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Research article

Clinical Microbiology and Biomedical Sciences

Effect of Antisera from Clostridium difficile-Infected Mice on Toxin-A-Induced Colonic Epithelial Cell Death Signaling

Dae Hong Kim 1, Ik Hwan Lee 1, Seung Taek Nam 1, Hyo Jung Nam 1, Jin Ku Kang 1, Heon Seok 2, Jae Sam Hwang 3 and Ho Kim 1*

1Department of Life Science, College of Natural Science, Daejin University, Pocheon 487-711, Republic of Korea, 2Department of Biomedical Science, Jungwon University, Geosan 367-700, Republic of Korea, 3Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707, Republic of Korea

Received: February 3, 2014; Accepted: February 7, 2014

J. Microbiol. Biotechnol. 2014; 24(5): 696-703

Published May 28, 2014 https://doi.org/10.4014/jmb.1401.01059

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Clostridium difficile causes mucosal damage and diarrhea by releasing two exotoxins: toxin A and toxin B. C. difficile colitis is associated with alterations in bowel flora and the failure to mount an effective antibody response. The aim of the current study was to investigate whether antitoxin sera prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss in cultured colonic epithelial cells. Serum samples were isolated from mice that survived a C. difficile infection following antibiotic treatment, and the antitoxin effects of these samples were investigated in toxin-A-exposed HT29 colonic epithelial cells and a toxin-A-induced animal model of gut inflammation. Unchallenged mice did not produce IgG against toxin A, whereas serum (antiserum) from C. difficile-challenged mice showed significant IgG responses against toxin A. Treatment with the antiserum markedly inhibited mucosal damage and inflammation in the toxin-A-treated mouse model. In contrast to control mouse serum, the antiserum also markedly inhibited toxin-A-induced DNA fragmentation, dephosphorylation of paxillin and Epo receptor (EpoR), deacetylation of tubulin, and upregulation of p21(WAF1/CIP1) and p53. Taken together, these results reveal that the generated antitoxin serum has biotherapeutic effects in preventing various C. difficile toxin-Ainduced cellular toxicities.

Keywords

clostridium difficile, toxin A, antitoxin serum, apoptosis, gut inflammation, cell stress signals

References

  1. Chen X, Katchar K, Goldsmith JD, Nanthakumar N, Cheknis A, Gerding DN, Kelly CP. 2008. A mouse model of Clostridium difficile-associated disease. Gastroenterology 135:1984-1992.
    CrossRef
  2. Giannasca PJ, Zhang ZX, Lei WD, Boden JA, Giel MA, Monath TP, Thomas WD. 1999. Serum antitoxin antibodies mediate systemic and mucosal protection from Clostridium difficile disease in hamsters. Infect. Immun. 67: 527-538.
  3. Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375: 500-503.
    CrossRef
  4. Kang JK, Hwang JS, Nam HJ, Ahn KJ, Seok H, Kim SK, et al. 2011. The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity. Antimicrob. Agents Chemother. 55: 4850-4857.
    CrossRef
  5. Kaur S , Vaishnavi C, K ochhar R , Prasad KK, R ay P . 2012. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection. Indian J. Med. Microbiol. 30: 431-436.
    CrossRef
  6. Kelly CP, Pothoulakis C, LaMont JT. 1994. Clostridium difficile colitis. N. Engl. J. Med. 330: 257-262.
    CrossRef
  7. Kim H, Kokkotou E, Na X, Rhee SH, Moyer MP, Pothoulakis C, Lamont JT. 2005. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 129: 1875-1888.
    CrossRef
  8. Kim H, Rhee SH, Kokkotou E, Na X, Savidge T, Moyer MP, et al. 2005. Clostridium difficile t oxin A r egulates ind ucible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J. Biol. Chem. 280: 21237-21245.
    CrossRef
  9. Kim H, Rhee SH, Pothoulakis C, Lamont JT. 2007. Inflammation and apoptosis in Clostridium difficile enteritis is mediated by PGE2 up-regulation of Fas ligand. Gastroenterology 133: 875886.
    CrossRef
  10. Kyne L, Warny M, Qamar A, Kelly CP. 2001. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet 357: 189-193.
    CrossRef
  11. Kyne L, Warny M, Qamar A, Kelly CP. 2000. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N. Engl. J. Med. 342: 390-397.
    CrossRef
  12. Lai KK, Melvin ZS, Menard MJ, Kotilainen HR, Baker S. 1997. Clostridium difficile-associated diarrhea: epidemiology, risk factors, and infection control. Infect. Control Hosp. Epidemiol. 18: 628-632.
    CrossRef
  13. Nam HJ, Kang JS, Kim SK, Ahn KJ, Seok H, Park SJ, et al. 2010. Clostridium difficile toxin A decreases acetylation of tubulin, leading to microtubule depolymerization through activation of histone deacetylase 6, and this mediates acute inflammation. J. Biol. Chem. 285: 32888-32896.
    CrossRef
  14. Nam ST, Seok H, Kim DH, Nam HJ, Kang JK, Eom JH, et al. 2012. Clostridium difficile toxin A inhibits erythropoietin receptor-mediated colonocyte focal adhesion through inactivation of Janus Kinase-2. J. Microbiol. Biotechnol. 22: 1629-1635.
    CrossRef
  15. Nandy RK, Albert MJ, Ghose AC. 1996. Serum antibacterial and antitoxin r esponses in clinical cholera c aused by Vibrio cholerae O139 Bengal and evaluation of their importance in protection. Vaccine 14: 1137-1142.
    CrossRef
  16. Pothoulakis C, Gilbert RJ, Cladaras C, Castagliuolo I, Semenza G, Hitti Y, et al. 1996. Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A. J. Clin. Invest. 98: 641-649.
    CrossRef
  17. Pothoulakis C, LaMont JT. 1993. Clostridium difficile colitis and diarrhea. Gastroenterol. Clin. North Am. 22: 623-637.
  18. Pothoulakis C, Lamont JT. 2001. Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins. Am. J. Physiol. Gastrointest. Liver Physiol. 280: G178G183.
  19. Pothoulakis C, Triadafilopoulos G, Clark M, Franzblau C, LaMont JT. 1986. Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa. Gastroenterology 91: 1147-1153.
  20. Sun X, Wang H, Zhang Y, Chen K, Davis B, Feng H. 2011. Mouse relapse model of Clostridium difficile infection. Infect. Immun. 79: 2856-2864.
    CrossRef
  21. Warny M, Vaerman JP, Avesani V, Delmee M. 1994. Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection. Infect. Immun. 62: 384-389.

