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. 1995 Oct;63(10):4063–4071. doi: 10.1128/iai.63.10.4063-4071.1995

Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin.

M Giry 1, M R Popoff 1, C von Eichel-Streiber 1, P Boquet 1
PMCID: PMC173571  PMID: 7558320

Abstract

The bacterial pathogen Clostridium difficle synthesizes two high-molecular-weight toxins (A and B), which exhibit toxic effects in vivo and in vitro. Here, we present evidence that the major intracellular targets of these two toxins are the Rho GTPases. Overexpression of RhoA, RhoB, or RhoC GTPases in transfected HeLa cells conferred an increased resistance to toxins A and B, indicating that these toxins cause their cytopathic effects primarily by affecting Rho proteins. In addition, toxin A and B treatment appeared to result in modification of Rho, since Rho isolated from toxin-treated cells had a decreased ability to be ADP-ribosylated by Clostridium botulinum C3 exoenzyme. In contrast, the lethal toxin (LT) of Clostridium sordellii, although structurally and immunologically related to C. difficile toxin B, appeared to induce cytopathic effects independently of the Rho GTPases. Overexpression of RhoA in transfected HeLa cells did not protect them from the effect of LT, and Rho isolated from lysates of LT-treated cells was not resistant to modification by C3. Immunofluorescence studies showed that LT treatment caused a cytopathic effect that was very different from those described for C. difficile toxins A and B, resulting in an increase in cortical F-actin, with a concomitant decrease in the number of stress fibers, and in the formation of numerous microvilli containing the actin-bundling protein fimbrin/plastin.

