title: Caspase and bid involvement in Clostridium difficile toxin A-induced apoptosis and modulation of toxin A effects by glutamine and alanyl-glutamine in vivo and in vitro.
abstract: Clostridium difficile is the leading cause of nosocomial bacterial diarrhea. Glutamine and its stable and highly soluble derivative alanyl-glutamine, have been beneficial in models of intestinal injury. In this study, we extend our work on the mechanisms of Clostridium difficile toxin A (TxA)-induced apoptosis in human intestinal epithelial T84 cells and evaluate the effects of glutamine and alanyl-glutamine on TxA-induced apoptosis in vitro and disruption of ileal mucosa in vivo. T84 cells were incubated with TxA (100 ng/ml) in medium with or without glutamine or alanyl-glutamine (3 to 100 mM). Apoptosis was evaluated by DNA fragmentation in vitro and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method in vivo. Caspase and Bid involvement were investigated by Western blotting. Ligated rabbit ileal loops were used for the evaluation of intestinal secretion, mucosal disruption, and apoptosis. TxA induced caspases 6, 8, and 9 prior to caspase 3 activation in T84 cells and induced Bid cleavage by a caspase-independent mechanism. Glutamine or alanyl-glutamine significantly reduced TxA-induced apoptosis of T84 cells by 47% and inhibited activation of caspase 8. Both glutamine and alanyl-glutamine reduced TxA-induced ileal mucosal disruption and secretion. Altogether, we further delineated the apoptosis-signaling cascade induced by TxA in T84 cells and demonstrated the protective effects of glutamine and alanyl-glutamine. Glutamine and alanyl-glutamine inhibited the apoptosis of T84 cells by preventing caspase 8 activation and reduced TxA-induced intestinal secretion and disruption.
authors.affiliation: Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia, MR4, Lane Road, Room 3148, Charlottesville, VA 22908, USA.
authors.forename: Benedito A
authors.initials: BA
authors.lastname: Carneiro
authors.forename: Jun
authors.initials: J
authors.lastname: Fujii
authors.forename: Gerly A C
authors.initials: GA
authors.lastname: Brito
authors.forename: Cirle
authors.initials: C
authors.lastname: Alcantara
authors.forename: Reinaldo B
authors.initials: RB
authors.lastname: Oriá
authors.forename: Aldo A M
authors.initials: AA
authors.lastname: Lima
authors.forename: Tom
authors.initials: T
authors.lastname: Obrig
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D001427:Bacterial Toxins; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D016360:Clostridioides difficile / Q000472:pathogenicity / Q000502:physiology*; D004151:Dipeptides / Q000494:pharmacology / Q000502:physiology*; D004768:Enterotoxins / Q000502:physiology*; D005973:Glutamine / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D007082:Ileum; D007413:Intestinal Mucosa / Q000378:metabolism / Q000382:microbiology; D011817:Rabbits
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D001427:Bacterial Toxins; D004151:Dipeptides; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D005973:Glutamine; D020169:Caspases; C054122:alanylglutamine