. Aurass P. Flieger A. Int. J. Med. Microbiol. 2008; 298: 169-181Crossref PubMed Scopus (43) Google Scholar). Although variability in amino acid homology between phospholipases is quite elevated, we propose PlaB and homologs to represent a novel division of lipolytic proteins. To understand the biochemical nature of L. pneumophila PlaB, in this study we (a) determined expression of PlaB during host cell infection, (b) identified the catalytically important residues and further protein domains crucial for lipid hydrolysis, (c) analyzed the contribution of the catalytic and other important sites to hemolytic activity, and (d) investigated lipid substrate specificity with respect to fatty acid chain length. We additionally addressed the questions of whether cell-associated phospholipase activity is found in various clinical isolates and whether PlaB is unique to L. pneumophila or also present within non-pneumophila species. L. pneumophila, environmental and clinical isolates, and different E. coli strains used in this study are listed in Table 1 and were grown as described previously (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar, 18Edelstein P.H. J. Clin. Microbiol. 1981; 14: 298-303Crossref PubMed Google Scholar).TABLE 1Bacterial strains and plasmids used in this study Amplification of L. pneumophila Corby plaB (lpc_1029) was carried out by using primers plaB_SalIF and plaB_EagIr (oligonucleotides were purchased from TIB MOLBIOL (Berlin) or Eurofins MWG Operon (Ebersberg)). The resulting fragment was cloned into SalI and EagI (New England Biolabs) restricted pBCKS vector to yield pJB04. This additional complementation vector was constructed because the plaB gene within pKH192 harbors an amino acid conversion, although not influencing lipolytic activities. Mutagenesis of important amino acids was carried out by utilizing the combined chain reaction method, as described elsewhere (19Bi W. Stambrook P.J. Anal. Biochem. 1998; 256: 137-140Crossref PubMed Scopus (45) Google Scholar). Minor modifications included performance of PCR with 20 ng of each primer and 20 ng of pJB04 plasmid DNA (Table 1), a 0.25 mm concentration of each dNTP, 2.5 units of Pfu polymerase (Fermentas), and 3 units of Ampligase (Epicenter). Mutagenesis primers were phosphorylated in advance by T4 polynucleotide kinase (New England Biolabs). Plasmid pJB04, carrying wild-type plaB, served as template for single amino acid exchanges, whereas pJB06, which harbors already plaB S85A, was used for creating loss of function double mutants S85A/D203N and S85A/H251N. Amplification of the designated fragments was performed by using plaB_SalIF and plaB_EagIr primers and the appropriate mutagenesis primer (Table 2). Restriction endonucleases SalI and EagI were used for site-specific integration of the desired fragments into vector pBCKS. Mutants were verified by sequencing both DNA strands, and resulting plasmids were electroporated into L. pneumophila plaB mutant strain. Table 1 gives a summary of all generated plasmids and respective plaB mutants.TABLE 2Primers used for site-directed mutagenesis, cloning, and sequence analysis For construction of full-length glutathione S-transferase-PlaB fusion protein, pGEX-5X-1 vector backbone was linearized by EcoRI and EagI restriction enzymes and ligated to the appropriate digested plaB fragment, amplified by using gene-specific primers plaB_EagIr and plaB_Ecof2 (Table 2). Truncated versions were cloned into vector pGEX-6P-1. Genes for PlaB-(1–459) and PlaB-(1–388) were amplified by PlaB_truncC1_P1 and PlaB_truncC2_P2, respectively, in combination with plaB_TOPf2. Both fragments were inserted over SmaI restriction sites into vector pGEX-6P-1. The truncated version PlaB-(1–307) was amplified by means of plaB_Ecof2 and plaB_trunc3Eag primers and ligated to pGEX-6P-1 via EcoRI and EagI restriction sites. All clones were verified by sequencing before electroporation into protein expression host E. coli BL21. The plaB gene from L. spiritensis was amplified from genomic DNA using primers plaB_pBADf and plaB_c1-r. Subsequently, the product was cloned into pGEMT-EZ, resulting in vector pKR8 before primers PlaBspir_BamHI and PlaBspir_NotI were used to precisely ligate the amplicon into digested pBCKS backbone vector, yielding pJB37 (Tables 1 and 2). plaB gene sequences from strains L. spiritensis and partially for L. gormanii were determined from pKR8 and pKR10, respectively. The latter vector resulted from an amplification product generated from L. gormanii genomic DNA by using primers plaB_pBADf and plaB_g1_r and cloned into pGEMT-EZ. Samples were obtained from cultures grown to an A660 of 2. For assessment of lipolytic activities, via release of