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Selected References

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  1. Adamson P., Paterson H. F., Hall A. Intracellular localization of the P21rho proteins. J Cell Biol. 1992 Nov;119(3):617–627. doi: 10.1083/jcb.119.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aktories K., Bärmann M., Ohishi I., Tsuyama S., Jakobs K. H., Habermann E. Botulinum C2 toxin ADP-ribosylates actin. Nature. 1986 Jul 24;322(6077):390–392. doi: 10.1038/322390a0. [DOI] [PubMed] [Google Scholar]
  3. Aktories K., Weller U., Chhatwal G. S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 1987 Feb 9;212(1):109–113. doi: 10.1016/0014-5793(87)81566-1. [DOI] [PubMed] [Google Scholar]
  4. Arseculeratne S. N., Panabokké R. G., Wijesundera S. The toxins responsible for the lesions of Clostridium sordelli gas gangrene. J Med Microbiol. 1969 Feb;2(1):37–53. doi: 10.1099/00222615-2-1-37. [DOI] [PubMed] [Google Scholar]
  5. Bartlett J. G., Chang T. W., Gurwith M., Gorbach S. L., Onderdonk A. B. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978 Mar 9;298(10):531–534. doi: 10.1056/NEJM197803092981003. [DOI] [PubMed] [Google Scholar]
  6. Bartlett J. G. Clostridium difficile: clinical considerations. Rev Infect Dis. 1990 Jan-Feb;12 (Suppl 2):S243–S251. doi: 10.1093/clinids/12.supplement_2.s243. [DOI] [PubMed] [Google Scholar]
  7. Bowman E. P., Uhlinger D. J., Lambeth J. D. Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein. J Biol Chem. 1993 Oct 15;268(29):21509–21512. [PubMed] [Google Scholar]
  8. Bretscher A., Weber K. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol. 1980 Jul;86(1):335–340. doi: 10.1083/jcb.86.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang T. W., Lauermann M., Bartlett J. G. Cytotoxicity assay in antibiotic-associated colitis. J Infect Dis. 1979 Nov;140(5):765–770. doi: 10.1093/infdis/140.5.765. [DOI] [PubMed] [Google Scholar]
  10. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chardin P., Madaule P., Tavitian A. Coding sequence of human rho cDNAs clone 6 and clone 9. Nucleic Acids Res. 1988 Mar 25;16(6):2717–2717. doi: 10.1093/nar/16.6.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Didsbury J., Weber R. F., Bokoch G. M., Evans T., Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem. 1989 Oct 5;264(28):16378–16382. [PubMed] [Google Scholar]
  14. Donta S. T., Sullivan N., Wilkins T. D. Differential effects of Clostridium difficile toxins on tissue-cultured cells. J Clin Microbiol. 1982 Jun;15(6):1157–1158. doi: 10.1128/jcm.15.6.1157-1158.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fiorentini C., Arancia G., Paradisi S., Donelli G., Giuliano M., Piemonte F., Mastrantonio P. Effects of Clostridium difficile toxins A and B on cytoskeleton organization in HEp-2 cells: a comparative morphological study. Toxicon. 1989;27(11):1209–1218. doi: 10.1016/0041-0101(89)90029-9. [DOI] [PubMed] [Google Scholar]
  16. Fiorentini C., Thelestam M. Clostridium difficile toxin A and its effects on cells. Toxicon. 1991;29(6):543–567. doi: 10.1016/0041-0101(91)90050-2. [DOI] [PubMed] [Google Scholar]
  17. Florin I., Thelestam M. Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B. Microb Pathog. 1986 Aug;1(4):373–385. doi: 10.1016/0882-4010(86)90069-0. [DOI] [PubMed] [Google Scholar]
  18. Gumbiner B. M. Breaking through the tight junction barrier. J Cell Biol. 1993 Dec;123(6 Pt 2):1631–1633. doi: 10.1083/jcb.123.6.1631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54. doi: 10.1146/annurev.cb.10.110194.000335. [DOI] [PubMed] [Google Scholar]
  20. Hecht G., Koutsouris A., Pothoulakis C., LaMont J. T., Madara J. L. Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. Gastroenterology. 1992 Feb;102(2):416–423. doi: 10.1016/0016-5085(92)90085-d. [DOI] [PubMed] [Google Scholar]
  21. Hecht G., Pothoulakis C., LaMont J. T., Madara J. L. Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers. J Clin Invest. 1988 Nov;82(5):1516–1524. doi: 10.1172/JCI113760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Just I., Fritz G., Aktories K., Giry M., Popoff M. R., Boquet P., Hegenbarth S., von Eichel-Streiber C. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J Biol Chem. 1994 Apr 8;269(14):10706–10712. [PubMed] [Google Scholar]
  23. Just I., Richter H. P., Prepens U., von Eichel-Streiber C., Aktories K. Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes. J Cell Sci. 1994 Jun;107(Pt 6):1653–1659. doi: 10.1242/jcs.107.6.1653. [DOI] [PubMed] [Google Scholar]
  24. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem. 1993 Nov 25;268(33):24535–24538. [PubMed] [Google Scholar]
  26. Madara J. L., Barenberg D., Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol. 1986 Jun;102(6):2125–2136. doi: 10.1083/jcb.102.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Madara J. L. Tight junction dynamics: is paracellular transport regulated? Cell. 1988 May 20;53(4):497–498. doi: 10.1016/0092-8674(88)90562-4. [DOI] [PubMed] [Google Scholar]
