1887

Abstract

A membrane-bound, haemolytic phospholipase A (PLA) activity was detected in clinical strains of isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of gene (phospholipase A gene) from amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in . The detection of haemolytic PLA activity in indicates the presence of a potential virulence factor in this species and supports the hypothesis that is a possible opportunistic pathogen.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.45554-0
2004-06-01
2019-11-14
Loading full text...

Full text loading...

/deliver/fulltext/jmm/53/6/JM530603.html?itemId=/content/journal/jmm/10.1099/jmm.0.45554-0&mimeType=html&fmt=ahah

References

  1. Aabenhus, R., Permin, H., On, S. L. & Andersen, L. P. ( 2002;). Prevalence of Campylobacter concisus in diarrhoea of immunocompromised patients. Scand J Infect Dis 34, 248–252.[CrossRef]
    [Google Scholar]
  2. Bastyns, K., Chapelle, S., Vandamme, P., Goossens, H. & De Wachter, R. ( 1995;). Specific detection of Campylobacter concisus by PCR amplification of 23S rDNA areas. Mol Cell Probes 9, 247–250.[CrossRef]
    [Google Scholar]
  3. Brok, R. G., Boots, A. P., Dekker, N., Verheij, H. M. & Tommassen, J. ( 1998;). Sequence comparison of outer membrane phospholipases A: implications for structure and for the catalytic mechanism. Res Microbiol 149, 703–710.[CrossRef]
    [Google Scholar]
  4. Coelho, A., Andrade, J. R. C., Vicente, A. P. & Di Rita, V. J. ( 2000;). Cytotoxic cell vacuolating activity from Vibrio cholerae hemolysin. Infect Immun 68, 1700–1705.[CrossRef]
    [Google Scholar]
  5. de Geus, P., van Die, I., Bergmans, H., Tommassen, J. & de Haas, G. ( 1983;). Molecular cloning of pldA, the structural gene of outer membrane phospholipase of E.coli K12. Mol Gen Genet 190, 150–155.[CrossRef]
    [Google Scholar]
  6. Dekker, N. ( 2000;). Outer-membrane phospholipase A: known structure, unknown biological function. Mol Microbiol 35, 711–717.[CrossRef]
    [Google Scholar]
  7. Deshpande, R., Khan, M., Bhat, D. & Navalkar, R. ( 1997;). Isolation of a contact-dependent hemolysin from Mycobacterium tuberculosis. J Med Microbiol 46, 233–238.[CrossRef]
    [Google Scholar]
  8. Dorrell, N., Martino, M. C., Stabler, R. A., Ward, S. J., Zhang, Z. W., McColm, A. A., Farthing, M. J. G. & Wren, B. W. ( 1999;). Characterization of Helicobacter pylori PldA, a phospholipase with a role in colonization of the gastric mucosa. Gastroenterology 117, 1098–1104.[CrossRef]
    [Google Scholar]
  9. Engberg, J., On, S. L. W., Harrington, C. S. & Gerner-Smidt, P. ( 2000;). Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp.in human fecal samples as estimated by a re-evaluation of isolation methods for campylobacters. J Clin Microbiol 38, 286–291.
    [Google Scholar]
  10. Field, H. F., Headley, V. L., Payne, S. M. & Berry, L. J. ( 1986;). Influence of iron on growth, morphology, outer membrane protein composition, and synthesis of siderophores in Campylobacter jejuni. Infect Immun 54, 126–132.
    [Google Scholar]
  11. Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. ( 1973;). Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115, 717–722.
    [Google Scholar]
  12. Flieger, A., Gong, S., Faigle, M., Deeg, M., Bartmann, P. & Neumeister, B. ( 2000;). Novel phospholipase A activity secreted by Legionella species. J Bacteriol 182, 1321–1327.[CrossRef]
    [Google Scholar]
  13. Grant, K. A., Belandia, I. U., Dekker, N., Richardson, P. T. & Park, S. F. ( 1997;). Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis. Infect Immun 65, 1172–1180.
    [Google Scholar]
  14. Hertle, R., Hilger, M., Weingard-Kocher, S. & Walev, I. ( 1999;). Cytotoxic action of Serratia marcescens hemolysin on human epithelial cells. Infect Immun 67, 817–825.
    [Google Scholar]
