Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Aug;65(8):3112–3117. doi: 10.1128/iai.65.8.3112-3117.1997

Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae.

A E Fiore 1, J M Michalski 1, R G Russell 1, C L Sears 1, J B Kaper 1
PMCID: PMC175439  PMID: 9234762

Abstract

Phospholipases are associated with virulence in bacterial diseases. Vibrio cholerae produces a phospholipase (lecithinase), with enzyme production visualized as a zone of clearing around colonies plated on egg yolk agar. The role of phospholipase in gut colonization or disease pathogenesis is unknown. We used the egg yolk agar assay to clone and characterize a gene encoding a phospholipase from V. cholerae El Tor strain E7946. Sequence analysis revealed a 1,254-bp open reading frame (lec) encoding a 418-amino-acid protein with a predicted molecular weight of 47,600. The predicted sequence exhibits DNA homology to other Vibrionaceae phospholipases. A potential signal sequence exists in the predicted amino acid sequence, as does a lipid binding motif found in prokaryotic and eukaryotic phospholipases and lipases. Polyacrylamide gel electrophoresis combined with an egg yolk agarose overlay demonstrated phospholipase activity migrating at a relative molecular weight of 45,000 in preparations of V. cholerae and the Escherichia coli clone. Restriction mapping and Southern blot analysis revealed that lec, hlyA (hemolysin), and hlyC (lipase) are adjacent on the V. cholerae chromosome, and chromosomal digests of several El Tor, classical, and O139 (Bengal) strains demonstrated conservation of this gene arrangement. An in-frame internal deletion of the lec gene was constructed and recombined into the chromosome of attenuated V. cholerae El Tor strain CVD 110. The resulting mutant lacked lecithinase activity on egg yolk agar but had undiminished reactivity in rabbit ligated ileal loop assays.