fatty acids, bacterial cell lysates and supernatants were prepared and incubated with deduced lipids as described previously (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). Briefly, incubation of bacterial cell lysates or supernatants with different lipids (dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), 1-monopalmitoyllysophosphatidylcholine (MPLPC), 1-monopalmitoyllysophosphatidylglycerol (MPLPG), 1-monopalmitoylglycerol, 1-monolauroyllysophosphatidylcholine, or 1-monooctanoyllysophosphatidylcholine) was performed at 37 °C with continuous agitation at 100 rpm for overnight incubations and at 150 rpm for various shorter time points. Final concentration of each lipid was set to 6.7 mm. Free fatty acids as a marker of lipid hydrolysis by PLA/LPLA/lipase were determined by means of the NEFA-C-kit® (WAKO Chemicals, Neuss, Germany) according to the instructions of the manufacturer. Depending upon the nature of the experiment, BYE-broth, LB-broth, or 40 mm Tris-HCl, pH 7.5 (25 °C), was incubated, treated like the cultures, and subsequently used as a negative control. All lipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma. E. coli BL21 cells harboring the designated vectors were grown overnight prior to inoculation into fresh media. At an A660 of 0.6–0.8, protein expression was induced by using isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 1 mm. Bacterial cells were harvested at an A660 of 2 and centrifuged for 5 min at 5,000 × g, and pellets were stored overnight at −20 °C. Cell lysates were obtained according to our previously published protocol and subsequently used for determination of lipolytic activity (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). For detection of hemolytic activity, E. coli strains were grown for 1 day on LB agar plates. Bacteria were inoculated into PBS medium and set to a final A660 of 0.3. As described by Kirby et al. (20Kirby J.E. Vogel J.P. Andrews H.L. Isberg R.R. Mol. Microbiol. 1998; 27: 323-336Crossref PubMed Scopus (190) Google Scholar), human red blood cells were mixed with an equal volume of bacterial suspension, centrifuged at 16,000 × g for 2 min before incubation was carried out at 37 °C. At the desired time points, the suspension was vortexed and repelleted by centrifugation, and release of hemoglobin into the supernatant was determined at an optical density of 415 nm. 106/ml U937 macrophages were infected with L. pneumophila Corby (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar, 21Cianciotto N.P. Fields B.S. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 5188-5191Crossref PubMed Scopus (227) Google Scholar, 22Liles M.R. Edelstein P.H. Cianciotto N.P. Mol. Microbiol. 1999; 31: 959-970Crossref PubMed Scopus (99) Google Scholar). For detection of enzymatic activities, samples were taken from co-incubations as well as from uninfected U937 cells and divided into culture supernatant and cell pellet. Culture supernatant was furthermore passed through a 0.2-μm filter, and cell pellet was treated for 10 min by ultrasonication, stored at −20 °C for several days, thawed, and then resuspended in 20 mm Tris-HCl, pH 7.5 (25 °C), to original culture volume of the U937 infections. Culture supernatants and cell lysates were then incubated for 3 h with different lipid substrates, and enzymatic activities were determined. Hydrolysis in the presence of 6.25 mm EDTA accounted for bacterial PLA and LPLA activities. Although PlaB was shown to be dispensable for intracellular replication (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar), we were interested whether PlaB-dependent lipolytic activities are expressed during the infection process. To address this question, U937 macrophages were infected with wild-type L. pneumophila Corby, plaB mutant plaB1, and the complementing strain plaB1 (pKH192). Both replication of bacteria and PLA (via hydrolysis of DPPG)/LPLA (via hydrolysis of monopalmitoyllysophosphatidylcholine (MPLPC)) activities associated with cells and present in the culture supernatant were monitored. Although bacterial replication was comparable for all three strains (data not shown), wild-type infection comprised high PLA/LPLA activities, whereas the plaB mutant infection or uninfected U937 cells did not exhibit these activities (Fig. 1, A and B). A similar pattern was observed for cell culture supernatants (data not shown). The introduction of a functional copy of plaB into the plaB1 mutant complemented demonstrated activity defects (Fig. 1, A and B). Expression of plaB was additionally confirmed by reverse transcription-PCR analysis (not shown). Comparable data were obtained both for infections of U937 macrophages and Acanthamoeba castellanii amoebae