Saccharomyces boulardii Produces in Rat Small Intestine a Novel Protein Phosphatase that Inhibits Escherichia coli Endotoxin by Dephosphorylation

Abstract

Using a polyclonal antibody raised against a highly conserved sequence of 38 amino acids containing the activation site (VTDSAAGAT) common to mammalian and yeast alkaline phosphatases (AP), we identified in decapsidated Saccharomyces boulardii a protein phosphatase detected by autoradiography as a single signal (63 kD). Using an affinity chromatography column, the protein phosphatase could be concentrated 39.1-fold and presented as a doublet of two subunits. Compared with rat and bovine purified intestinal AP, the enzyme from S. boulardii had a greater ability to dephosphorylate the lipopolysaccharide (LPS) of Escherichia coli 055B5. When tested in vivo, intraperitoneal injection of intact LPS to rats produced, after 9 h, 100 ng/mL of circulating tumor necrosis factor-␣ with inflammatory lesions and apoptotic bodies in the liver and the heart, whereas rats injected with partially dephosphorylated LPS produced only 40 ng/mL tumor necrosis factor-␣ without organic lesions. In conclusion, S. boulardii is able to inhibit toxicity of E. coli surface endotoxins by the release of a protein phosphatase exhibiting a great capacity of dephosphorylation. S accharomyces boulardii, a nonpathogenic yeast, exerts therapeutic properties in acute and chronic enterocolopathies, antibiotic-associated diarrhea, and enterotoxigenic Clostridium difficile infections (1-4). In human volunteers (4,5) and in growing rats (4), several studies have documented that oral treatment with a lyophilized preparation of S. boulardii produces trophic intestinal effects, including increases in the activities of BBM enzymes (4,5) and enhanced secretion of s-IgA in intestinal fluid (6). After oral treatment of rats with S. boulardii, there is a marked stimulation of sodium-dependent D-glucose uptake into BBM vesicles with a corresponding accumulation of the sodium D-glucose co-transporter-1 (SGLT-1) (7). These trophic effects are, at least in part, mediated by endoluminal release of polyamines, as yeast cells contain substantial amounts of spermine and spermidine (4,8,9). In a recent work (10), we found that S. boulardii enhanced N-terminal peptide hydrolysis in suckling rat small intestine by endoluminal release of a zinc-binding metalloprotease. In the present study, we have analyzed whether oral treatment with S. boulardii could enhance the endoluminal activity of IAP. We also have purified a protein phosphatase secreted by S. boulardii in the rat intestinal lumen and have compared some of its properties with rat and bovine IAP. Finally, we have assessed whether the protein phosphatase released from S. boulardii can inhibit the toxicity of LPS from O55B5 Escherichia coli by dephosphorylation of its two phosphorylation sites. METHODSMedia and culture conditions. S. boulardii cells were inoculated in YPD (yeast extract, 0.5%; peptone, 2%; glucose, 2%; DIFCO, Detroit, MI) media and were grown at 30°C with moderate shaking to exponential growth as described (10).To disrupt the external capside, yeast cells were concentrated (1.45-1.50 ϫ 10 10...

Anti-inflammatory effects ofSaccharomyces boulardiimediated by myeloid dendritic cells from patients with Crohn's disease and ulcerative colitis

“…One study in an animal model identified in Sb a protein phosphatase that had a greater ability to dephosphorylate LPS of E. coli, which when injected in rats produced substantially less TNF-␣ and no organic lesions compared with the non-Sb-exposed LPS (11,12). However, this observation would not explain the other novel immunological and epithelial restitution effects demonstrated by us.…”

Efficacy and safety of the probiotic Saccharomyces boulardii for the prevention and therapy of gastrointestinal disorders

