Molecular mechanism of bacterial infection based on the interaction between hosts and pathogens

基于宿主与病原体相互作用的细菌感染分子机制

• 批准号: 17591038
• 负责人: NISHIOKA Hiroaki
• 金额: $ 2.24万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591038/
• 关键词: gram-negative bacteria; Shigella flexneri; effector proteins; Ipa proteins; host cell; mechanism of infection

Gram-negative bacteria causes many infectious diseases. Type III secretons (TTSSs) are essential determinants of the interaction of many Gram-negative bacteria with animal and plant hosts. Their major function is to translocate bacterial 'effector' proteins into eukaryotic host cells to manipulate them during infection. Shigella flexneri is the etiological gram-negative bacterium of the endemic form of bacillary dysentery. It also uses a type III secretion apparatus and secretes effector proteins in order to invade eukaryotic cells. The essential proteins of Shigella are IpaB, IpaC, and IpaD. We tried to characterize these Ipaproteins.Purified secreted IpaB and IpaC formed a stable soluble complex of approximately 200kD. The complex contained a high level of secondary structure. It was monodispersed in solution and of roughly spherical shape with a diameter of 10-20 nm. We could visualize the IpaB/IpaC complex by electron microscopy. The IpaB/IpaC complex displayed a molar ratio of〜1:3-5, which correspond to 176-252 kD. The purified complex bound to red blood cell membranes in a cholesterol-dependent manner. It also bound to cultured mammalian cell membranes in a CD44-and cholesterol-dependent manner. The complex localized to lipid rafts in a cholesterol-dependent but CD44-independent manner. The purified IpaB/IpaC complex interacted with lipid membranes as a function of lipid composition, inserted, and formed pores within liposomes.As for IpaD, it possessed distinct functional domains. Some ipaD mutants resulted in increased secretion of IpaC. Others showed constitutive secretionf of IpaC and IpaB. Moreover, IpaB and IpaC associated with erythrocyte membranes less effectively in the presence of IpaD mutants. Thus, we showed that IpaD is required for regulation of secretion of IpaB and IpaC and effective insertion of them into host membranes.

革兰氏阴性菌引起许多传染病。III型分泌子（TTSS）是许多革兰氏阴性菌与动植物宿主相互作用的重要决定因素。它们的主要功能是将细菌“效应”蛋白转运到真核宿主细胞中，以在感染过程中操纵它们。福氏志贺菌是引起细菌性痢疾流行的革兰氏阴性菌。它还使用III型分泌装置并分泌效应蛋白以侵入真核细胞。志贺氏菌的必需蛋白质是IpaB、IpaC和iPad。纯化的分泌型IpaB和IpaC可形成约200 kD的稳定可溶性复合物。该复合物含有高水平的二级结构。它在溶液中是单分散的，并且具有直径为10-20 nm的大致球形形状。我们可以通过电子显微镜观察到IpaB/IpaC复合物。IpaB/IpaC复合物的摩尔比为1：3-5，相当于176-252 kD。纯化的复合物以胆固醇依赖的方式与红细胞膜结合。它也结合到培养的哺乳动物细胞膜的CD 44和胆固醇依赖性的方式。该复合物以胆固醇依赖但不依赖于CD 44的方式定位于脂筏。纯化的IpaB/IpaC复合物可与脂质膜相互作用，插入脂质体并形成孔，而iPad具有独特的功能结构域。一些iPad突变体导致IpaC分泌增加。其他细胞显示IpaC和IpaB的组成性分泌。此外，IpaB和IpaC与红细胞膜有效地在iPad突变体的存在下。因此，我们表明iPad是调节IpaB和IpaC的分泌以及将它们有效插入宿主膜所必需的。

项目成果

IpaD of Shigella is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes.

志贺氏菌的 IpaD 是调节 Ipa 蛋白分泌以及将 IpaB 和 IpaC 有效插入宿主细胞膜所独立需要的。

• 发表时间: 2005
• 期刊: Infection and Immunity 73 (3)
• 作者: Chang X;Yamada R;Sawada T;Suzuki A;Kochi Y;Yamamoto K.;三崎義堅;Urasaki Y;W.Picking
• 通讯作者: W.Picking