Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans.

[1]  M. Delmée,et al.  Saccharomyces boulardii for Clostridium difficile-associated enteropathies in infants. , 1993, Journal of pediatric gastroenterology and nutrition.

[2]  C. Pothoulakis,et al.  Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein. , 1991, The Journal of clinical investigation.

[3]  J. Hotz.  [Prevention of antibiotic-associated diarrhea by Saccharomyces boulardii: a prospective study]. , 1990, Zeitschrift fur Gastroenterologie.

[4]  N. Sullivan,et al.  Purification and characterization of toxins A and B of Clostridium difficile , 1982, Infection and immunity.

[5]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[6]  W. Stamm,et al.  Nosocomial acquisition of Clostridium difficile infection. , 1989, The New England journal of medicine.

[7]  L. Mcfarland,et al.  Suppression by Saccharomyces boulardii of toxigenic Clostridium difficile overgrowth after vancomycin treatment in hamsters , 1987, Antimicrobial Agents and Chemotherapy.

[8]  R. Eliakim,et al.  Ketotifen inhibits Clostridium difficile toxin A-induced enteritis in rat ileum. , 1993, Gastroenterology.

[9]  C. Surawicz,et al.  A randomized placebo-controlled trial of Saccharomyces boulardii in combination with standard antibiotics for Clostridium difficile disease. , 1994, JAMA.

[10]  R Sedivy,et al.  Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. , 1995, The Journal of clinical investigation.

[11]  E. Jennische,et al.  Enterotoxins from Clostridium difficile; diarrhoeogenic potency and morphological effects in the rat intestine. , 1990, Gut.

[12]  J. Bartlett,et al.  Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. , 1977, The Journal of infectious diseases.

[13]  G. Elmer,et al.  Prevention of clindamycin-induced mortality in hamsters by Saccharomyces boulardii , 1984, Antimicrobial Agents and Chemotherapy.

[14]  I. Lönnroth,et al.  Comparison of methods for the production and purification of toxin A from Clostridium difficile , 1988 .

[15]  G. Corthier,et al.  Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A , 1991, Infection and immunity.

[16]  J. Johnson,et al.  Molecular characterization of the Clostridium difficile toxin A gene , 1990, Infection and immunity.

[17]  C. Giampaolo,et al.  Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum. , 1989, Gastroenterology.

[18]  L. Mcfarland.  Saccharomyces boulardii is not Saccharomyces cerevisiae. , 1996, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[19]  C. Pothoulakis,et al.  CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Tweten,et al.  Purification and characterization of toxin B from Clostridium difficile , 1988, Infection and immunity.

[21]  Esther Jacobowitz Israel,et al.  Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is associated with decreased toxin A receptor. , 1992, The Journal of clinical investigation.

[22]  E. Wierbicki,et al.  Assay of proteolytic enzyme activity using a 14C-labeled hemoglobin. , 1971, Analytical biochemistry.

[23]  G. Corthier,et al.  Prevention of Clostridium difficile-induced experimental pseudomembranous colitis by Saccharomyces boulardii: a scanning electron microscopic and microbiological study. , 1990, Journal of general microbiology.

[24]  G. Corthier,et al.  Modulation of Clostridium difficile induced mortality as a function of the dose and the viability of the Saccharomyces boulardii used as a preventative agent in gnotobiotic mice. , 1991, Canadian journal of microbiology.

[25]  M. Craynest,et al.  Response of Human and Rat Small Intestinal Mucosa to Oral Administration of Saccharomyces boulardii , 1986, Pediatric Research.

[26]  T. Wilkins,et al.  Susceptibility of Clostridium difficile Toxins A and B to Trypsin and Chymotrypsin , 1989 .

[27]  C. Pothoulakis,et al.  Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum. , 1993, Gastroenterology.

[28]  R. Laskey,et al.  Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. , 1975, European journal of biochemistry.

[29]  G. Corthier,et al.  Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii. , 1986, Canadian journal of microbiology.

[30]  M. O'brien,et al.  Differential effects of Clostridium difficile toxins A and B on rabbit ileum. , 1987, Gastroenterology.

[31]  J. Bartlett,et al.  Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. , 1978, The New England journal of medicine.

[32]  A. Parodi,et al.  Bacterio-pharmacological activity of Saccharomyces boulardii in clindamycin-induced colitis in the hamster. , 1984, Arzneimittel-Forschung.

[33]  J. Powers,et al.  Proteases--structures, mechanism and inhibitors. , 1993, Agents and actions. Supplements.

[34]  C. Pothoulakis,et al.  Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum. , 1994, Gastroenterology.