1887

Abstract

The mechanism by which causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of , but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an -helix bundle and a unique subdomain containing a hydrophobic -hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/014134-0
2008-03-01
2020-04-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/3/693.html?itemId=/content/journal/micro/10.1099/mic.0.2007/014134-0&mimeType=html&fmt=ahah

References

  1. Arnaud M., Chastanet A., Débarbouillé M.. 2004; New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol70:6887–6891
    [Google Scholar]
  2. Beecher D. J., Macmillan J. D.. 1991; Characterization of the components of hemolysin-BL from Bacillus cereus . Infect Immun59:1778–1784
    [Google Scholar]
  3. del Castillo F. J., Leal S. C., Moreno F., del Castillo I.. 1997; The Escherichia coli K-12 sheA gene encodes a 34-kDa secreted haemolysin. Mol Microbiol25:107–115
    [Google Scholar]
  4. Dietmann S., Holm L.. 2001; Identification of homology in protein structure classification. Nat Struct Biol8:953–957
    [Google Scholar]
  5. Dietrich R., Fella C., Strich S., Märtlbauer E.. 1999; Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus . Appl Environ Microbiol65:4470–4474
    [Google Scholar]
  6. Dietrich R., Moravek M., Burk C., Granum P. E., Märtlbauer E.. 2005; Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex. Appl Environ Microbiol71:8214–8220
    [Google Scholar]
  7. Ehling-Schulz M., Svensson B., Guinebretiere M. H., Lindbäck T., Andersson M., Schulz A., Fricker M., Christiansson A., Granum P. E.. other authors 2005; Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology151:183–197
    [Google Scholar]
  8. Eifler N., Vetsch M., Gregorini M., Ringler P., Chami M., Philippsen A., Fritz A., Müller S. A., Glockshuber R.. other authors 2006; Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state. EMBO J25:2652–2661
    [Google Scholar]
  9. Estacion M., Weinberg J. S., Sinkins W. G., Schilling W. P.. 2003; Blockade of maitotoxin-induced endothelial cell lysis by glycine and l-alanine. Am J Physiol Cell Physiol284:C1006–C1020
    [Google Scholar]
  10. Fink S. L., Cookson B. T.. 2006; Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cell Microbiol8:1812–1825
    [Google Scholar]
  11. Gibbons S. J., Washburn K. B., Talamo B. R.. 2001; P2X7 receptors in rat parotid acinar cells: formation of large pores. J Auton Pharmacol21:181–190
    [Google Scholar]
  12. Gibrat J. F., Madej T., Bryant S. H.. 1996; Surprising similarities in structure comparison. Curr Opin Struct Biol6:377–385
    [Google Scholar]
  13. Granum P. E., O'Sullivan K., Lund T.. 1999; The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus . FEMS Microbiol Lett177:225–229
    [Google Scholar]
  14. Green J., Baldwin M. L.. 1997; The molecular basis for the differential regulation of the hlyE -encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved Activating Region 1 contact of HlyX. Microbiology143:3785–3793
    [Google Scholar]
  15. Guérout-Fleury A. M., Shazand K., Frandsen N., Stragier P.. 1995; Antibiotic-resistance cassettes for Bacillus subtilis . Gene167:335–336
    [Google Scholar]
  16. Hardy S. P., Lund T., Granum P. E.. 2001a; CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiol Lett197:47–51
    [Google Scholar]
  17. Hardy S. P., Ritchie C., Allen M. C., Ashley R. H., Granum P. E.. 2001b; Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins. Biochim Biophys Acta1515:38–43
    [Google Scholar]
  18. Hauge S.. 1955; Food poisoning caused by aerobic spore-forming bacilli. J Appl Bacteriol18:591–595
    [Google Scholar]
  19. Holm L., Park J.. 2000; DaliLite workbench for protein structure comparison. Bioinformatics16:566–567
    [Google Scholar]
  20. Koradi R., Billeter M., Wuthrich K.. 1996; MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph14:51–55
    [Google Scholar]
  21. Lindbäck T., Granum P. E.. 2006; Detection and purification of Bacillus cereus enterotoxins. In Methods in Biotechnology, Volume 21: Food-borne pathogens: Methods and protocols pp15–26 Edited by Adley C. C. Totowa, NJ: Humana Press;
    [Google Scholar]
  22. Lindbäck T., Fagerlund A., Rødland M. S., Granum P. E.. 2004; Characterization of the Bacillus cereus Nhe enterotoxin. Microbiology150:3959–3967
    [Google Scholar]
  23. Ludwig A., Bauer S., Benz R., Bergmann B., Goebel W.. 1999; Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12. Mol Microbiol31:557–567
    [Google Scholar]
