1887

Microbiology Society logo

Volume 148, Issue 7

Proteins released during high toxin production in Free

Abstract

The mechanism by which toxins A and B are released by is unknown and information about the other extracellular proteins of this bacterium is limited. The authors identified exported proteins from strain VPI 10463 during conditions promoting high toxin production. Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis. A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC. Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in , a protein supposedly exported via phage-specific holins. The two most abundant extracellular proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins. As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene in different serogroup reference strains. To conclude, uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): extracellular proteins , outer membrane efflux proteins , PBSX prophage and S-layer
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-7-2245
2002-07-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/7/1482245a.html?itemId=/content/journal/micro/10.1099/00221287-148-7-2245&mimeType=html&fmt=ahah

References

  1. Antelmann H., Tjalsma H., Voigt B., Ohlmeier S., Bron S., van Dijl J. M., Hecker M. 2001; A proteomic view on genome-based signal peptide predictions. Genome Res 11:1484–1502 [CrossRef]
     [Google Scholar]
  2. Braun V., Hundsberger T., Leukel T., Sauerborn M., von Eichel-Streiber C. 1996; Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181:29–38
     [Google Scholar]
  3. Calabi E., Ward S., Wren B., Paxton T., Panico M., Morris H., Dell A., Dougan G., Fairweather N. 2001; Molecular characterization of the surface layer proteins from Clostridium difficile . Mol Microbiol 40:1187–1199 [CrossRef]
     [Google Scholar]
  4. Cerquetti M., Molinari A., Sebastianelli A., Diociaiuti M., Petruzzelli R., Capo C., Mastrantonio P. 2000; Characterization of surface layer proteins from different Clostridium difficile clinical isolates. Microb Pathog 28:363–372 [CrossRef]
     [Google Scholar]
  5. Dove C. H., Wang S.-Z., Price S. B., Phelps C. J., Lyerly D. M., Wilkins T. D., Johnson J. L. 1990; Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun 58:480–488
     [Google Scholar]
  6. Dupuy B., Sonenshein A. L. 1998; Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol 27:107–120 [CrossRef]
     [Google Scholar]
  7. Dworkin J., Blaser M. J. 1997; Molecular mechanisms of Campylobacter fetus surface layer protein expression. Mol Microbiol 26:433–440 [CrossRef]
     [Google Scholar]
  8. Economou A. 1999; Following the leader: bacterial protein export through the Sec-pathway. Trends Microbiol 7:315–319 [CrossRef]
     [Google Scholar]
  9. Hammond G. A., Johnson J. L. 1995; The toxigenic element of Clostridium difficile strain VPI 10463. Microb Pathog 19:203–213 [CrossRef]
     [Google Scholar]
  10. Hammond G. A., Lyerly D. M., Johnson J. L. 1997; Transcriptional analysis of the toxigenic element of Clostridium difficile. Microb Pathog 22:143–154 [CrossRef]
     [Google Scholar]
  11. Haslam S. C., Ketley J. M., Mitchell T. J., Stephen J., Burdon D. W., Candy D. C. A. 1986; Growth of Clostridium difficile and production of toxins A and B in complex and defined media. J Med Microbiol 21:293–297 [CrossRef]
     [Google Scholar]
  12. Hundsberger H., Braun V., Weidmann M., Leukel P., Sauerborn M., von Eichel-Streiber C. 1997; Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur J Biochem 244:735–742 [CrossRef]
     [Google Scholar]
  13. Izard J. W., Kendall D. A. 1994; Signal peptides: exquisitely designed transport promoters. Mol Microbiol 13:765–773 [CrossRef]
     [Google Scholar]
  14. Kamiya S., Ogura H., Meng X. Q., Nakamura S. 1992; Correlation between cytotoxin production and sporulation in Clostridium difficile . J Med Microbiol 37:206–210 [CrossRef]
     [Google Scholar]
  15. Karjalainen T., Waligora-Dupriet A. J., Cerquetti M., Spigaglia P., Maggioni A., Mauri P., Mastrantonio P. 2001; Molecular and genomic analysis of genes encoding surface-anchored proteins from Clostridium difficile . Infect Immun 69:3442–3446 [CrossRef]
     [Google Scholar]
  16. Karlsson S., Burman L. G., Åkerlund T. 1999; Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids. Microbiology 145:1683–1693 [CrossRef]
     [Google Scholar]
  17. Karlsson S., Lindberg A., Norin E., Burman L. G., Åkerlund T. 2000; Toxins, butyric acid and other short chain fatty acids are co-ordinately expressed and down regulated by cysteine in Clostridium difficile. Infect Immun 68:5881–5888 [CrossRef]
     [Google Scholar]
  18. Ketley J. M., Haslam S. C., Mitchell T. J., Stephen J., Candy D. C. A., Burdon D. W. 1984; Production and release of toxins A and B by Clostridium difficile. J Med Microbiol 18:385–391 [CrossRef]
     [Google Scholar]
  19. Ketley J. M., Mitchell T. J., Haslam S. C., Stephen J., Candy D. C. A., Burdon D. W. 1986; Sporogenesis and toxin A production by Clostridium difficile. J Med Microbiol 22:33–38 [CrossRef]
