Functional analysis of Legionella Dot/Icm T4SS component DotA.

军团菌 Dot/Icm T4SS 组件 DotA 的功能分析。

• 批准号: 18590420
• 负责人: NAGAI Hiroki
• 金额: $ 2.57万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590420/
• 关键词: Legionella; type IV secretion; protein secretion; Dot; Icm; IV型分泌系; 膜タンパク質

Protein secretion plays a central role in bacterial pathogenesis. Legionella pneumophlia, the causative agent of Legionnaires' disease, translocated a large array of effector proteins via the Dot/Icm type IV secretion system (T4SS). Dot/Icm T4SS is composed of>20 proteins and essential for Legionella infection. However, molecular basis of type IV secretion remains largely unknown. We had demonstrated that vast majority of DotA, a Dot/Icm T4SS component, was secreted into extracellular milieu via the Dot/Icm T4SS. In bacteria, DotA is integrated into inner membrane with seven trans-membrane regions. To address how such integral inner membrane protein is secreted by T4SS, we isolated DotA point mutants defective either in DotA secretion or in DotA function. DotA function was assessed by the ability of Legionella to grow within environmental model host Acanthamoeba castellanii We established a high-throughput screening system and isolated 57 DotA mutants. All of these mutants are defective both in DotA secretion and in DotA function, suggesting that DotA secretion is indispensable for DotA function. Fine mapping of these mutants unveiled 22 independent point mutations. No specific region of DotA rich of mutations was found. Notably, these point mutations were mapped both at cytoplasmic and periplasmic regions of DotA, implying that DotA secretion involve multi-step processes. Additonally, we found that DotF, a putative core component of Dot/Icm T4SS, is dispensable for DotA secretion. DotA secretion from DotF KO strain was reduced by 10-fold. Because DotF is thought to be a core component of Dot/Icm T4SS, we examined effector translocation from DotF KO strain. The data indicated that Dot F is dispensable both for DotA secretion and for effector translocation.

蛋白质分泌在细菌致病中起着核心作用。嗜肺军团菌（Legionella pneumophlia）是军团病的病原体，通过Dot/Icm IV型分泌系统（T4 SS）转运大量效应蛋白。Dot/Icm T4 SS由>20种蛋白质组成，并且是军团菌感染所必需的。然而，IV型分泌的分子基础在很大程度上仍然未知。我们已经证明绝大多数DotA，一种Dot/Icm T4 SS组分，通过Dot/Icm T4 SS分泌到细胞外环境中。在细菌中，DotA被整合到具有七个跨膜区域的内膜中。为了解决这种完整的内膜蛋白是如何分泌的T4 SS，我们分离DotA点突变体缺陷的DotA分泌或DotA功能。DotA功能的评估是通过军团菌在环境模式宿主卡氏阿米巴中的生长能力来进行的。我们建立了一个高通量筛选系统，分离了57个DotA突变体。所有这些突变体都在DotA分泌和DotA功能上有缺陷，这表明DotA分泌对于DotA功能是必不可少的。这些突变体的精细作图揭示了22个独立的点突变。未发现DotA基因突变丰富的区域，但这些点突变分布在DotA的胞质和周质区域，提示DotA的分泌涉及多步过程。此外，我们发现DotF，一个推定的Dot/Icm T4 SS的核心成分，是DotA分泌的关键。来自DotF KO菌株的DotA分泌减少了10倍。因为DotF被认为是Dot/Icm T4 SS的核心组分，所以我们检查了来自DotF KO菌株的效应子易位。数据表明，Dot F对于DotA分泌和效应子易位都是不稳定的。

项目成果

Screening of novel protein substrates of the Legionella Dot/Icm type IV secretion system based on the properties of C-terminal translocation signals.

基于C端易位信号的特性筛选军团菌Dot/Icm IV型分泌系统的新型蛋白质底物。

• 发表时间: 2006
• 作者: Mun H-S.;Aosai F;Fang H;Piao LX;Winn T;Norose K;Yano A.;永井宏樹
• 通讯作者: 永井宏樹

Screening of novel protein substrates of the Legionella Dot/Icm type IV secretion system based on the properties of C-terminal translocation signals

基于C端易位信号特性筛选军团菌Dot/Icm IV型分泌系统的新型蛋白底物

• 发表时间: 2006
• 作者: 川本;進;Hiroki Nagai
• 通讯作者: Hiroki Nagai

Molecular analysis of Legionella type IV effectors..

IV 型军团菌效应子的分子分析..

• 发表时间: 2008
• 作者: 喜多 英二;東 伸岳;岡山 明子;百武晃宏;久堀智子;Akihiro Hyakutake;Tomoko Kubori;Hiroki Nagai
• 通讯作者: Hiroki Nagai

IV型分泌装置の機能または自己分泌能を失った変異型DotAの単離と解析

失去IV型分泌器功能或自分泌能力的突变体DotA的分离和分析

• 发表时间: 2008
• 作者: 喜多 英二;東 伸岳;岡山 明子;百武晃宏
• 通讯作者: 百武晃宏

Isolation and characterization of DotA mutants defective either in DotA secretion or in DotA function

DotA 分泌或 DotA 功能缺陷的 DotA 突变体的分离和表征

• 发表时间: 2008
• 作者: 喜多 英二;東 伸岳;岡山 明子;百武晃宏;久堀智子;Akihiro Hyakutake
• 通讯作者: Akihiro Hyakutake