人および家畜に下痢症を引き起こす新規細菌毒素の構造解析と阻害剤の開発

引起人类和牲畜腹泻的新型细菌毒素抑制剂的结构分析和开发

• 批准号: 16J09051
• 负责人: TONITI Waraphan (2017)
• 金额: $ 0.83万
• 依托单位: Kyoto Sangyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2016
• 资助国家: 日本
• 起止时间: 2016-04-22 至 2018-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16J09051/
• 关键词: enterotoxin; food-poisoning; Clostridium; binary toxin; CPILE; x-ray crystallography

The W5052 strain isolated from unusual Tokyo food-poisoning outbreaks did not harbor the cpe nor produce C. perfringens enterotoxin (CPE). On the other hand, they secreted a novel enterotoxin, CPILE. It is consisting of two components: CPILE-a, which acts as an enzymatic ADP-ribosyltransferase and CPILE-b, a membrane binding component.The enterotoxicity of CPILE relies on endocytosis of the CPILE complex into the cytosol. CPILE-b binds to the specific cell receptor and assists translocation of CPILE-a into the target cell. How CPILE-b binds to the particular cell receptor is still unclear.The purified CPILE-b was subjected to cleavage pro-sequence region by trypsin to trigger oligomerization. It should be noted that the SDS-PAGE of the purified CPILE-b showed both pro-sequence and CPILE-b bands. Then, the purified CPILE-b was concentrated and crystalized using standard crystallization kits via hanging drop vapor diffusion method. CPILE-b crystals found after one month screening and SDS-PAGE of selected crystals confirmed that they were CPILE-b crystals. However, poor data set was collected and failed to assign the correct space group even though the good shapes of CPILE-b crystals were used. The pro-sequence probably nicked to CPILE-b and disturbed good quality of crystal formation.In conclusion, N-terminal GST-tag CPILE-b with regular purification procedure failed to separate pro-sequence region from CPILE-b body. The condition that triggers oligomerization of CPILE-b should be further investigation.

从不寻常的东京食物中毒爆发中分离的W5052菌株不携带cpe，也不产生C。产气荚膜杆菌肠毒素（CPE）。另一方面，它们分泌一种新的肠毒素，CPILE。它由两种组分组成：CPILE-a，其充当酶促ADP-核糖基转移酶，和CPILE-b，膜结合组分XPILE的肠毒性依赖于CPILE复合物内吞到胞质溶胶中。CPILE-b与特异性细胞受体结合，并协助CPILE-a易位到靶细胞中。CPILE-b是如何与特定的细胞受体结合的尚不清楚，纯化的CPILE-b经胰蛋白酶切割前序列区，引发寡聚化。应该注意的是，纯化的CPILE-b的SDS-PAGE显示前序列和CPILE-b条带。然后，将纯化的CPILE-b浓缩并使用标准结晶试剂盒通过悬滴气相扩散法结晶。经过一个月的筛选发现了CPILE-b晶体，并对所选晶体进行SDS-PAGE，证实它们是CPILE-b晶体。然而，收集到的数据集很差，即使使用了良好形状的CPILE-b晶体，也未能分配正确的空间群。因此，用常规的纯化方法分离CPILE-b的N端GST标签，不能将CPILE-b的前序列区从CPILE-b体中分离出来。 引发CPILE-b寡聚化的条件有待进一步研究。

项目成果

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

• DOI: 10.1371/journal.pone.0171278
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Toniti W;Yoshida T;Tsurumura T;Irikura D;Monma C;Kamata Y;Tsuge H
• 通讯作者: Tsuge H

Characterization of Actin ADP-ribosyltransferase from Clostridium perfringens iota-like enterotoxin

产气荚膜梭菌 iota 样肠毒素中肌动蛋白 ADP-核糖基转移酶的表征

• 发表时间: 2016
• 作者: Waraphan Toniti;Toru Yoshida;Toshiharu Tsurumura;Daisuke Irikura;Chie Monma;Yoichi Kamata;and Hideaki Tsuge
• 通讯作者: and Hideaki Tsuge

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