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.

蛋白质分泌在细菌发病机制中起着核心作用。肺炎军团菌是军团病的病原体，它通过 Dot/Icm IV 型分泌系统 (T4SS) 转运大量效应蛋白。 Dot/Icm T4SS 由超过 20 种蛋白质组成，对于军团菌感染至关重要。然而，IV 型分泌的分子基础仍然很大程度上未知。我们已经证明，绝大多数 DotA（Dot/Icm T4SS 成分）通过 Dot/Icm T4SS 分泌到细胞外环境中。在细菌中，DotA 整合到具有七个跨膜区域的内膜中。为了解决 T4SS 如何分泌这种完整的内膜蛋白，我们分离了 DotA 分泌或 DotA 功能缺陷的 DotA 点突变体。通过军团菌在环境模型宿主卡氏棘阿米巴中生长的能力来评估 DotA 功能。我们建立了高通量筛选系统并分离了 57 个 DotA 突变体。所有这些突变体在DotA分泌和DotA功能方面均存在缺陷，表明DotA分泌对于DotA功能是不可或缺的。这些突变体的精细定位揭示了 22 个独立的点突变。没有发现富含突变的 DotA 特定区域。值得注意的是，这些点突变同时定位在 DotA 的细胞质和周质区域，这意味着 DotA 的分泌涉及多步骤过程。此外，我们发现 DotF（Dot/Icm T4SS 的假定核心组件）对于 DotA 分泌来说是可有可无的。 DotF KO 菌株的 DotA 分泌减少了 10 倍。由于 DotF 被认为是 Dot/Icm T4SS 的核心组成部分，因此我们检查了 DotF KO 菌株的效应子易位。数据表明Dot F对于DotA分泌和效应器易位都是可有可无的。

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