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Volume 148, Issue 7

genes that encode lipase and phospholipase C activities

aThe GenBank accession numbers for the and sequences are AF454863, AF454864 and AF454865, respectively.

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Abstract

, the agent of Legionnaires’ disease, is an intracellular parasite of aquatic protozoans and human macrophages. The type II protein secretion system of the Gram-negative organism promotes intracellular infection. A lipase activity and a -nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in type II secretion mutants. The lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol. In a number of other bacterial systems, the release of -nitrophenol from pNPPC is due to a phospholipase C. In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in strain 130b that were predicted to encode either a secreted lipase or a phospholipase C. The first two genes, which were designated and , encoded proteins containing the lipase consensus sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]. Mutations in in particular reduced supernatant activity against mono- and triacylglycerols. However, loss of and/or did not impair the ability of to infect amoebae or U937 cell macrophages. The third gene, which was denoted , encoded a protein that was highly homologous with a phospholipase C from . Inactivation of diminished secreted pNPPC hydrolase activity but did not influence infection of host cells. Taken together, these data indicate that has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection.

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Keyword(s): 1,2-DG, 1,2-dipalmitoylglycerol , 1-MG, 1-monopalmitoylglycerol , FFA, free fatty acid , intracellular bacteria , Legionaires’ disease , lipolytic enzymes , macrophages , PLC, phospholipase C , PNP, p-nitrophenol , pNPPC, p-nitrophenylphosphorylcholine and type II secretion
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2002-07-01
2024-04-19
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References