with L. pneumophila strain 130b, plaB mutant, and the complementing strain (data not shown). These data demonstrate that L. pneumophila PlaB is indeed expressed and presumably the most prominent PLA/LPLA of the pathogen during intracellular infection of eukaryotic host cells. Lipase or phospholipase families typically contain a catalytic triad of Ser, Asp, and His, mostly arranged within conserved amino acid blocks. Assuming that PlaB exhibits a catalytic triad as well, we determined conserved and reasonable residues of PlaB and homologs to be used for site-directed mutagenesis. Interestingly, none of the potential catalytically active site serines is located within the typical pentapeptide GXSXG of true lipases or patatin-like proteins (12Arpigny J.L. Jaeger K.E. Biochem. J. 1999; 343: 177-183Crossref PubMed Scopus (1021) Google Scholar, 16Banerji S. Flieger A. Microbiology. 2004; 150: 522-525Crossref PubMed Scopus (92) Google Scholar); however, five serines were found to be part of similar amino acid motifs, such as Ser-85 (THSTG), Ser-129 (GKSRL), Ser-200 (GESGS), Ser-229 (GESLV), and Ser-250 (GLSHS). On the basis of this reasoning, serines were replaced by alanine, whereas reasonable aspartates and histidines were replaced by asparagine residues (Table 1). Resulting genes were expressed in a L. pneumophila plaB-deficient background, and cell lysates were subsequently tested for their ability to release fatty acids from various lipid substrates. In particular, dipalmitoylphosphatidylcholine (DPPC) and DPPG were used to check for PLA activity, and MPLPC and MPLPG were used to examine LPLA activity. All of those activities are usually comprised by the PlaB enzyme. We thereby could identify Ser-85, Asp-203, and His-251 to form the catalytic triad, since corresponding mutants were reduced in >99% of activity against all tested substrates (Fig. 2A). Further, mutant S129A was severely reduced in hydrolysis of choline-containing phospholipids, such as both DPPC and MPLPC, but not remarkably against substrates containing glycerol esterified to the phosphate residue instead (Fig. 2A). Moreover, we found that H270N mutants retained only ∼10% of their original hydrolytic properties and found His-7 to be particularly linked to substrate specificity regarding choline-containing lipids as well, since the mutant was about 10-fold less active against DPPC when compared with wild-type PlaB (Fig. 2A). Other mutants (S200A, S229A, S250A, D75N, D167N, D342N, D381N, H339N, and H433N) retained their ability to hydrolyze deduced substrates (data not shown). Reduction of enzymatic activity of described PlaB mutants was not due to second site mutations or decreased expression levels, as determined by sequencing and reverse transcription-PCR analysis (data not shown). Together, amino acids crucial for PlaB enzymatic activity constitute a typical lipase Ser-Asp-His triad, arranged within the N-terminal part of the protein, thus leaving an extension of about 174 amino acids at the C-terminal domain, a function which still requires further investigation (Fig. 2B). Amino acid sequence alignment to related proteins (ClustalW2; EMBL-EBI) revealed that the designated residues composing the catalytic triad and embedding amino acid motifs are well conserved throughout PlaB homologs (Fig. 2C), underlining their necessity for enzymatic activity. Interestingly, the crucial Ser-85 in PlaB is surrounded by similar amino acids, as is the case for most lipases; however, a threonine substitutes for the usually comprised glycine residue within the GXSXG motif. Omitting Thr in the immediate vicinity of the active site Ser through Gly substitution strongly reduces PlaB activity by 95%, revealing its particular importance for PlaB enzyme activity (Fig. 2A). Additional replacement of Thr-83 by Val, a residue found in a PlaB homolog of P. aeruginosa (Fig. 2C) underlined this observation, since mutant T83V was reduced in DPPC/MPLPC hydrolysis to 27 or 50% of wild-type activity, respectively (Fig. 2A). Ultimately, PlaB utilizes a well known catalytic triad, although conserved motifs embedding the crucial catalytic amino acids, designated THSTG, GSDGVV, and SHS (Fig. 2C), are unique among described phospholipases. PlaB thus represents the first described member of a new class of lipolytic families. PlaB has been shown to contribute to hemolytic activity of L. pneumophila (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar). Although it seems reasonable to assume that extensive phospholipase activity correlates with cytolytic activity, we aimed to clarify whether the identified catalytic domains are indeed essential for hemolysis. Corresponding PlaB site-directed mutant genes were