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

大肠杆菌是一种经常存在于温血动物肠道中的细菌。大肠杆菌的亚群已经进化出了致病的能力。肠出血性大肠杆菌(EHEC)就是其中之一，它可以引起人类的血性腹泻。感染可能涉及影响肾脏的危及生命的并发症，并经常通过食物链和农场环境从反刍动物那里感染。牛是EHEC的主要宿主，包括近年来导致严重疫情的O157：H7形式。致肠性大肠杆菌(EPEC)是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”在肠道内定植并产生疾病。这种注射器由一组名为肠细胞消失点(Lee)的基因编码，用于将一组名为效应器的细菌蛋白质注入肠道内的细胞。这一过程被称为III型分泌，使细菌能够控制宿主细胞内的过程，为自己的利益服务。我们的研究表明，III型分泌物对于EHEC和EPEC与肠道衬里的黏附至关重要，并干扰宿主反应的诱导，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维持感染细胞的活性。此外，效应器ESPG靶向高尔基体，MAP和ESPF靶向线粒体；ESPF也靶向核仁。重要的是，到目前为止还没有发现EHEC效应器以细胞核为靶点。最近，一个名为NleG的III型EHEC效应器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、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。本项目的目标是研究两种在C末端含有PDZ结合基序(PDZ结合基序介导这些NleG与宿主细胞蛋白相互作用的基序)的NleG效应器，NleG-EDL和NleG7-EDL。重要的是，当NleG-EDL靶向于细胞核时，发现NleG7-EDL弥漫在胞浆中。具体来说，我们的目标是：1.确定NleG-EDL和NleG7-EDL的细胞内转运以及PDZ结合基序的作用。鉴定NleG-EDL和NleG7-EDL的宿主细胞伴侣和底物蛋白，并研究细胞信号转导。确定NleG-EDL对核蛋白质组、核蛋白周转和转录的影响。确定NleG7-EDL对细胞蛋白质组和蛋白质周转的影响6.利用体外(牛肠道体外器官杂乱)和体内(类EHEC鼠病原体罗氏柠檬酸杆菌)模型确定NleGs在感染过程中的作用了解NleGs如何调节受感染细胞的功能可能有助于合理设计控制EHEC和EPEC感染和家畜携带的策略。

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.