     [Google Scholar]
  20. Krogh S., O’Reilly M., Nolan N., Devine K. M. 1996; The phage-like element PBSX and part of the skin element, which are resident at different locations on the Bacillus subtilis chromosome, are highly homologous. Microbiology 142:2031–2040 [CrossRef]
     [Google Scholar]
  21. Lazarevic V., Margot P., Suldo B., Karamata D. 1992; Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N -acetylmuramoyl-l-alanine amidase and its modifier. J Gen Microbiol 138:1949–1961 [CrossRef]
     [Google Scholar]
  22. Luckevich M. D., Beveridge T. J. 1989; Characterization of a dynamic S-layer on Bacillus thuringiensis . J Bacteriol 171:6656–6667
     [Google Scholar]
  23. Lupas A., Engelhardt H., Peters J., Santarius U., Volker S., Baumeister W. 1994; Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis. J Bacteriol 176:1224–1233
     [Google Scholar]
  24. Lyerly D. M., Wilkins T. D. 1986; Commercial latex test for Clostridium difficile toxin A does not detect toxin A. J Clin Microbiol 23:622–623
     [Google Scholar]
  25. Lyerly D. M., Wilkins T. D. 1995; Clostridium difficile. In Infections of the Gastrointestinal Tract pp 867–891 Edited by Blaser M. J., Smith P. D., Ravdin J. I., Greenberg H. B., Guerrant R. L. New York: Raven Press;
     [Google Scholar]
  26. Mani N., Dupuy B. 2001; Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci USA 98:5844–5849 [CrossRef]
     [Google Scholar]
  27. Mauri P. L., Pietta P. G., Maggioni A., Cerquetti M., Sebastianelli A., Mastrantonio P. 1999; Characterization of surface layer proteins from Clostridium difficile by liquid chromatography/ electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 13:695–703 [CrossRef]
     [Google Scholar]
  28. Moncrief J. S., Barroso L. A., Wilkins T. D. 1997; Positive regulation of Clostridium difficile toxins. Infect Immun 65:1105–1108
     [Google Scholar]
  29. Okamoto K., Mudd J. A., Marmur J. 1968; Conversion of Bacillus subtilis DNA to phage DNA following mitomycin C. J Mol Biol 137:261–267
     [Google Scholar]
  30. Onderdonk A. B., Lowe B. R., Bartlett J. G. 1979; Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation. Appl Environ Microbiol 38:637–641
     [Google Scholar]
  31. Paulsen I. T., Brown M. H., Skurray R. A. 1996; Proton-dependent multidrug efflux systems. Microbiol Rev 60:575–608
     [Google Scholar]
  32. Sa’ra M., Kuen B., Mayer H. F., Mandl F., Schuster K. C., Sleytr U. B. 1996; Dynamics in oxygen induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer deficient variant T5 in continuous culture and studies on the cell wall composition. J Bacteriol 178:2108–2117
     [Google Scholar]
  33. Seaman E., Tarmy E., Marmur J. 1964; Inducible phages of Bacillus subtilis . Biochemistry 3:607–612 [CrossRef]
     [Google Scholar]
  34. Stubbs S. L., Brazier J. S., O’Neill G. L., Duerden B. I. 1999; PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol 37:461–463
     [Google Scholar]
  35. Takeoka A., Takumi K., Koga T., Kawata T. 1991; Purification and characterization of S-layer proteins from Clostridium difficile GAI 0714. J Gen Microbiol 137:261–267 [CrossRef]
     [Google Scholar]
  36. Tan K. S., Wee B. Y., Song K. P. 2000; Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile . J Med Microbiol 50:613–619
     [Google Scholar]
  37. von Eichel-Streiber C., Laufenberg-Feldman R., Sartingen S., Schulze J., Sauerborn M. 1992; Comparative sequence analysis of the Clostridium difficile toxins A and B. Mol Gen Genet 233:260–268 [CrossRef]
     [Google Scholar]
  38. von Eichel-Streiber C., Boquet P., Sauerborn M., Thelestam M. 1996; Large clostridial cytotoxins – a family of glycosyltransferase modifying small GTP-binding proteins. Trends Microbiol 4:375–382 [CrossRef]
     [Google Scholar]
  39. Waligora A. J., Hennequin C., Mullany P., Bourlioux P., Collignon A., Karjalainen T. 2001; Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infect Immun 69:2144–2153 [CrossRef]
     [Google Scholar]
  40. Yamakawa K., Kamiya S., Meng X. Q., Karasawa T., Nakamura S. 1994; Toxin production by Clostridium difficile in a defined medium with limited amino acids. J Med Microbiol 41:319–323 [CrossRef]
     [Google Scholar]
  41. Yamakawa K., Karasawa T., Ikoma S., Nakamura S. 1996; Enhancement of Clostridium difficile toxin production in biotin-limited conditions. J Med Microbiol 44:111–114 [CrossRef]
     [Google Scholar]
  42. Zgurskaya H. I., Nikaido H. 2000; Cross-linked complex between oligomeric periplasmic lipoprotein AcrA and the inner-membrane-associated multidrug efflux pump AcrB from Escherichia coli. J Bacteriol 182:4264–4267
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-7-2245
Loading
Proteins released during high toxin production in Clostridium difficile
Microbiology 148, 2245 (2002); https://doi.org/10.1099/00221287-148-7-2245
/content/journal/micro/10.1099/00221287-148-7-2245
/content/journal/micro/10.1099/00221287-148-7-2245
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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