Brewer's Yeast and Saccharomyces boulardii Both Attenuate Clostridium difficile-Induced Colonic Secretion in the Rat

Saccharomyces boulardii (Sb), a nonpathogenicyeast, has been used to prevent recurrences ofClostridium difficile (C.diff)-associated diarrhea. Asingle report suggested that treatment withSaccharomyces cerevisiae (Sc), commonly called brewer's yeast(BY), facilitates treatment of persistent C.diffinfection. We conducted this experiment to determinewhether C.diff toxin A-induced colonic secretion in the rat is blunted by pretreatment with eitherSb or BY. We employed closed cecal pouches in two groupsof five adult male Sprague-Dawley rats fed with standardchow for five days prior to the experiment, another group whose water was supplemented with20 × 10⁹ colony-forming units (CFU) ofSb per day for five days, and another group whose waterwas supplemented with 20 × 10⁹ CFU ofSc per day for five days. Cecal pouches were infused for 3 hr with one ofthe following: (1) normal saline alone for a controlgroup, or (2) normal saline plus 5 g of C.diff toxinA (for the other control group and for the two experimental groups). Water movement wasmeasured by a nonabsorbable marker technique. Sodiummovement and permeability to mannitol were alsomeasured. Prior to the infusion, cecal contents werequantitatively cultured. In the three animals whose ceca werecolonized with less than 10⁶ CFU of eitheryeast per gram wet cecal content, toxin A-inducedsecretion could not be attenuated. In contrast, animalswhose ceca were colonized with more than10⁶ CFU of either yeast per gram of wet cecalcontent showed significantly less secretion after toxinA application than those which were not fed yeast. S.cerevisiae reduced secretion by half (N = 5, P = 0.039 forwater, 0.044 for sodium) and Sb by 75% (N = 4, P = 0.015for water, 0.034 for sodium). Toxin-induced increases inpermeability to [³H]mannitol from systemic circulation to cecum could not be blunted byeither yeast. We conclude that rat ceca can be colonizedby either organism and that both organisms reduce C.difftoxin A-mediated secretion. We speculate that both organisms might have benefit in humanC.diff-associated enterocolitis. Further studies oftheir mechanisms of action as well as clinical trialsfor the prevention and treatment of human C.diffinfections should be pursued.     

啤酒酵母鲨烯合酶基因的克隆及其基因表达载体的构建

[目的] 克隆啤酒酵母鲨烯合酶基因及构建其基因表达载体,为在微生物体内重建青蒿素合成途径打下基础.[方法]采用聚合酶链反应(PCR)扩增、扩增片段与T-载体连接和克隆片段的序列分析;酶切并回收目的基因,反向插入酵母表达载体pGAPZαA,双酶切鉴定重组子.[结果] 通过PCR扩增获得1 335 bp片段,克隆后经PCR和酶切反应进行了鉴定.初步测序结果与GenBank上已登录的酵母鲨烯合酶基因序列基本吻合,相差仅数个碱基对;将此片段反向插入酵母表达载体pGAPZαA,构建了反义酵母表达载体,并通过双酶切加以鉴定.[结论] 成功克隆了啤酒酵母鲨烯合酶基因并进行了测序,同时构建了反义酵母表达载体.     

Effect of Saccharomyces boulardii on cAMP- and Ca2+-dependent Cl- Secretion in T84 Cells

Several reports have confirmed that thecooperative interaction between cAMP- andCa²⁺-mediated transduction pathways maycontribute to the stimulatory or inhibitory regulationof Cl^- secretion in intestinal epithelium.Saccharomyces boulardii has been shown to inhibitcholera toxin-induced secretion in rat jejunum. We haveidentified a 120-kDa protein in medium conditioned bySaccharomyces boulardii that reduces cholera toxin-inducedcAMP in intestinal cells. The present study evaluatedthe effect of medium conditioned by Saccharomycesboulardii on cAMP- and Ca²⁺-mediatedCl^- secretion in T84 cells. Experiments performedwith cAMP agonists revealed that 1 hr of preincubationof cells with medium conditioned by Saccharomycesboulardii was necessary to elicit a 40-50% reduction in receptor (cholera toxin, prostaglandinE₂, and vasoactive intestinal polypeptide)and nonreceptor (forskolin) mediated cAMP synthesis and¹²⁵I^- efflux. Secretion induced by carbachol was inhibited when cells werepretreated for 1 hr with medium conditioned bySaccharomyces boulardii despite the absence ofinhibition of Ins (1,4,5)P₃. From this studywe conclude that Saccharomyces boulardii exerts aninhibitory effect in vitro on Cl^- secretionmediated through both cAMP- and Ca²⁺-mediatedsignaling pathways.