Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells.

This study is an investigation into the mechanism of Clostridium difficile toxin A-induced apoptosis in human intestinal epithelial cells. Toxin A induced apoptosis of T84 cells in a dose- and time-dependent fashion. Toxin A-induced apoptosis was completely inhibited by blocking toxin enzymatic activity on Rho GTPases with uridine 5'-diphosphate-2',3'-dialdehyde by a nonspecific caspase inhibitor and was partially inhibited by caspase-1, -3, -6, -8, and -9 inhibitors. Caspases 3, 6, 8, and 9 and Bid activation were detected. Toxin A also induced changes in mitochondrial membrane potential and cytochrome c release at 18-24 h, a time course similar to caspase-9 activation. In conclusion, toxin A induces apoptosis by a mechanism dependent on inactivation of Rho, activation of caspases 3, 6, 8, and 9 and Bid, and mitochondrial damage followed by cytochrome c release. Toxin A proapoptotic activity may contribute to the mucosal disruption seen in toxin A-induced enteritis.

[1]  Y. Lazebnik,et al.  Caspases: enemies within. , 1998, Science.

[2]  L. R. Mccarthy,et al.  Relative frequency of Clostridium difficile in patients with diarrheal disease , 1981, Journal of clinical microbiology.

[3]  R. Guerrant,et al.  Clostridium difficile toxin A. Interactions with mucus and early sequential histopathologic effects in rabbit small intestine. , 1989, Laboratory investigation; a journal of technical methods and pathology.

[4]  Guido Kroemer,et al.  Mitochondria and programmed cell death: back to the future , 1996, FEBS letters.

[5]  D. Green,et al.  The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis , 1997, Science.

[6]  Y. Lazebnik,et al.  Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  V. A. Asst. Prof. W. L. George M. D.,et al.  Clostridium difficile and cytotoxin in feces of patients with antimicrobial agent-associated pseudomembranous colitis , 2005, Infection.

[8]  J. Cohen,et al.  Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. , 1987, Journal of immunology.

[9]  S. Colgan,et al.  Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. , 1998, Gastroenterology.

[10]  T. Wilkins,et al.  Clostridium difficile: its disease and toxins , 1988, Clinical Microbiology Reviews.

[11]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[12]  T. Wilkins,et al.  Biological activities of toxins A and B of Clostridium difficile , 1982, Infection and immunity.

[13]  K. Aktories,et al.  New Method To Generate Enzymatically DeficientClostridium difficile Toxin B as an Antigen for Immunization , 2000, Infection and Immunity.

[14]  C. Humphrey,et al.  Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine. , 1978, Gastroenterology.

[15]  R. Guerrant,et al.  Clostridium difficile toxin A induces the release of neutrophil chemotactic factors from rat peritoneal macrophages: role of interleukin-1beta, tumor necrosis factor alpha, and leukotrienes , 1997, Infection and immunity.

[16]  G. Kroemer,et al.  Bid acts on the permeability transition pore complex to induce apoptosis , 2000, Oncogene.

[17]  Jean-Claude Martinou,et al.  Bid-induced Conformational Change of Bax Is Responsible for Mitochondrial Cytochrome c Release during Apoptosis , 1999, The Journal of cell biology.

[18]  T. Gray,et al.  Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment. , 1996, Gut.

[19]  M. Wilm,et al.  The Enterotoxin from Clostridium difficile (ToxA) Monoglucosylates the Rho Proteins(*) , 1995, The Journal of Biological Chemistry.

[20]  C. Martínez-A,et al.  Rho prevents apoptosis through Bcl‐2 expression: Implications for interleukin‐2 receptor signal transduction , 1997, European journal of immunology.

[21]  C. Hahn,et al.  Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis , 1997, Oncogene.

[22]  C Haanen,et al.  A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. , 1995, Journal of immunological methods.

[23]  R. Guerrant,et al.  Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro , 1988, Infection and immunity.

[24]  J C Reed,et al.  Pro-caspase-3 Is a Major Physiologic Target of Caspase-8* , 1998, The Journal of Biological Chemistry.

[25]  J. Gray,et al.  Clostridium difficile and its toxin in healthy neonates. , 1983, British medical journal.

[26]  S. Hafiz.  Clostridium difficile and its toxins , 1974 .

[27]  Dean P. Jones,et al.  Superoxide in Apoptosis , 1998, The Journal of Biological Chemistry.

[28]  V. Dixit,et al.  Death receptors: signaling and modulation. , 1998, Science.

[29]  A. Befus,et al.  Effects of Toxin A from Clostridium difficile on Mast Cell Activation and Survival , 1998, Infection and Immunity.

[30]  C. Pothoulakis,et al.  Clostridium difficile toxin A causes early damage to mitochondria in cultured cells. , 2000, Gastroenterology.