  24. Ludwig A., von Rhein C., Bauer S., Huttinger C., Goebel W.. 2004; Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains. J Bacteriol186:5311–5320
    [Google Scholar]
  25. Lund T., Granum P. E.. 1996; Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol Lett141:151–156
    [Google Scholar]
  26. Lund T., Granum P. E.. 1997; Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus . Microbiology143:3329–3336
    [Google Scholar]
  27. Lund T., De Buyser M. L., Granum P. E.. 2000; A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol38:254–261
    [Google Scholar]
  28. Masson L., Prefontaine G., Brousseau R.. 1989; Transformation of Bacillus thuringiensis vegetative cells by electroporation. FEMS Microbiol Lett60:273–278
    [Google Scholar]
  29. Miles G., Bayley H., Cheley S.. 2002; Properties of Bacillus cereus hemolysin II: a heptameric transmembrane pore. Protein Sci11:1813–1824
    [Google Scholar]
  30. Moravek M., Dietrich R., Buerk C., Broussolle V., Guinebretiere M. H., Granum P. E., Nguyen-The C., Märtlbauer E.. 2006; Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses. FEMS Microbiol Lett257:293–298
    [Google Scholar]
  31. Oscarsson J., Mizunoe Y., Uhlin B. E., Haydon D. J.. 1996; Induction of haemolytic activity in Escherichia coli by the slyA gene product. Mol Microbiol20:191–199
    [Google Scholar]
  32. Oscarsson J., Mizunoe Y., Li L., Lai X. H., Wieslander A., Uhlin B. E.. 1999; Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli . Mol Microbiol32:1226–1238
    [Google Scholar]
  33. Oscarsson J., Westermark M., Löfdahl S., Olsen B., Palmgren H., Mizunoe Y., Wai S. N., Uhlin B. E.. 2002; Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars Typhi and Paratyphi A. Infect Immun70:5759–5769
    [Google Scholar]
  34. Ouhib O., Clavel T., Schmitt P.. 2006; The production of Bacillus cereus enterotoxins is influenced by carbohydrate and growth rate. Curr Microbiol53:222–226
    [Google Scholar]
  35. Parker M. W., Feil S. C.. 2005; Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol88:91–142
    [Google Scholar]
  36. Pelegrin P., Surprenant A.. 2006; Pannexin-1 mediates large pore formation and interleukin-1 β release by the ATP-gated P2X7 receptor. EMBO J25:5071–5082
    [Google Scholar]
  37. Pelegrin P., Surprenant A.. 2007; Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1 β release through a dye uptake-independent pathway. J Biol Chem282:2386–2394
    [Google Scholar]
  38. Planchot V., Roger P., Colonna P.. 2000; Suitability of starch granule porosity for biosynthesis and amylolysis susceptibility. Starch52:333–339
    [Google Scholar]
  39. Ramarao N., Lereclus D.. 2006; Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively. Microbes Infect8:1483–1491
    [Google Scholar]
  40. Ryan P. A., Macmillan J. D., Zilinskas B. A.. 1997; Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus . J Bacteriol179:2551–2556
    [Google Scholar]
  41. Scherrer R., Gerhardt P.. 1971; Molecular sieving by Bacillus megaterium cell wall and protoplast. J Bacteriol107:718–735
    [Google Scholar]
  42. Schilling W. P., Sinkins W. G., Estacion M.. 1999; Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts. Am J Physiol Cell Physiol277:C755–C765
    [Google Scholar]
  43. Schoeni J. L., Wong A. C. L.. 2005; Bacillus cereus food poisoning and its toxins. J Food Prot68:636–648
    [Google Scholar]
  44. Schwede T., Kopp J., Guex N., Peitsch M. C.. 2003; SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res31:3381–3385
    [Google Scholar]
  45. Song L., Hobaugh M. R., Shustak C., Cheley S., Bayley H., Gouaux J. E.. 1996; Structure of staphylococcal α -hemolysin, a heptameric transmembrane pore. Science274:1859–1866
    [Google Scholar]
  46. Tzokov S. B., Wyborn N. R., Stillman T. J., Jamieson S., Czudnochowski N., Artymiuk P. J., Green J., Bullough P. A.. 2006; Structure of the hemolysin E (HlyE, ClyA, and SheA) channel in its membrane-bound form. J Biol Chem281:23042–23049
    [Google Scholar]
  47. Wallace A. J., Stillman T. J., Atkins A., Jamieson S. J., Bullough P. A., Green J., Artymiuk P. J.. 2000; E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy. Cell100:265–276
    [Google Scholar]
  48. Whitaker J. R., Granum P. E.. 1980; An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal Biochem109:156–159
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/014134-0
Loading
/content/journal/micro/10.1099/mic.0.2007/014134-0
Loading

Data & Media loading...

Most cited this month

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