  1. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
     [Google Scholar]
  2. Anguita J., Rodriguez Aparicio L. B., Naharro G. 1993; Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate. Appl Environ Microbiol 59:2411–2417
     [Google Scholar]
  3. Aragon V., Kurtz S., Flieger A., Neumeister B., Cianciotto N. P. 2000; Secreted enzymatic activities of wild-type and pilD -deficient Legionella pneumophila . Infect Immun 68:1855–1863 [CrossRef]
     [Google Scholar]
  4. Aragon V., Kurtz S., Cianciotto N. P. 2001; Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect Immun 69:177–185 [CrossRef]
     [Google Scholar]
  5. Atlas R. M. 1999; Legionella : from environmental habitats to disease pathology, detection and control. Environ Microbiol 1:283–293 [CrossRef]
     [Google Scholar]
  6. Baine W. B. 1985; Cytolytic and phospholipase C activity in Legionella species. J Gen Microbiol 131:1383–1391
     [Google Scholar]
  7. Baine W. B. 1988; 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 134:489–498
     [Google Scholar]
  8. Cianciotto N. P. 2001; Pathogenicity of Legionella pneumophila . Int J Med Microbiol 291:331–343 [CrossRef]
     [Google Scholar]
  9. Cianciotto N. P., Fields B. S. 1992; Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci USA 89:5188–5191 [CrossRef]
     [Google Scholar]
  10. Cianciotto N. P., Long R., Eisenstein B. I., Engleberg N. C. 1988; Site-specific mutagenesis in Legionella pneumophila by allelic exchange using counterselectable ColE1 vectors. FEMS Microbiol Lett 56:203–208 [CrossRef]
     [Google Scholar]
  11. Cianciotto N., Eisenstein B. I., Engleberg N. C., Shuman H. 1989a; Genetics and molecular pathogenesis of Legionella pneumophila , an intracellular parasite of macrophages. Mol Biol Med 6:409–424
     [Google Scholar]
  12. Cianciotto N. P., Eisenstein B. I., Mody C. H., Toews G. B., Engleberg N. C. 1989b; A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun 57:1255–1262
     [Google Scholar]
  13. Dowling J. N., Saha A. K., Glew R. H. 1992; Virulence factors of the family Legionellaceae . Microbiol Rev 56:32–60
     [Google Scholar]
  14. Edelstein P. H. 1981; Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J Clin Microbiol 14:298–303
     [Google Scholar]
  15. El Khattabi M., Ockhuijsen C., Bitter W., Jaeger K. E., Tommassen J. 1999; Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor. Mol Gen Genet 261:770–776 [CrossRef]
     [Google Scholar]
  16. Engleberg N. C., Drutz D. J., Eisenstein B. I. 1984; Cloning and expression of Legionella pneumophila antigens in Escherichia coli . Infect Immun 44:222–227
     [Google Scholar]
  17. Fields B. S. 1996; The molecular ecology of legionellae. Trends Microbiol 4:286–290 [CrossRef]
     [Google Scholar]
  18. Filloux A., Murgier M., Wretlind B., Lazdunski A. 1987; Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants. FEMS Microbiol Lett 40:159–163 [CrossRef]
     [Google Scholar]
  19. Flieger A., Gong S., Faigle M., Deeg P., Bartmann P., Neumeister B. 2000; Characterization of a novel phospholipase A (PLA) activity secreted by Legionella species. J Bacteriol 182:1321–1327 [CrossRef]
     [Google Scholar]
  20. Flieger A., Gong S., Faigle M., Stevanovic S., Cianciotto N. P., Neumeister B. 2001; Novel lysophospholipase A secreted by Legionella pneumophila . J Bacteriol 183:2121–2124 [CrossRef]
     [Google Scholar]
  21. Gribbon E. M., Cunliffe W. J., Holland K. T. 1993; Interaction of Propionibacterium acnes with skin lipids in vitro . J Gen Microbiol 139:1745–1751 [CrossRef]
     [Google Scholar]
  22. Hacker J., Ott M., Wintermeyer E., Ludwig B., Fischer G. 1993; Analysis of virulence factors of Legionella pneumophila . Zentbl Backteriol 278:348–358
     [Google Scholar]
  23. Hales L. M., Shuman H. A. 1999; Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect Immun 67:3662–3666
     [Google Scholar]
  24. Harb O. S., Gao L.-Y., Abu Kwaik Y. 2000; From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ Microbiol 2:251–265 [CrossRef]
     [Google Scholar]
  25. Higgins D. G., Sharp P. M. 1988; CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237–244 [CrossRef]
     [Google Scholar]
  26. Hoffmann G. E., Schmidt D., Bastian B., Guder W. G. 1986; Photometric determination of phospholipase A. J Clin Chem Clin Biochem 24:871–875
     [Google Scholar]
  27. Hofmann K., Bucher P., Falquet L., Bairoch A. 1999; The PROSITE database, its status in 1999. Nucleic Acids Res 27:215–219 [CrossRef]
     [Google Scholar]
  28. Horwitz M. A. 1992; Interactions between macrophages and Legionella pneumophila . Curr Topics Microbiol Immunol 181:265–282
     [Google Scholar]
  29. Jaeger K. E., Ransac S., Dijkstra B. W., Colson C., van Heuvel M., Misset O. 1994; Bacterial lipases. FEMS Microbiol Rev 15:29–63 [CrossRef]
     [Google Scholar]
  30. Jaeger K. E., Dijkstra B. W., Reetz M. T. 1999; Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351 [CrossRef]
     [Google Scholar]
  31. Jepson M., Howells A., Bullifent H. L., Bolgiano B., Crane D., Miller J., Holley J., Jayasekera P., Titball R. W. 1999; Differences in the carboxy-terminal (putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes. Infect Immun 67:3297–3301
     [Google Scholar]
  32. Konig B., Jaeger K. E., Sage A. E., Vasil M. L., Konig W. 1996; Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes). Infect Immun 64:3252–3258
     [Google Scholar]
  33. Koster M., Bitter W., Tommassen J. 2000; Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol 290:325–331 [CrossRef]
     [Google Scholar]
  34. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M., Peterson K. M. 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [CrossRef]
     [Google Scholar]
  35. Kurioka S., Matsuda M. 1976; Phospholipase C assay using p -nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Anal Biochem 75:281–289 [CrossRef]
     [Google Scholar]
  36. Lee V. T., Schneewind O. 2001; Protein secretion and the pathogenesis of bacterial infections. Genes Dev 15:1725–1752 [CrossRef]
     [Google Scholar]