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(5): 696-703

Published online May 28, 2014 https://doi.org/10.4014/jmb.1401.01059

Copyright © The Korean Society for Microbiology and Biotechnology.

Effect of Antisera from Clostridium difficile-Infected Mice on Toxin-A-Induced Colonic Epithelial Cell Death Signaling

Dae Hong Kim 1, Ik Hwan Lee 1, Seung Taek Nam 1, Hyo Jung Nam 1, Jin Ku Kang 1, Heon Seok 2, Jae Sam Hwang 3 and Ho Kim 1*

1Department of Life Science, College of Natural Science, Daejin University, Pocheon 487-711, Republic of Korea, 2Department of Biomedical Science, Jungwon University, Geosan 367-700, Republic of Korea, 3Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707, Republic of Korea

Received: February 3, 2014; Accepted: February 7, 2014

Abstract

Clostridium difficile causes mucosal damage and diarrhea by releasing two exotoxins: toxin A
and toxin B. C. difficile colitis is associated with alterations in bowel flora and the failure to
mount an effective antibody response. The aim of the current study was to investigate whether
antitoxin sera prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment,
and tight junction loss in cultured colonic epithelial cells. Serum samples were isolated from
mice that survived a C. difficile infection following antibiotic treatment, and the antitoxin
effects of these samples were investigated in toxin-A-exposed HT29 colonic epithelial cells and
a toxin-A-induced animal model of gut inflammation. Unchallenged mice did not produce IgG
against toxin A, whereas serum (antiserum) from C. difficile-challenged mice showed
significant IgG responses against toxin A. Treatment with the antiserum markedly inhibited
mucosal damage and inflammation in the toxin-A-treated mouse model. In contrast to control
mouse serum, the antiserum also markedly inhibited toxin-A-induced DNA fragmentation,
dephosphorylation of paxillin and Epo receptor (EpoR), deacetylation of tubulin, and
upregulation of p21(WAF1/CIP1) and p53. Taken together, these results reveal that the
generated antitoxin serum has biotherapeutic effects in preventing various C. difficile toxin-Ainduced
cellular toxicities.

