Castagliuolo I, Riegler M F, Valenick L, LaMont J T, Pothoulakis C
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Infect Immun. 1999 Jan;67(1):302-7. doi: 10.1128/IAI.67.1.302-307.1999.
Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225-5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.
布拉酵母菌是一种非致病性酵母，用于治疗艰难梭菌腹泻和结肠炎。我们曾报道，布拉酵母菌通过释放一种54 kDa的蛋白酶来抑制大鼠的艰难梭菌毒素A肠炎，该蛋白酶可消化毒素A分子及其刷状缘膜（BBM）受体（I. 卡斯塔廖洛、J. T. 拉蒙特、S. T. 尼古拉斯松和C. 波托拉基斯，《感染与免疫》64:5225 - 5232，1996年）。本研究的目的是进一步评估布拉酵母菌蛋白酶在预防大鼠回肠艰难梭菌毒素A肠炎中的作用，并确定其是否能保护人结肠黏膜免受艰难梭菌毒素的侵害。针对纯化的布拉酵母菌丝氨酸蛋白酶制备的兔多克隆抗血清，在体外可抑制布拉酵母菌条件培养基中73%的蛋白水解活性。抗蛋白酶免疫球蛋白G（IgG）可阻止布拉酵母菌对毒素A诱导的肠道分泌以及大鼠回肠肠袢中黏膜对[3H]甘露醇通透性的作用，而对照兔IgG则无此效果。抗蛋白酶IgG还可阻止布拉酵母菌蛋白酶对毒素A和B的消化作用以及[3H]毒素A和[3H]毒素B与纯化的人结肠BBM的结合。纯化的布拉酵母菌蛋白酶可逆转毒素A和毒素B对人结肠（HT - 29）细胞蛋白质合成的抑制作用。此外，将毒素预先暴露于布拉酵母菌蛋白酶后，在尤斯灌流小室中安装的人结肠黏膜中，毒素A和B诱导的跨上皮电阻下降分别被逆转了60%和68%。我们得出结论，布拉酵母菌对人类艰难梭菌诱导的炎症性腹泻的保护作用至少部分归因于其分泌的蛋白酶对毒素A和B分子的蛋白水解消化作用。
