1887

Abstract

is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one gene inactivated, and one double mutant with both genes inactivated. The single mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.

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2007-06-01
2024-03-28
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References

  1. Alexeyev M. F. 1999; The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. Biotechniques 26:824–826 828
    [Google Scholar]
  2. de Lorenzo V., Timmis K. N. 1994; Analysis and construction of stable phenotypes in gram-negative bacteria with Tn 5 - and Tn 10 -derived minitransposons. Methods Enzymol 235:386–405
    [Google Scholar]
  3. Geoffroy C., Raveneau J., Beretti J. L., Lecroisey A., Vazquez-Boland J. A., Alouf J. E., Berche P. 1991; Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.. Infect Immun 59:2382–2388
    [Google Scholar]
  4. Grundling A., Gonzalez M. D., Higgins D. E. 2003; Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. J Bacteriol 185:6295–6307 [CrossRef]
    [Google Scholar]
  5. Holden M. T., Titball R. W., Peacock S. J., Cerdeno-Tarraga A. M., Atkins T., Crossman L. C., Pitt T., Churcher C., Mungall K. other authors 2004; Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A 101:14240–14245 [CrossRef]
    [Google Scholar]
  6. Jepson M., Titball R. 2000; Structure and function of clostridial phospholipases C. Microbes Infect 2:1277–1284 [CrossRef]
    [Google Scholar]
  7. Kespichayawattana W., Rattanachetkul S., Wanun T., Utaisincharoen P., Sirisinha S. 2000; Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreading. Infect Immun 68:5377–5384 [CrossRef]
    [Google Scholar]
  8. Korbsrisate S., Suwanasai N., Leelaporn A., Ezaki T., Kawamura Y., Sarasombath S. 1999; Cloning and characterization of a nonhemolytic phospholipase C gene from Burkholderia pseudomallei. J Clin Microbiol 37:3742–3745
    [Google Scholar]
  9. Korbsrisate S., Vanaporn M., Kerdsuk P., Kespichayawattana W., Vattanaviboon P., Kiatpapan P., Lertmemongkolchai G. 2005; The Burkholderia pseudomallei RpoE (AlgU) operon is involved in environmental stress tolerance and biofilm formation. FEMS Microbiol Lett 252:243–249 [CrossRef]
    [Google Scholar]
  10. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Peterson K. M., Roop R. M. II 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [CrossRef]
    [Google Scholar]
  11. Krug E. L., Kent C. 1984; Phospholipase C from Clostridium perfringens : preparation and characterization of homogeneous enzyme. Arch Biochem Biophys 231:400–410 [CrossRef]
    [Google Scholar]
  12. Kurioka S., Matsuda M. 1976; Phospholipase C assay using p -nitrophenylphosphoryl-choline together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Anal Biochem 75:281–289 [CrossRef]
    [Google Scholar]
  13. Loprasert S., Sallabhan R., Whangsuk W., Mongkolsuk S. 2002; The Burkholderia pseudomallei oxyR gene: expression analysis and mutant characterization. Gene 296:161–169 [CrossRef]
    [Google Scholar]
  14. Mengaud J., Braun-Breton C., Cossart P. 1991; Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes : a novel type of virulence factor?. Mol Microbiol 5:367–372 [CrossRef]
    [Google Scholar]
  15. Meyers D. J., Palmer K. C., Bale L. A., Kernacki K., Preston M., Brown T., Berk R. S. 1992; In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa. Toxicon 30:161–169 [CrossRef]
    [Google Scholar]
  16. Nierman W. C., DeShazer D., Kim H. S., Tettelin H., Nelson K. E., Feldblyum T., Ulrich R. L., Ronning C. M., Brinkac L. M. other authors 2004; Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci U S A 101:14246–14251 [CrossRef]
    [Google Scholar]
  17. Ochsner U. A., Snyder A., Vasil A. I., Vasil M. L. 2002; Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc Natl Acad Sci U S A 99:8312–8317 [CrossRef]
    [Google Scholar]
  18. Ostroff R. M., Vasil A. I., Vasil M. L. 1990; Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa. J Bacteriol 172:5915–5923
    [Google Scholar]
  19. Raveneau J., Geoffroy C., Beretti J. L., Gaillard J. L., Alouf J. E., Berche P. 1992; Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene. Infect Immun 60:916–921
    [Google Scholar]
  20. Raynaud C., Guilhot C., Rauzier J., Bordat Y., Pelicic V., Manganelli R., Smith I., Gicquel B., Jackson M. 2002; Phospholipases C are involved in the virulence of Mycobacterium tuberculosis. Mol Microbiol 45:203–217 [CrossRef]
    [Google Scholar]
  21. Smith G. A., Marquis H., Jones S., Johnston N. C., Portnoy D. A., Goldfine H. 1995; The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect Immun 63:4231–4237
    [Google Scholar]
  22. 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. other authors 2002; A novel class of microbial phosphocholine-specific phospholipases C. Mol Microbiol 46:661–676 [CrossRef]
    [Google Scholar]
  23. Suparak S., Kespichayawattana W., Haque A., Easton A., Damnin S., Lertmemongkolchai G., Bancroft G. J., Korbsrisate S. 2005; Multinucleated giant cell formation and apoptosis in infected host cells is mediated by Burkholderia pseudomallei type III secretion protein BipB. J Bacteriol 187:6556–6560 [CrossRef]
    [Google Scholar]
  24. 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]
  25. Titball R. W. 1993; Bacterial phospholipases C. Microbiol Rev 57:347–366
    [Google Scholar]
  26. Titball R. W. 1998; Bacterial phospholipases. Symp Ser Soc Appl Microbiol 27:127S–137S
    [Google Scholar]
  27. Tuanyok A., Tom M., Dunbar J., Woods D. E. 2006; Genome-wide expression analysis of Burkholderia pseudomallei infection in a hamster model of acute melioidosis. Infect Immun 74:5465–5476 [CrossRef]
    [Google Scholar]
  28. van Echten-Deckert G. 2000; Sphingolipid extraction and analysis by thin-layer chromatography. Methods Enzymol 312:64–79
    [Google Scholar]
  29. Vasil M. L., Berka R. M., Gray G. L., Nakai H. 1982; Cloning of a phosphate-regulated hemolysin gene (phospholipase C) from Pseudomonas aeruginosa. J Bacteriol 152:431–440
    [Google Scholar]
  30. Vazquez-Boland J. A., Kocks C., Dramsi S., Ohayon H., Geoffroy C., Mengaud J., Cossart P. 1992; Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun 60:219–230
    [Google Scholar]
  31. von Heijne G. 1985; Signal sequences. The limits of variation. J Mol Biol 184:99–105 [CrossRef]
    [Google Scholar]
  32. Voulhoux R., Ball G., Ize B., Vasil M. L., Lazdunski A., Wu L. F., Filloux A. 2001; Involvement of the twin-arginine translocation system in protein secretion via the type II pathway. EMBO J 20:6735–6741 [CrossRef]
    [Google Scholar]
  33. White N. J. 2003; Melioidosis. Lancet 361:1715–1722 [CrossRef]
    [Google Scholar]
  34. Wong K. T., Puthucheary S. D., Vadivelu J. 1995; The histopathology of human melioidosis. Histopathology 26:51–55 [CrossRef]
    [Google Scholar]
  35. Woods D. E., DeShazer D., Moore R. A., Brett P. J., Burtnick M. N., Reckseidler S. L., Senkiw M. D. 1999; Current studies on the pathogenesis of melioidosis. Microbes Infect 1:157–162 [CrossRef]
    [Google Scholar]
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