  15. Hossain, A., Stewart-Tull, D. E. S. & Freer, J. H. ( 1993;). Heat-labile and heat-stable haemolysins of Campylobacter jejuni. FEMS Immunol Med Microbiol 6, 331–339.[CrossRef]
    [Google Scholar]
  16. Istivan, T. S., Ward, P. W., Lee, A., Coloe, P. J. & Smith, S. C. ( 1998;). Hemolysins of Campylobacter concisus. In Campylobacter, Helicobacter and Related Organisms, pp. 440–444. Edited by A. J. Lastovica, D. G. Newell & E. E. Lastovica. Cape Town: University of Cape Town Press.
  17. Kamma, J. J., Nakou, M. & Manti, F. A. ( 1994;). Microbiota of rapidly progressive periodontitis lesions in association with clinical parameters. J Periodontol 65, 1073–1078.[CrossRef]
    [Google Scholar]
  18. Kingma, R. L. & Egmond, M. R. ( 2002;). Substrate interferes with dimerisation of outer membrane phospholipase A. FEBS Lett 516, 31–34.[CrossRef]
    [Google Scholar]
  19. Laemmli, U. K. ( 1970;). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  20. Lastovica, A. J. & le Roux, E. ( 2000;). Efficient isolation of Campylobacteria from stools. J Clin Microbiol 38, 2798–2799.
    [Google Scholar]
  21. Leach, S. A. ( 1997;). Growth, survival and pathogenicity of enteric campylobacters. Rev Med Microbiol 8, 113–124.[CrossRef]
    [Google Scholar]
  22. le Roux, E. & Lastovica, A. J. ( 1998;). The Cape Town protocol: how to isolate the most campylobacters for your dollar, pound, franc, yen, etc. In Campylobacter, Helicobacter and Related Organisms, pp. 30–33. Edited by A. J. Lastovica, D. G. Newell & E. E. Lastovica. Cape Town: University of Cape Town Press.
  23. Lindblom, G. B., Sjogren, E., Hansson-Westerberg, J. & Kaijser, B. ( 1995;). Campylobacter upsaliensis, C.sputorum sputorum and C. concisus as common causes of diarrhoea in Swedish children. Scand J Infect Dis 27, 187–188.[CrossRef]
    [Google Scholar]
  24. Maher, M., Finnegan, C., Collins, E., Ward, B., Carroll, C. & Cormican, M. ( 2003;). Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces. J Clin Microbiol 41, 2980–2986.[CrossRef]
    [Google Scholar]
  25. Maniatis, T., Fritsch, E. F. & Sambrook, J. ( 1982;). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  26. Markwell, M. A. K., Haas, S. M., Bieber, L. L. & Tolbert, N. E. ( 1987;). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87, 206–210.
    [Google Scholar]
  27. Massari, P., Manetti, R., Burroni, D., Nuti, S., Norais, N., Rappuoli, R. & Telford, J. L. ( 1998;). Binding of the Helicobacter pylori vacuolating cytotoxin to target cells. Infect Immun 66, 3981–3984.
    [Google Scholar]
  28. Matoba, Y., Katsube, Y. & Sugiyama, M. ( 2002;). The crystal structure of prokaryotic phospholipase A2. J Biol Chem 277, 20059–20069.[CrossRef]
    [Google Scholar]
  29. Matsheka, M. I., Lastovica, A. J. & Elisha, B. G. ( 2001;). Molecular identification of Campylobacter concisus. J Clin Microbiol 39, 3684–3689.[CrossRef]
    [Google Scholar]
  30. Matsheka, M. I., Elisha, B. G., Lastovica, A. L. & On, S. L. ( 2002;). Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling. FEMS Microbiol Lett 211, 17–22.[CrossRef]
    [Google Scholar]
  31. Merino, S., Aguilar, A., Nogueras, M., Regue, M., Swift, S. & Tomas, J. ( 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]
  32. Moore, L. V., Moore, W. E., Cato, E. P., Smibert, R. M., Burmeister, J. A., Best, A. M. & Ranney, R. R. ( 1987;). Bacteriology of human gingivitis. J Dent Res 66, 989–995.[CrossRef]
    [Google Scholar]
  33. Musmanno, R. A., Russi, M., Figura, N., Guglielmetti, P., Zanchi, A., Signori, R. & Rossolini, A. ( 1998;). Unusual species of campylobacters isolated in the Siena Tuscany area, Italy. New Microbiol 21, 15–22.
    [Google Scholar]
  34. On, S. L. ( 1994;). Confirmation of human Campylobacter concisus isolates misidentified as Campylobacter mucosalis and suggestions for improved differentiation between the two species. J Clin Microbiol 32, 2305–2306.