expressed in non-hemolytic E. coli DH5α. Disrupting catalytically important amino acids, such as Ser-85 or Ser-85/Asp-203, a double knock-out without residual lipolytic activity, diminished hemolytic potential of plaB to the level of the empty vector-containing strain (Fig. 3). Mutant H433N did not show altered lipolytic quantities and profiles and therefore lysed human erythrocytes to the same extent as E. coli DH5α cells containing wild-type plaB. Interestingly, when we expressed mutant Ser-129, which was shown to hydrolyze DPPC/MPLPC less efficiently, hemolysis was comparable with vector control level (Fig. 3). Thus, PC-hydrolyzing activity of PlaB was directly responsible for its hemolytic effects. As demonstrated in Fig. 2B, PlaB catalytically important residues are present within the N-terminal region; therefore, the protein exhibits an extended C terminus of additional 174 amino acids, a function that remains to be elucidated. To this end, we constructed three truncated versions of the protein, ranging from 15 to 167 amino acids less than wild-type PlaB. Deletion of only 15 or more amino acids completely abolished enzymatic activities of PlaB, whereas protein expression levels of the truncated enzymes were comparable with the full-length construct (data not shown). Thus, catalytic activity of PlaB depends on both the N-terminal sequence harboring the crucial amino acids and the C-terminal region. To further examine biochemical properties, we analyzed acyl chain length selectivity of wild-type PlaB enzyme. To this end, cell lysates of L. pneumophila Corby wild-type, the plaB mutant, and complementing strain were exposed to comparable concentrations of lysophophatidylcholines varying in chain length from C8 to C16, and the release of fatty acids was quantified. We demonstrate here that PlaB almost solely hydrolyzed long fatty acid chained substrates, such as MPLPC (C16) and 1-monolauroyllysophosphatidylcholine (C12) (Fig. 4). 1-Monooctanoyllysophosphatidylcholine (C8) was not affected by PlaB-dependent lipolysis under our experimental conditions (Fig. 4). This observation was verified by using p-nitrophenyl-based substrates esterified to carboxylic acids from C4, C12, and C16 in chain length (data not shown). Thus, PlaB acts mostly against long-chain fatty acid substrates with preferences for lipids containing fatty acyl residues larger than eight carbon atoms. We demonstrated above that PlaB is a potent hemolytic agent and is expressed during intracellular replication, which probably contributes to extensive lung destruction during Legionnaires' disease. Thus, we investigated the distribution of cell-associated PLA/LPLA activity within nine clinical isolates. We observed comparable hydrolytic activities for various L. pneumophila strains, including different serogroups (Fig. 5A). Investigation of putative plaB expression by reverse transcription-PCR analysis revealed that these isolates indeed possess and actively transcribe plaB-like genes (not shown). This suggests that PlaB is widely spread among L. pneumophila bacteria isolated from various patients. Interestingly, an environmental L. pneumophila strain (Lpn 257), obtained from a water conduit, showed lipolytic potential comparable with that of the clinical isolates tested (Fig. 5A). L. pneumophila has been most often associated with Legionella infections, and therefore researchers seek to identify virulence factors that are present only in pathogenic but not in less pathogenic species (23Fang G.D. Yu V.L. Vickers R.M. Medicine. 1989; 68: 116-132Crossref PubMed Scopus (137) Google Scholar, 24Muder R.R. Yu V.L. Clin. Infect. Dis. 2002; 35: 990-998Crossref PubMed Scopus (225) Google Scholar). Although we previously determined plaB genetically to be restricted to L. pneumophila (9Flieger A. Rydzewski K. Banerji S. Broich M. Heuner K. Infect. Immun. 2004; 72: 2648-2658Crossref PubMed Scopus (52) Google Scholar), we now wanted to verify whether non-pneumophila strains possess cell-associated PLA/LPLA activities. To this end, we tested L. pneumophila 130b and 12 non-pneumophila (Table 1) cell lysates for fatty acid release from phospholipids, lysophospholipids, and a lipase substrate, 1-MPG. Only L. gormanii and L. spiritensis possessed a spectrum and quantity of PLA/LPLA/lipase activities comparable with that of L. pneumophila 130b (Fig. 5B). However, both L. gormanii and L. spiritensis showed a lower PLA activity toward DPPC than L. pneumophila strains (Fig. 5, A and B). Additionally, by increasing the incubation time, Legionella anisa, Legionella jordanis, Legionella parisiensis, and Legionella sainthelensis very predominantly hydrolyz
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