  28. Madaule P., Axel R. A novel ras-related gene family. Cell. 1985 May;41(1):31–40. doi: 10.1016/0092-8674(85)90058-3. [DOI] [PubMed] [Google Scholar]
  29. Martinez R. D., Wilkins T. D. Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C. difficile. J Med Microbiol. 1992 Jan;36(1):30–36. doi: 10.1099/00222615-36-1-30. [DOI] [PubMed] [Google Scholar]
  30. Moore R., Pothoulakis C., LaMont J. T., Carlson S., Madara J. L. C. difficile toxin A increases intestinal permeability and induces Cl- secretion. Am J Physiol. 1990 Aug;259(2 Pt 1):G165–G172. doi: 10.1152/ajpgi.1990.259.2.G165. [DOI] [PubMed] [Google Scholar]
  31. Munemitsu S., Innis M. A., Clark R., McCormick F., Ullrich A., Polakis P. Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol Cell Biol. 1990 Nov;10(11):5977–5982. doi: 10.1128/mcb.10.11.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Müller H., von Eichel-Streiber C., Habermann E. Morphological changes of cultured endothelial cells after microinjection of toxins that act on the cytoskeleton. Infect Immun. 1992 Jul;60(7):3007–3010. doi: 10.1128/iai.60.7.3007-3010.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ottlinger M. E., Lin S. Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells. Exp Cell Res. 1988 Jan;174(1):215–229. doi: 10.1016/0014-4827(88)90156-5. [DOI] [PubMed] [Google Scholar]
  34. Paterson H. F., Self A. J., Garrett M. D., Just I., Aktories K., Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990 Sep;111(3):1001–1007. doi: 10.1083/jcb.111.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Popoff M. R., Milward F. W., Bancillon B., Boquet P. Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells. Infect Immun. 1989 Aug;57(8):2462–2469. doi: 10.1128/iai.57.8.2462-2469.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Popoff M. R. Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin. Infect Immun. 1987 Jan;55(1):35–43. doi: 10.1128/iai.55.1.35-43.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Popoff M. R., Rubin E. J., Gill D. M., Boquet P. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect Immun. 1988 Sep;56(9):2299–2306. doi: 10.1128/iai.56.9.2299-2306.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ridley A. J., Hall A. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 1994 Jun 1;13(11):2600–2610. doi: 10.1002/j.1460-2075.1994.tb06550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  40. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  41. Rubin E. J., Gill D. M., Boquet P., Popoff M. R. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol Cell Biol. 1988 Jan;8(1):418–426. doi: 10.1128/mcb.8.1.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shinjo K., Koland J. G., Hart M. J., Narasimhan V., Johnson D. I., Evans T., Cerione R. A. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9853–9857. doi: 10.1073/pnas.87.24.9853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taylor N. S., Thorne G. M., Bartlett J. G. Comparison of two toxins produced by Clostridium difficile. Infect Immun. 1981 Dec;34(3):1036–1043. doi: 10.1128/iai.34.3.1036-1043.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thelestam M., Brönnegård M. Interaction of cytopathogenic toxin from Clostridium difficile with cells in tissue culture. Scand J Infect Dis Suppl. 1980;(Suppl 22):16–29. [PubMed] [Google Scholar]
  45. Vincent S., Jeanteur P., Fort P. Growth-regulated expression of rhoG, a new member of the ras homolog gene family. Mol Cell Biol. 1992 Jul;12(7):3138–3148. doi: 10.1128/mcb.12.7.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wegner A., Aktories K. ADP-ribosylated actin caps the barbed ends of actin filaments. J Biol Chem. 1988 Sep 25;263(27):13739–13742. [PubMed] [Google Scholar]
  47. Yeramian P., Chardin P., Madaule P., Tavitian A. Nucleotide sequence of human rho cDNA clone 12. Nucleic Acids Res. 1987 Feb 25;15(4):1869–1869. doi: 10.1093/nar/15.4.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang J., King W. G., Dillon S., Hall A., Feig L., Rittenhouse S. E. Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. J Biol Chem. 1993 Oct 25;268(30):22251–22254. [PubMed] [Google Scholar]
  49. von Eichel-Streiber C., Harperath U., Bosse D., Hadding U. Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related. Microb Pathog. 1987 May;2(5):307–318. doi: 10.1016/0882-4010(87)90073-8. [DOI] [PubMed] [Google Scholar]
  50. von Eichel-Streiber C., Laufenberg-Feldmann R., Sartingen S., Schulze J., Sauerborn M. Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B. Med Microbiol Immunol. 1990;179(5):271–279. doi: 10.1007/BF00192465. [DOI] [PubMed] [Google Scholar]

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