Study for Inhibitory Factors of El Tor Hemolysin Produced by Vibrio cholerae Ol in Mouse Small Intestine

小鼠小肠霍乱弧菌O1产生El Tor溶血素抑制因子的研究

• 批准号: 11670282
• 负责人: IKIGAI Hajime
• 金额: $ 2.11万
• 依托单位: Suzuka National College of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670282/
• 关键词: Vibrio cholerae; El Tor hemolysin; mouse intestinal mucus; Inhibitory factors; membrane damage; oligomer; cholesterol; protease; 溶血阻害因子; トリプシン; 限定分解; 細胞溶解; 蛋白分解酵素; マウス腸管粘膜

Vibrio cholerae 01 produces El Tor hemolysin (ETH ; 65 kDa), which expresses obvious strong cytotoxic effects. The ETH forms pore-forming transmembrane oligomers in lipid bilayer membranes that are permeable to smaller molecules less than 600 Da. We have shown that mouse small intestinal mucus (MSIM) inhibits hemolysis induced by ETH. Mechanism of the inhibition is not yet clear, but this leads to a speculation about a possible cytoprotective role of small intestinal mucus against toxin. Thus, we studied inhibitory factors of ETH in mouse small intestine and its inhibition mechanism.We found that the toxin oligomers are formed by only MSIM under condition without membranes. Interestingly, we also found that ETH monomers were converted to oligomers in a suspension of cholesterol. Hemolysis of rabbit erythrocytes was not caused by oligomers isolated from artificial lipid bilayer membranes. These results suggest that hemolysis of rabbit erythrocytes was induced by ETH monomers, but not by the oligomers. The inhibition of hemolysis was induced by the formation of ETH oligomers by MSIM prior to encountering erythrocyte membranes, suggesting the possibility that cholesterol composed of mouse small intestine is an inhibitory factor.We demonstrated that a 50-kDa truncated protein was yielded by the digestion of ETH monomers with trypsin or a-chymotrypsin, which are the major proteases in the mouse small intestines. These proteolytic enzymes inhibited ETH-induced hemolysis, suggesting that hemolytic activity of the 50-kDa truncated monomer was lower than that of the 65-kDa ETH monomer. It is clearly that proteases composed of MSIM are another inhibitory factor. Therefore, alternatively, hemolysis inhibition might be induced by proteolytic digestion of ETH monomers.

Vibrio Cholerae 01产生El Tor Hemolysin（ETH; 65 kDa），表达明显的强细胞毒性作用。 ETH形成脂质双层膜中孔形成的跨膜寡聚物，可渗透到小于600 Da的较小分子。我们已经表明，小鼠小肠粘液（MSIM）抑制ETH诱导的溶血。抑制的机制尚不清楚，但这导致人们对小肠粘液对毒素的可能细胞保护作用的猜测。因此，我们研究了小鼠小肠及其抑制机制中ETH的抑制因子。我们发现毒素低聚物仅由MSIM在没有膜的情况下形成。有趣的是，我们还发现，乙醇单体被转换为胆固醇暂停的低聚物。兔红细胞的溶血不是由从人工脂质双层膜中分离出的低聚物引起的。这些结果表明，兔子红细胞的溶血是由ETH单体诱导的，而不是由低聚物诱导的。在遇到红细胞膜之前通过MSIM形成ETH寡聚物的抑制作用是诱导的肠。这些蛋白水解酶抑制了伦理学诱导的溶血，表明50 kDa截短的单体的溶血活性低于65 kDa ETH单体的溶血活性。显然，由MSIM组成的蛋白酶是另一个抑制因素。因此，替代地，溶血抑制可能是由乙醇单体的蛋白水解消化引起的。

项目成果

Hajime Ikigai: "Application for Bionanotechnology of Pore-Forming Toxins : Conversion of Injurious Bacterial Toxins to Nanomachineries"Chemistry Today. April. 32-37 (2002)

Hajime Ikigai：“成孔毒素生物纳米技术的应用：将有害细菌毒素转化为纳米机械”《今日化学》。

生貝 初: "細胞膜に穴をあける毒素のバイオナノテクノロジーへの利用-有害で無益な毒素を役に立つ道具に変える-"現代化学. 4月号. 32-37 (2002)

Hajime Namagai：“利用在细胞膜上产生孔洞的毒素进行生物纳米技术 - 将有害和无用的毒素转化为有用的工具 -”Gendai Kagaku 4 月号（2002 年）。