Full Text

The Full Text of this article is available as a PDF (1,002.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alm R. A., Mayrhofer G., Kotlarski I., Manning P. A. Amino-terminal domain of the El Tor haemolysin of Vibrio cholerae O1 is expressed in classical strains and is cytotoxic. Vaccine. 1991 Aug;9(8):588–594. doi: 10.1016/0264-410x(91)90247-4. [DOI] [PubMed] [Google Scholar]
  2. Alm R. A., Stroeher U. H., Manning P. A. Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B. Mol Microbiol. 1988 Jul;2(4):481–488. doi: 10.1111/j.1365-2958.1988.tb00054.x. [DOI] [PubMed] [Google Scholar]
  3. Brown R. C., Taylor R. K. Organization of tcp, acf, and toxT genes within a ToxT-dependent operon. Mol Microbiol. 1995 May;16(3):425–439. doi: 10.1111/j.1365-2958.1995.tb02408.x. [DOI] [PubMed] [Google Scholar]
  4. Butterton J. R., Stoebner J. A., Payne S. M., Calderwood S. B. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992 Jun;174(11):3729–3738. doi: 10.1128/jb.174.11.3729-3738.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casanova T. B., Peterson K. M. The Vibrio cholerae hlyC gene encodes a protein that is related to lipases of pseudomonas species. DNA Seq. 1995;5(3):181–184. doi: 10.3109/10425179509029360. [DOI] [PubMed] [Google Scholar]
  6. Daugherty S., Low M. G. Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor. Infect Immun. 1993 Dec;61(12):5078–5089. doi: 10.1128/iai.61.12.5078-5089.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Diener M., Eglème C., Rummel W. Phospholipase C-induced anion secretion and its interaction with carbachol in the rat colonic mucosa. Eur J Pharmacol. 1991 Aug 6;200(2-3):267–276. doi: 10.1016/0014-2999(91)90581-a. [DOI] [PubMed] [Google Scholar]
  8. Felsenfeld O. The Lecithinase Activity of Vibrio comma and the El Tor Vibrio. J Bacteriol. 1944 Aug;48(2):155–157. doi: 10.1128/jb.48.2.155-157.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujii Y., Sakurai J. Contraction of the rat isolated aorta caused by Clostridium perfringens alpha toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism. Br J Pharmacol. 1989 May;97(1):119–124. doi: 10.1111/j.1476-5381.1989.tb11931.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. GANGAROSA E. F., BEISEL W. R., BENYAJATI C., SPRINZ H., PIYARATN P. The nature of the gastrointestinal lesion in asiatic cholera and its relation to pathogenesis: a biopsy study. Am J Trop Med Hyg. 1960 Mar;9:125–135. doi: 10.4269/ajtmh.1960.9.125. [DOI] [PubMed] [Google Scholar]
  11. Goldberg M. B., Boyko S. A., Calderwood S. B. Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1125–1129. doi: 10.1073/pnas.88.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda T., Finkelstein R. A. Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios. Infect Immun. 1979 Dec;26(3):1020–1027. doi: 10.1128/iai.26.3.1020-1027.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Isakov N. Activation of murine lymphocytes by exogenous phosphatidylethanolamine- and phosphatidylcholine-specific phospholipase C. Cell Immunol. 1993 Nov;152(1):72–81. doi: 10.1006/cimm.1993.1268. [DOI] [PubMed] [Google Scholar]
  14. Jones B. D., Mobley H. L. Genetic and biochemical diversity of ureases of Proteus, Providencia, and Morganella species isolated from urinary tract infection. Infect Immun. 1987 Sep;55(9):2198–2203. doi: 10.1128/iai.55.9.2198-2203.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kald B., Boll R. M., Gustafson-Svärd C., Sjödahl R., Tagesson C. Phospholipase C from Clostridium perfringens stimulates acetyltransferase-dependent formation of platelet-activating factor in cultured intestinal epithelial cells (INT 407). Scand J Gastroenterol. 1994 Mar;29(3):243–247. doi: 10.3109/00365529409090471. [DOI] [PubMed] [Google Scholar]
  16. Levine M. M., Kaper J. B., Herrington D., Ketley J., Losonsky G., Tacket C. O., Tall B., Cryz S. Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet. 1988 Aug 27;2(8609):467–470. doi: 10.1016/s0140-6736(88)90120-1. [DOI] [PubMed] [Google Scholar]
  17. Michalski J., Galen J. E., Fasano A., Kaper J. B. CVD110, an attenuated Vibrio cholerae O1 El Tor live oral vaccine strain. Infect Immun. 1993 Oct;61(10):4462–4468. doi: 10.1128/iai.61.10.4462-4468.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mundy L. M., Sears C. L. Detection of toxin production by Bacteroides fragilis: assay development and screening of extraintestinal clinical isolates. Clin Infect Dis. 1996 Aug;23(2):269–276. doi: 10.1093/clinids/23.2.269. [DOI] [PubMed] [Google Scholar]
  20. Neena, Asnani P. J. Structural and functional changes in rabbit ileum by purified extracellular phospholipase A of Salmonella newport. Folia Microbiol (Praha) 1991;36(6):572–577. doi: 10.1007/BF02884039. [DOI] [PubMed] [Google Scholar]
  21. Nishibuchi M., Fasano A., Russell R. G., Kaper J. B. Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin. Infect Immun. 1992 Sep;60(9):3539–3545. doi: 10.1128/iai.60.9.3539-3545.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peterson J. W., Ochoa L. G. Role of prostaglandins and cAMP in the secretory effects of cholera toxin. Science. 1989 Aug 25;245(4920):857–859. doi: 10.1126/science.2549637. [DOI] [PubMed] [Google Scholar]
  23. Shaw J. F., Chang R. C., Chuang K. H., Yen Y. T., Wang Y. J., Wang F. G. Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli. Biochem J. 1994 Mar 15;298(Pt 3):675–680. doi: 10.1042/bj2980675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shinoda S., Matsuoka H., Tsuchie T., Miyoshi S., Yamamoto S., Taniguchi H., Mizuguchi Y. Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene. J Gen Microbiol. 1991 Dec;137(12):2705–2711. doi: 10.1099/00221287-137-12-2705. [DOI] [PubMed] [Google Scholar]
  25. Silva T. M., Schleupner M. A., Tacket C. O., Steiner T. S., Kaper J. B., Edelman R., Guerrant R. New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae. Infect Immun. 1996 Jun;64(6):2362–2364. doi: 10.1128/iai.64.6.2362-2364.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stoll B. J., Glass R. I., Banu H., Huq M. I., Khan M. U., Ahmed M. Value of stool examination in patients with diarrhoea. Br Med J (Clin Res Ed) 1983 Jun 25;286(6383):2037–2040. doi: 10.1136/bmj.286.6383.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tacket C. O., Losonsky G., Nataro J. P., Comstock L., Michalski J., Edelman R., Kaper J. B., Levine M. M. Initial clinical studies of CVD 112 Vibrio cholerae O139 live oral vaccine: safety and efficacy against experimental challenge. J Infect Dis. 1995 Sep;172(3):883–886. doi: 10.1093/infdis/172.3.883. [DOI] [PubMed] [Google Scholar]
  28. Tacket C. O., Losonsky G., Nataro J. P., Cryz S. J., Edelman R., Fasano A., Michalski J., Kaper J. B., Levine M. M. Safety and immunogenicity of live oral cholera vaccine candidate CVD 110, a delta ctxA delta zot delta ace derivative of El Tor Ogawa Vibrio cholerae. J Infect Dis. 1993 Dec;168(6):1536–1540. doi: 10.1093/infdis/168.6.1536. [DOI] [PubMed] [Google Scholar]
  29. Taniguchi H., Hirano H., Kubomura S., Higashi K., Mizuguchi Y. Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus. Microb Pathog. 1986 Oct;1(5):425–432. doi: 10.1016/0882-4010(86)90004-5. [DOI] [PubMed] [Google Scholar]
  30. Taniguchi H., Ohta H., Ogawa M., Mizuguchi Y. Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes. J Bacteriol. 1985 May;162(2):510–515. doi: 10.1128/jb.162.2.510-515.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thelestam M., Möllby R. Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. Infect Immun. 1975 Aug;12(2):225–232. doi: 10.1128/iai.12.2.225-232.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thornton J., Howard S. P., Buckley J. T. Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases. Biochim Biophys Acta. 1988 Mar 25;959(2):153–159. doi: 10.1016/0005-2760(88)90026-4. [DOI] [PubMed] [Google Scholar]
  33. Titball R. W. Bacterial phospholipases C. Microbiol Rev. 1993 Jun;57(2):347–366. doi: 10.1128/mr.57.2.347-366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trucksis M., Galen J. E., Michalski J., Fasano A., Kaper J. B. Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5267–5271. doi: 10.1073/pnas.90.11.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Van Loon F. P., Rabbani G. H., Bukhave K., Rask-Madsen J. Indomethacin decreases jejunal fluid secretion in addition to luminal release of prostaglandin E2 in patients with acute cholera. Gut. 1992 May;33(5):643–645. doi: 10.1136/gut.33.5.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Waldor M. K., Mekalanos J. J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 1996 Jun 28;272(5270):1910–1914. doi: 10.1126/science.272.5270.1910. [DOI] [PubMed] [Google Scholar]
  37. Weikel C. S., Grieco F. D., Reuben J., Myers L. L., Sack R. B. Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology. Infect Immun. 1992 Feb;60(2):321–327. doi: 10.1128/iai.60.2.321-327.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES