The NleG type III secretion system effectors of E. coli O157

大肠杆菌 O157 的 NleG III 型分泌系统效应子

• 批准号: BB/K001515/1
• 负责人: Gad Frankel
• 金额: $ 49.53万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK001515%2F1
• 关键词: NleG; type; III; secretion; system

Escherichia coli is a bacterium that often inhabits the intestines of warm-blooded animals. Subsets of E. coli have evolved the ability to cause disease. One such group are enterohaemorrhagic E. coli (EHEC), which can cause bloody diarrhoea in humans. Infections can involve life-threatening complications affecting the kidneys and are frequently acquired via the food chain and farm environment from ruminants. Cattle are a major reservoir of EHEC, including the O157:H7 form that has caused serious outbreaks in recent years. Enteropathogenic E. coli (EPEC) are a related subset of bacteria that cause acute watery diarrhoea in infants in the developing world. Both types of E. coli rely on a 'molecular syringe' to colonise the intestines and produce disease. This syringe, encoded by a cluster of genes called the locus of enterocyte effacement (LEE), serves to inject a set of bacterial proteins termed effectors into cells lining the intestines. This process, known as Type III secretion, enables the bacteria to take control of processes inside host cells for their own benefit. Our research has shown that Type III secretion is vital for adherence of EHEC and EPEC to the gut lining and to interfere with the induction of host responses that might otherwise resolve the infection. The type III secretion system effectors target diverse signalling pathways and the main cellular organelles. For example, the effectors Tir, Map, EspH and EspM modulate the cellular cytoskeleton, NleB, NleC and NleE block innate immune responses and NleH maintains viability of infected cells. Moreover, the effector EspG is targeted to the Golgi, Map and EspF are targeted to the mitochondria; EspF also targets the nucleolus. Importantly, no EHEC effectors were thus far found to target the nucleus. Recently, a large family of Type III EHEC effectors named NleG was shown to have a ubiquitin ligase activity. Ubiquitin and deubiquitylating enzymes are key regulators of protein turnover, cell signaling, transcription, cell cycle, and DNA damage repair. It is therefore not surprising that bacterial pathogens hijack the cellular ubiquitylation machinery as part of their infection strategies. The goals of this project are to study two NleG effectors that contain a PDZ-binding motif at the C-terminus (PDZ binding motif mediate the interaction of these NleGs with host cellular proteins), NleG-EDL and NleG7-EDL. Importantly, while NleG-EDL is targeted to the nucleus, NleG7-EDL is found diffused in the cytosol. In particular we aim to:1. Determine the intracellular trafficking of NleG-EDL and NleG7-EDL and the role of the PDZ-binding motif2. Identify the host cell partner and substrate proteins of NleG-EDL and NleG7-EDL and study cell signaling3. Determine the effect of NleG-EDL on the nuclear proteome, nuclear proteins' turnover and the transcriptome5. Determine the effect of NleG7-EDL on the cytocsolic proteome and proteins' turnover 6. Determine the role of NleGs during infection using ex vivo (bovine intestinal in vitro organ clutters) and in vivo (the EHEC-like moue pathogen Citrobacter rodetnium) modelsAn understanding of how the NleGs modulate the function of infected cells is likely to aid in rational design of strategies to control EHEC and EPEC infections and carriage by farm animals.

大肠杆菌是一种通常栖息在温血动物肠道中的细菌。大肠杆菌的亚群已经进化出了引起疾病的能力。其中一组是肠出血性大肠杆菌（EHEC），它可以引起人类血性腹泻。感染可能涉及影响肾脏的危及生命的并发症，并且经常通过反刍动物的食物链和农场环境获得。牛是肠出血性大肠杆菌的主要宿主，其中包括近年来引起严重疫情的 O157:H7 型肠出血性大肠杆菌。肠病性大肠杆菌 (EPEC) 是导致发展中国家婴儿急性水样腹泻的相关细菌亚群。这两种类型的大肠杆菌都依靠“分子注射器”在肠道中定殖并产生疾病。这种注射器由一组称为肠细胞消失位点（LEE）的基因编码，用于将一组称为效应器的细菌蛋白注入肠道内壁细胞中。这一过程被称为 III 型分泌，使细菌能够为了自身利益而控制宿主细胞内的过程。我们的研究表明，III 型分泌对于 EHEC 和 EPEC 粘附到肠壁并干扰宿主反应的诱导至关重要，否则宿主反应可能会解决感染。 III 型分泌系统效应器针对不同的信号通路和主要细胞器。例如，效应器 Tir、Map、EspH 和 EspM 调节细胞骨架，NleB、NleC 和 NleE 阻断先天免疫反应，NleH 维持受感染细胞的活力。此外，效应器EspG针对高尔基体，Map和EspF针对线粒体； EspF 还靶向核仁。重要的是，迄今为止还没有发现以细胞核为目标的肠出血性大肠杆菌效应子。最近，一个名为 NleG 的 III 型 EHEC 效应子大家族被证明具有泛素连接酶活性。泛素和去泛素化酶是蛋白质周转、细胞信号传导、转录、细胞周期和 DNA 损伤修复的关键调节因子。因此，细菌病原体劫持细胞泛素化机制作为其感染策略的一部分也就不足为奇了。该项目的目标是研究两种 NleG 效应子，它们在 C 末端含有 PDZ 结合基序（PDZ 结合基序介导这些 NleG 与宿主细胞蛋白的相互作用）：NleG-EDL 和 NleG7-EDL。重要的是，虽然 NleG-EDL 靶向细胞核，但发现 NleG7-EDL 扩散在细胞质中。我们的具体目标是：1。确定 NleG-EDL 和 NleG7-EDL 的细胞内运输以及 PDZ 结合基序 2 的作用。鉴定 NleG-EDL 和 NleG7-EDL 的宿主细胞伴侣和底物蛋白并研究细胞信号传导3。确定 NleG-EDL 对核蛋白质组、核蛋白质周转和转录组的影响5。确定 NleG7-EDL 对胞质蛋白质组和蛋白质周转的影响 6. 使用离体（牛肠道体外器官杂乱）和体内（类 EHEC 小鼠病原体柠檬酸杆菌）模型确定 NleG 在感染过程中的作用了解 NleG 如何调节受感染细胞的功能可能有助于合理设计策略 控制农场动物的 EHEC 和 EPEC 感染和携带。