[31]  L. Peso,et al.  Rho-regulated signals induce apoptosis in vitro and in vivo by a p53-independent, but Bcl2 dependent pathway , 1998, Oncogene.

[32]  M. Cohen,et al.  Fas-mediated apoptosis in human prostatic carcinoma cell lines occurs via activation of caspase-8 and caspase-7. , 1998, Cancer research.

[33]  D. Green,et al.  Granzyme B–Mediated Cytochrome C Release Is Regulated by the Bcl-2 Family Members Bid and Bax , 2000, The Journal of experimental medicine.

[34]  Andreas Bergmann,et al.  When cells die , 2000 .

[35]  G. Núñez,et al.  Caspases: the proteases of the apoptotic pathway , 1998, Oncogene.

[36]  D. Gerding,et al.  Clostridium difficile--associated diarrhea. , 1998, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[37]  S. Srinivasula,et al.  Cascades of Mammalian Caspase Activation in the YeastSaccharomyces cerevisiae * , 1999, The Journal of Biological Chemistry.

[38]  Zhenbiao Yang,et al.  RHO Gtpases and the Actin Cytoskeleton , 2000 .

[39]  R. Brettle,et al.  Clostridium difficile-associated diarrhoea. , 1984, The Journal of infection.

[40]  H. Sewell,et al.  Effect of Clostridium difficile Toxin A on Human Colonic Lamina Propria Cells: Early Loss of Macrophages Followed by T-Cell Apoptosis , 1998, Infection and Immunity.

[41]  S. Cory,et al.  The Bcl-2 protein family: arbiters of cell survival. , 1998, Science.

[42]  C. Hahn,et al.  Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4. , 1996, Journal of immunology.

[43]  K. Nishida,et al.  Anti-apoptotic function of Rac in hematopoietic cells , 1999, Oncogene.

[44]  J. Abastado.  Apoptosis: function and regulation of cell death. , 1996, Research in immunology.

[45]  M. Kagnoff,et al.  A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. , 1995, The Journal of clinical investigation.

[46]  Ruedi Aebersold,et al.  Molecular characterization of mitochondrial apoptosis-inducing factor , 1999, Nature.

[47]  W. E. Golden,et al.  Pseudomembranous Colitis , 2018 .

[48]  J. Bartlett,et al.  Clinical and laboratory observations in Clostridium difficile colitis. , 1980, The American journal of clinical nutrition.

[49]  T. Tsuruo,et al.  Actin cleavage by CPP-32/apopain during the development of apoptosis , 1997, Oncogene.

[50]  C. Fiorentini,et al.  Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis. , 1998, Experimental cell research.

[51]  D. Green,et al.  Suicidal Tendencies: Apoptotic Cell Death by Caspase Family Proteinases* , 1999, The Journal of Biological Chemistry.

[52]  P. Auberger,et al.  Sustained Polymorphonuclear Leukocyte Transmigration Induces Apoptosis in T84 Intestinal Epithelial Cells , 2000, The Journal of cell biology.

[53]  C. Potten,et al.  Epithelial cell growth and differentiation. II. Intestinal apoptosis. , 1997, The American journal of physiology.

[54]  S. Srinivasula,et al.  Cytochrome c and dATP-mediated Oligomerization of Apaf-1 Is a Prerequisite for Procaspase-9 Activation* , 1999, The Journal of Biological Chemistry.

[55]  A. Wyllie,et al.  Induction of apoptosis in NIH3T3 cells after serum deprivation by overexpression of rho-p21, a GTPase protein of the ras superfamily. , 1995, Oncogene.

[56]  Xiaodong Wang,et al.  DFF, a Heterodimeric Protein That Functions Downstream of Caspase-3 to Trigger DNA Fragmentation during Apoptosis , 1997, Cell.

[57]  C. Fiorentini,et al.  Effects of Clostridium difficile toxins A and B on cytoskeleton organization in HEp-2 cells: a comparative morphological study. , 1989, Toxicon : official journal of the International Society on Toxinology.

[58]  J. Rehg.  Cecal toxin(s) from guinea pigs with clindamycin-associated colitis, neutralized by Clostridium sordellii antitoxin , 1980, Infection and immunity.

[59]  Martin Raff,et al.  Cell suicide for beginners , 1998, Nature.

[60]  A. Chinnaiyan,et al.  The CED-3/ICE-like Protease Mch2 Is Activated during Apoptosis and Cleaves the Death Substrate Lamin A* , 1996, The Journal of Biological Chemistry.

[61]  坂平 英樹.  Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis , 2000 .

[62]  C. Pothoulakis,et al.  Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins. , 2001, American journal of physiology. Gastrointestinal and liver physiology.