Keywords: clostridium difficile, toxin A, antitoxin serum, apoptosis, gut inflammation, cell stress signals

References

  1. Chen X, Katchar K, Goldsmith JD, Nanthakumar N, Cheknis A, Gerding DN, Kelly CP. 2008. A mouse model of Clostridium difficile-associated disease. Gastroenterology 135:1984-1992.
    CrossRef
  2. Giannasca PJ, Zhang ZX, Lei WD, Boden JA, Giel MA, Monath TP, Thomas WD. 1999. Serum antitoxin antibodies mediate systemic and mucosal protection from Clostridium difficile disease in hamsters. Infect. Immun. 67: 527-538.
  3. Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375: 500-503.
    CrossRef
  4. Kang JK, Hwang JS, Nam HJ, Ahn KJ, Seok H, Kim SK, et al. 2011. The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity. Antimicrob. Agents Chemother. 55: 4850-4857.
    CrossRef
  5. Kaur S , Vaishnavi C, K ochhar R , Prasad KK, R ay P . 2012. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection. Indian J. Med. Microbiol. 30: 431-436.
    CrossRef
  6. Kelly CP, Pothoulakis C, LaMont JT. 1994. Clostridium difficile colitis. N. Engl. J. Med. 330: 257-262.
    CrossRef
  7. Kim H, Kokkotou E, Na X, Rhee SH, Moyer MP, Pothoulakis C, Lamont JT. 2005. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 129: 1875-1888.
    CrossRef
  8. Kim H, Rhee SH, Kokkotou E, Na X, Savidge T, Moyer MP, et al. 2005. Clostridium difficile t oxin A r egulates ind ucible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J. Biol. Chem. 280: 21237-21245.
    CrossRef
  9. Kim H, Rhee SH, Pothoulakis C, Lamont JT. 2007. Inflammation and apoptosis in Clostridium difficile enteritis is mediated by PGE2 up-regulation of Fas ligand. Gastroenterology 133: 875886.
    CrossRef
  10. Kyne L, Warny M, Qamar A, Kelly CP. 2001. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet 357: 189-193.
    CrossRef
  11. Kyne L, Warny M, Qamar A, Kelly CP. 2000. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N. Engl. J. Med. 342: 390-397.
    CrossRef
  12. Lai KK, Melvin ZS, Menard MJ, Kotilainen HR, Baker S. 1997. Clostridium difficile-associated diarrhea: epidemiology, risk factors, and infection control. Infect. Control Hosp. Epidemiol. 18: 628-632.
    CrossRef
  13. Nam HJ, Kang JS, Kim SK, Ahn KJ, Seok H, Park SJ, et al. 2010. Clostridium difficile toxin A decreases acetylation of tubulin, leading to microtubule depolymerization through activation of histone deacetylase 6, and this mediates acute inflammation. J. Biol. Chem. 285: 32888-32896.
    CrossRef
  14. Nam ST, Seok H, Kim DH, Nam HJ, Kang JK, Eom JH, et al. 2012. Clostridium difficile toxin A inhibits erythropoietin receptor-mediated colonocyte focal adhesion through inactivation of Janus Kinase-2. J. Microbiol. Biotechnol. 22: 1629-1635.
    CrossRef
  15. Nandy RK, Albert MJ, Ghose AC. 1996. Serum antibacterial and antitoxin r esponses in clinical cholera c aused by Vibrio cholerae O139 Bengal and evaluation of their importance in protection. Vaccine 14: 1137-1142.
    CrossRef
  16. Pothoulakis C, Gilbert RJ, Cladaras C, Castagliuolo I, Semenza G, Hitti Y, et al. 1996. Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A. J. Clin. Invest. 98: 641-649.
    CrossRef
  17. Pothoulakis C, LaMont JT. 1993. Clostridium difficile colitis and diarrhea. Gastroenterol. Clin. North Am. 22: 623-637.
  18. Pothoulakis C, Lamont JT. 2001. Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins. Am. J. Physiol. Gastrointest. Liver Physiol. 280: G178G183.
  19. Pothoulakis C, Triadafilopoulos G, Clark M, Franzblau C, LaMont JT. 1986. Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa. Gastroenterology 91: 1147-1153.
  20. Sun X, Wang H, Zhang Y, Chen K, Davis B, Feng H. 2011. Mouse relapse model of Clostridium difficile infection. Infect. Immun. 79: 2856-2864.
    CrossRef
  21. Warny M, Vaerman JP, Avesani V, Delmee M. 1994. Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection. Infect. Immun. 62: 384-389.