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. 1997 Mar;65(3):1105–1108. doi: 10.1128/iai.65.3.1105-1108.1997

Positive regulation of Clostridium difficile toxins.

J S Moncrief 1, L A Barroso 1, T D Wilkins 1
PMCID: PMC175096  PMID: 9038324

Abstract

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.

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Selected References

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  1. Barroso L. A., Moncrief J. S., Lyerly D. M., Wilkins T. D. Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity. Microb Pathog. 1994 Apr;16(4):297–303. doi: 10.1006/mpat.1994.1030. [DOI] [PubMed] [Google Scholar]
  2. Barroso L. A., Wang S. Z., Phelps C. J., Johnson J. L., Wilkins T. D. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 1990 Jul 11;18(13):4004–4004. doi: 10.1093/nar/18.13.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartlett J. G. Clostridium difficile: history of its role as an enteric pathogen and the current state of knowledge about the organism. Clin Infect Dis. 1994 May;18 (Suppl 4):S265–S272. doi: 10.1093/clinids/18.supplement_4.s265. [DOI] [PubMed] [Google Scholar]
  4. Bartlett J. G., Moon N., Chang T. W., Taylor N., Onderdonk A. B. Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis. Gastroenterology. 1978 Nov;75(5):778–782. [PubMed] [Google Scholar]
  5. Borriello S. P., Wren B. W., Hyde S., Seddon S. V., Sibbons P., Krishna M. M., Tabaqchali S., Manek S., Price A. B. Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect Immun. 1992 Oct;60(10):4192–4199. doi: 10.1128/iai.60.10.4192-4199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dillon S. T., Rubin E. J., Yakubovich M., Pothoulakis C., LaMont J. T., Feig L. A., Gilbert R. J. Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B. Infect Immun. 1995 Apr;63(4):1421–1426. doi: 10.1128/iai.63.4.1421-1426.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dove C. H., Wang S. Z., Price S. B., Phelps C. J., Lyerly D. M., Wilkins T. D., Johnson J. L. Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun. 1990 Feb;58(2):480–488. doi: 10.1128/iai.58.2.480-488.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frey S. M., Wilkins T. D. Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A. Infect Immun. 1992 Jun;60(6):2488–2492. doi: 10.1128/iai.60.6.2488-2492.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garnier T., Cole S. T. Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens. Plasmid. 1988 Mar;19(2):134–150. doi: 10.1016/0147-619x(88)90052-2. [DOI] [PubMed] [Google Scholar]
  10. Garnier T., Cole S. T. Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro. Mol Microbiol. 1988 Sep;2(5):607–614. doi: 10.1111/j.1365-2958.1988.tb00069.x. [DOI] [PubMed] [Google Scholar]
  11. Hammond G. A., Johnson J. L. The toxigenic element of Clostridium difficile strain VPI 10463. Microb Pathog. 1995 Oct;19(4):203–213. doi: 10.1016/s0882-4010(95)90263-5. [DOI] [PubMed] [Google Scholar]
  12. Hauser D., Eklund M. W., Boquet P., Popoff M. R. Organization of the botulinum neurotoxin C1 gene and its associated non-toxic protein genes in Clostridium botulinum C 468. Mol Gen Genet. 1994 Jun 15;243(6):631–640. doi: 10.1007/BF00279572. [DOI] [PubMed] [Google Scholar]
  13. Just I., Selzer J., Wilm M., von Eichel-Streiber C., Mann M., Aktories K. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature. 1995 Jun 8;375(6531):500–503. doi: 10.1038/375500a0. [DOI] [PubMed] [Google Scholar]
  14. Just I., Wilm M., Selzer J., Rex G., von Eichel-Streiber C., Mann M., Aktories K. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J Biol Chem. 1995 Jun 9;270(23):13932–13936. doi: 10.1074/jbc.270.23.13932. [DOI] [PubMed] [Google Scholar]
  15. Krivan H. C., Clark G. F., Smith D. F., Wilkins T. D. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc. Infect Immun. 1986 Sep;53(3):573–581. doi: 10.1128/iai.53.3.573-581.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lyerly D. M., Barroso L. A., Wilkins T. D., Depitre C., Corthier G. Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect Immun. 1992 Nov;60(11):4633–4639. doi: 10.1128/iai.60.11.4633-4639.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lyerly D. M., Krivan H. C., Wilkins T. D. Clostridium difficile: its disease and toxins. Clin Microbiol Rev. 1988 Jan;1(1):1–18. doi: 10.1128/cmr.1.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lyerly D. M., Sullivan N. M., Wilkins T. D. Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. J Clin Microbiol. 1983 Jan;17(1):72–78. doi: 10.1128/jcm.17.1.72-78.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mullany P., Wilks M., Puckey L., Tabaqchali S. Gene cloning in Clostridium difficile using Tn916 as a shuttle conjugative transposon. Plasmid. 1994 May;31(3):320–323. doi: 10.1006/plas.1994.1036. [DOI] [PubMed] [Google Scholar]
  20. Nakamura S., Mikawa M., Tanabe N., Yamakawa K., Nishida S. Effect of clindamycin on cytotoxin production by Clostridium difficile. Microbiol Immunol. 1982;26(11):985–992. doi: 10.1111/j.1348-0421.1982.tb00248.x. [DOI] [PubMed] [Google Scholar]
  21. Onderdonk A. B., Lowe B. R., Bartlett J. G. Effect of environmental stress on Clostridium difficile toxin levels during continuous cultivation. Appl Environ Microbiol. 1979 Oct;38(4):637–641. doi: 10.1128/aem.38.4.637-641.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Phelps C. J., Lyerly D. L., Johnson J. L., Wilkins T. D. Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli. Infect Immun. 1991 Jan;59(1):150–153. doi: 10.1128/iai.59.1.150-153.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sullivan N. M., Pellett S., Wilkins T. D. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1032–1040. doi: 10.1128/iai.35.3.1032-1040.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Torres J. F. Purification and characterisation of toxin B from a strain of Clostridium difficile that does not produce toxin A. J Med Microbiol. 1991 Jul;35(1):40–44. doi: 10.1099/00222615-35-1-40. [DOI] [PubMed] [Google Scholar]
  25. Tucker K. D., Wilkins T. D. Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y. Infect Immun. 1991 Jan;59(1):73–78. doi: 10.1128/iai.59.1.73-78.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yamakawa K., Karasawa T., Ikoma S., Nakamura S. Enhancement of Clostridium difficile toxin production in biotin-limited conditions. J Med Microbiol. 1996 Feb;44(2):111–114. doi: 10.1099/00222615-44-2-111. [DOI] [PubMed] [Google Scholar]
  27. von Eichel-Streiber C., Laufenberg-Feldmann R., Sartingen S., Schulze J., Sauerborn M. Comparative sequence analysis of the Clostridium difficile toxins A and B. Mol Gen Genet. 1992 May;233(1-2):260–268. doi: 10.1007/BF00587587. [DOI] [PubMed] [Google Scholar]

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