“…Luminal action A) Antimicrobial activity 1) Inhibition of growth of bacteria and parasites Czerucka et al 1994;Czerucka and Rampal, 2002;Dahan et al 2003;Dalmasso et al 2006a;Gedek, 1999a;Rigothier et al 1994;Rodrigues et al 1996;Mumy et al 2008;Wu et al 2008] 2) Reduction of gut translocation of pathogens [Herek et al 2004;Geyik et al 2006] 3) Neutralization of bacterial virulence factors [Buts et al 1994;Jahn et al 1996] 4) Suppression of host cell adherence that interferes with bacterial colonization [Czerucka et al 2000;Rodrigues et al 1996;Wu et al 2008] B) Antitoxin effects 1) Inhibition of toxin receptor binding sites [Buts et al 2006;Castagliuolo et al 1996Castagliuolo et al , 1999Czerucka et al 2000;Tasteyre et al 2002;Wu et al 2008] 2) Stimulation of antibody production against Clostridium difficile toxin A [Brandao et al 1998;Qamar et al 2001] 3) Direct proteolysis of the pathogenic toxins/Secretion of enzymatic proteins [Buts et al 2006;Castagliuolo et al 1996;Pothoulakis et al 1993] a) Produces a serine protease that cleaves C. difficile toxin A [Pothoulakis et al 1993] b) Produces 63 kDa phosphatase that destroys the endotoxin of pathogenic Escherichia coli [Buts et al 2006;Castagliuolo et al 1996] c) Produces a 120 kDa protein that reduces the effects of cholera toxin [Czerucka et al 1994] C) Cross-talk with normal microbiota When S. boulardii is given to antibiotic-exposed mice or patients with diarrhea, normal microbiota is re-established rapidly [Buts et al 1986[Buts et al , 1999[Buts et al , 2006Buts, 2009;…”

“…Clostridium difficile infection 1) Inhibits toxin A-mediated diarrhea, intestinal inflammation and histological damage by reducing toxin A-receptor binding [Castagliuolo et al 1996;Pothoulakis et al 1993] 2) Releases a protease that cleaves C. difficile toxins and toxin intestinal receptors [Castagliuolo et al 1996;Pothoulakis et al 1993] 3) Stimulates specific intestinal antitoxin A immunoglobulin levels [Castagliuolo et al 1996[Castagliuolo et al , 1999Pothoulakis et al 1993] 4) Inhibits IL-8 production and activation of the MAP kinases Erk1/2 and JNK/SAPK induced by C. difficile toxin A in human colonocytes Qamar et al 2001] 5) Significantly fewer animals challenged with C. difficile died if given S. boulardii compared with controls [Castex et al 1990;Elmer and Corthier, 1991;Rodrigues et al 1996;Toothaker and Elmer, 1984] Helicobacter pylori infection Alters the structure of H. pylori Vibrio cholerae infection 1) Inhibits the effect of V. cholerae toxin and hydroelectrolytic secretions by reducing cAMP activity [Vidon et al 1986;Czerucka et al 1994] 2) S. boulardii and the mammalian CT receptors could be structurally and functionally similar and the yeast binds CT [Brandao et al 1998;Czerucka et al 1994] Amebic dysentery 1) Reduces the number of red cells adhering to amoebae [Rigothier et al 1994] 2) Decreases the number of amoebae bearing red cells [Rigothier et al 1994] Infection with EHEC 1) S. boulardii modifies host signaling such as NF-κB-associated pathways activated by bacterial invasion with EHEC [Dahan et al 2002[Dahan et al , 2003 2) Addition to T84 colonocyte monolayers diminishes MLC phosphorylation and decreases transepithelial resistance in response to EHEC [Dahan et al 2002[Dahan et al , 2003 Infection with EPEC 1) Modifies EPEC infection and acts as a receptor decoy for EPEC [Buts et al 2006;Canil et al 1993;Czerucka et al 2000;Gedek, 1999b] 2) Reduces the number of intracellular EPEC [Buts et al 2006;Canil et al 1993;Czerucka et al 2000;Gedek, 1999b] 3...…”

Saccharomyces boulardiieffects on gastrointestinal diseases

“…It has been shown that S. boulardii has the capacity to release factors which neutralize bacterial toxins and decrease the deleterious effects of infectious pathogens (Buts et al, 2006;Castagliuolo et al, 1996;Castagliuolo et al, 1999). C. difficile is responsible of AAD in humans and animals, and is one of the most common nosocomial pathogens.…”

“…S. boulardii produces also a 63 kDa protein phosphatase that inhibits the LPS toxicity of enteropathogenic E. coli O55B5 by endotoxin dephosphorylation (Buts et al, 2006) ( Figure 2C). Intraperitoneal injection of dephosphorylated LPS in rats decreases the level of circulating TNF-α and abolishes inflammatory lesions in comparison to rats injected with native LPS.…”