    [Google Scholar]
  35. On, S. L. & Harrington, C. S. ( 2000;). Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles. FEMS Microbiol Lett 193, 161–169.[CrossRef]
    [Google Scholar]
  36. Pickett, C. L., Auffenberg, T., Pesci, E. C., Sheen, V. L. & Jusuf, S. S. D. ( 1992;). Iron acquisition and hemolysin production by Campylobacter jejuni. Infect Immun 60, 3872–3877.
    [Google Scholar]
  37. Russell, J. ( 1995;). Campylobacter like organisms: investigation of clinical and phenotypical aspects. In Applied Biology and Biotechnology, pp. 15–41. Melbourne: RMIT University Press.
  38. Russell, J. & Ward, P. ( 1998;). Adhesion and invasion of HEp2 cells by Campylobacter concisus from children with diarrhoea. In Campylobacter, Helicobacter and Related Organisms pp. 327–330. Edited by A. J. Lastovica, D. G. Newell & E. E. Lastovica. Cape Town: University of Cape Town Press.
  39. Schmiel, D. H., Wagar, E., Karamanou, L., Weeks, D. & Miller, V. L. ( 1998;). Phospholipase A of Yersinia enterocolitica contributes to pathogenesis in a mouse model. Infect Immun 66, 3941–3951.
    [Google Scholar]
  40. Song, J. K., Kim, M. K. & Rhee, J. S. ( 1999;). Cloning and expression of the gene encoding phospholipase A1 from Serratia sp.MK1 in Escherichia coli. J Biotechnol 72, 103–114.[CrossRef]
    [Google Scholar]
  41. Songer, J. G. ( 1997;). Bacterial phospholipases and their role in virulence. Trends Microbiol 5, 156–161.[CrossRef]
    [Google Scholar]
  42. Spiro, R. ( 1966;). Analysis of sugars found in glycoproteins: determination of neutral sugars. Methods Enzymol 8, 4–6.
    [Google Scholar]
  43. Tannaes, T., Dekker, N., Bukholm, G., Bijlsma, J. & Appelmelk, B. J. ( 2001;). Phase variation in the Helicobacter pylori phospholipase A gene and its role in acid adaptation. Infect Immun 69, 7334–7340.[CrossRef]
    [Google Scholar]
  44. Towbin, H., Staehelin, T. & Gordon, J. ( 1979;). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[CrossRef]
    [Google Scholar]
  45. Ubarretxena-Belandia, I., Cox, R. C., Dijkman, R., Egmond, M. R., Verheij, M. H. & Dekker, N. ( 1999;). Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor. Eur J Biochem 260, 794–800.[CrossRef]
    [Google Scholar]
  46. Vandamme, P., Falsen, E., Pot, B., Hoste, B., Kersters, K. & De Ley, J. ( 1989;). Identification of EF group 22 campylobacters from gastroenteritis cases as Campylobacter concisus. J Clin Microbiol 27, 1775–1781.
    [Google Scholar]
  47. Waite, M. ( 1996;). Phospholipases. In Biochemistry of Lipids, Lipoproteins and Membranes, pp. 211–236. Edited by D. E. Vance & J. E. Vance. Amsterdam: Elsevier
  48. Wassenaar, T. M. ( 1997;). Toxin production by Campylobacter spp. Clin Microbiol Rev 10, 466–476.
    [Google Scholar]
  49. Weinberg, E. D. ( 1985;). Enzymes, nutrition, and virulence. In Bacterial Enzymes and Virulence, pp. 1–16. Edited by I. A. Holder. Boca Raton, FL: CRC Press.
  50. Wood, G., Dutro, S. & Totten, P. ( 1999;). Target cell range of Haemophilus ducreyi hemolysin and its involvement in invasion of human epithelial cells. Infect Immun 67, 3740–3749.
    [Google Scholar]
  51. Xerry, J. & Owen, R. J. ( 2001;). Conservation and microdiversity of phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries. FEMS Immunol Med Microbiol 32, 17–25.[CrossRef]
    [Google Scholar]
  52. Ziprin, R. L., Young, C. R., Byrd, J. A., Stanker, L. H., Hume, M. E., Gray, S. A., Kim, B. J. & Konkel, M. E. ( 2001;). Role of Campylobacter jejuni potential virulence genes in cecal colonization. Avian Dis 45, 549–557.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.45554-0
Loading
/content/journal/jmm/10.1099/jmm.0.45554-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error