  37. Liles M. R., Viswanathan V. K., Cianciotto N. P. 1998; Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect Immun 66:1776–1782
     [Google Scholar]
  38. Liles M. R., Edelstein P. H., Cianciotto N. P. 1999; The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila . Mol Microbiol 31:959–970 [CrossRef]
     [Google Scholar]
  39. Merino S., Aguilar A., Nogueras M. M., Regue M., Swift S., Tomas J. M. 1999; Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O: 34. Infect Immun 67:4008–4013
     [Google Scholar]
  40. Moffat J. F., Edelstein P. H., Regula D. P. Jr, Cirillo J. D., Tompkins L. S. 1994; Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model. Mol Microbiol 12:693–705 [CrossRef]
     [Google Scholar]
  41. Muller H. E. 1981; Enzymatic profile of Legionella pneumophila . J Clin Microbiol 13:423–426
     [Google Scholar]
  42. Nakai K., Kanehisa M. 1991; Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11:95–110 [CrossRef]
     [Google Scholar]
  43. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6 [CrossRef]
     [Google Scholar]
  44. Nolte F. S., Hollick G. E., Robertson R. G. 1982; Enzymatic activities of Legionella pneumophila and Legionella -like organisms. J Clin Microbiol 15:175–177
     [Google Scholar]
  45. Nunn D. 1999; Bacterial type II protein export and pilus biogenesis: more than just homologies?. Trends Cell Biol 9:402–408 [CrossRef]
     [Google Scholar]
  46. O’Connell W. A., Bangsborg J. M., Cianciotto N. P. 1995; Characterization of a Legionella micdadei mip mutant. Infect Immun 63:2840–2845
     [Google Scholar]
  47. Ollis D. L., Cheah E., Cygler M. 10 other authors 1992; The alpha/beta hydrolase fold. Protein Eng 5:197–211 [CrossRef]
     [Google Scholar]
  48. Polesky A. H., Ross J. T., Falkow S., Tompkins L. S. 2001; Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect Immun 69:977–987 [CrossRef]
     [Google Scholar]
  49. Pratt J., Cooley J. D., Purdy C. W., Straus D. C. 2000; Lipase activity from strains of Pasteurella multocida . Curr Microbiol 40:306–309 [CrossRef]
     [Google Scholar]
  50. Preuss I., Kaiser I., Gehring U. 2001; Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C. Eur J Biochem 268:5081–5091 [CrossRef]
     [Google Scholar]
  51. Robey M., O’Connell W., Cianciotto N. P. 2001; Identification of Legionella pneumophila rcp , a pagP -like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection. Infect Immun 69:4276–4286 [CrossRef]
     [Google Scholar]
  52. Rollof J., Braconier J. H., Soderstrom C., Nilsson-Ehle P. 1988; Interference of Staphylococcus aureus lipase with human granulocyte function. Eur J Clin Microbiol Infect Dis 7:505–510 [CrossRef]
     [Google Scholar]
  53. Rossier O., Cianciotto N. P. 2001; Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila . Infect Immun 69:2092–2098 [CrossRef]
     [Google Scholar]
  54. Roy C. R. 1999; Trafficking of the Legionella pneumophila phagosome. ASM News 65:416–421
     [Google Scholar]
  55. Russel M. 1998; Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J Mol Biol 279:485–499 [CrossRef]
     [Google Scholar]
  56. Sandkvist M. 2001a; Biology of type II secretion. Mol Microbiol 40:271–283 [CrossRef]
     [Google Scholar]
  57. Sandkvist M. 2001b; Type II secretion and pathogenesis. Infect Immun 69:3523–3535 [CrossRef]
     [Google Scholar]
  58. Schmiel D. H., Miller V. L. 1999; Bacterial phospholipases and pathogenesis. Microbes Infect 1:1103–1112 [CrossRef]
     [Google Scholar]
  59. Shuman H. A., Purcell M., Segal G., Hales L., Wiater L. A. 1998; Intracellular multiplication of Legionella pneumophila : human pathogen or accidental tourist?. Curr Topics Microbiol Immunol 225:99–112
     [Google Scholar]
  60. Songer J. G. 1997; Bacterial phospholipases and their role in virulence. Trends Microbiol 5:156–161 [CrossRef]
     [Google Scholar]
  61. Stone B. J., Abu Kwaik Y. 1999; Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181:1395–1402
     [Google Scholar]
  62. Strom M. S., Nunn D., Lory S. 1991; Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa . J Bacteriol 173:1175–1180
     [Google Scholar]
  63. Swanson M. S., Hammer B. K. 2000; Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54:567–613 [CrossRef]
     [Google Scholar]
  64. Szeto L., Shuman H. A. 1990; The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect Immun 58:2585–2592
     [Google Scholar]
  65. Terada L. S., Johansen K. A., Nowbar S., Vasil A. I., Vasil M. L. 1999; Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity. Infect Immun 67:2371–2376
     [Google Scholar]
  66. Thanassi D. G., Hultgren S. J. 2000; Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12:420–430 [CrossRef]
     [Google Scholar]
  67. Thorpe T. C., Miller R. D. 1981; Extracellular enzymes of Legionella pneumophila . Infect Immun 33:632–635
     [Google Scholar]
  68. Titball R. W. 1998; Bacterial phospholipases. Soc Appl Bacteriol Symp Ser 27:127S–137S
     [Google Scholar]
  69. Viswanathan V. K., Edelstein P. H., Pope C. D., Cianciotto N. P. 2000; The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence. Infect Immun 68:1069–1079 [CrossRef]
     [Google Scholar]
  70. Vogel J. P., Isberg R. R. 1999; Cell biology of Legionella pneumophila . Curr Opin Microbiol 2:30–34 [CrossRef]
     [Google Scholar]
  71. Weingart C. L., Hooke A. M. 1999; Regulation of expression of the nonhemolytic phospholipase C of Burkholderia cepacia . Curr Microbiol 39:336–341 [CrossRef]
     [Google Scholar]
  72. Winn W. C. Jr 1988; Legionnaires disease: historical perspective. Clin Microbiol Rev 1:60–81
     [Google Scholar]
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Legionella pneumophila genes that encode lipase and phospholipase C activitiesaaThe GenBank accession numbers for the L. pneumophila lipA, lipB and plcA sequences are AF454863, AF454864 and AF454865, respectively.
Microbiology 148, 2223 (2002); https://doi.org/10.1099/00221287-148-7-2223
/content/journal/micro/10.1099/00221287-148-7-2223
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