The type III secretion system 'translocation-stop' activity of EspZ

大肠杆菌是一种经常栖息在温血动物肠道内的细菌。E.大肠杆菌已经进化出致病的能力。肠出血性大肠杆菌就是其中之一。大肠杆菌（EHEC），可导致人类出血性腹泻。感染可能涉及危及生命的并发症，影响肾脏，并经常通过食物链和农场环境从反刍动物获得。牛是肠出血性大肠杆菌的主要宿主，包括近年来引起严重疫情的O157：H7型。肠致病性E.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。两种类型的E.大肠杆菌依靠一个“分子注射器”在肠道内定居并产生疾病。这种注射器由一组称为肠上皮细胞消失基因座（LEE）的基因编码，用于将一组称为效应子的细菌蛋白质注射到肠道细胞中。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粘附于肠道内壁至关重要，并干扰可能解决感染的宿主反应的诱导。为了协调宿主细胞途径，细菌必须以所需的顺序和量递送III型分泌的效应物。此外，有必要控制递送的时间、作用的持续时间和效应物在宿主细胞内的位置。我们最近的研究表明，效应蛋白EspZ在控制效应蛋白流入宿主细胞中起着重要作用。它似乎只在注射一次后才能做到这一点，并且已经被工程化以表达EspZ的宿主细胞对效应物的注射具有抗性。缺乏EspZ的细菌会对宿主细胞造成过度损伤，这可能是因为它们以较高的水平注射效应物。这些数据表明，EspZ是一种新型的III型分泌的天然抑制剂，一旦它被递送到宿主细胞中，就可以阻止注射过程。我们的试验数据表明，EspZ可能与其他LEE编码的蛋白质相互作用，以关闭注射器产生的孔，并且一旦进入宿主细胞，它可能会被修改。EspZ与其他蛋白质之间的相互作用以及EspZ的修饰的性质和后果尚不清楚。因此，我们建议：确定何时将EspZ注射到宿主细胞中，以及蛋白质靶向宿主细胞膜是否与III型分泌的停滞一致。鉴定与EspZ相互作用的蛋白质，并确定这种相互作用如何阻止III型分泌。定义EspZ在宿主细胞内修饰的性质和后果。在动物模型中确定EspZ在细菌持久性和疾病中的作用。了解EspZ如何调节III型分泌将有助于合理设计控制EHEC和EPEC感染和家畜携带的策略。该过程的抑制剂可用于治疗人类或储库宿主中的感染。此外，我们将探索建立转基因动物的可能性，这些转基因动物由于在肠细胞中表达EspZ而难以感染。

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. To orchestrate host cell pathways, the bacteria must deliver Type III secreted effectors in the required order and amounts. Moreover, it is necessary to control the timing of delivery, and the duration of action and location of effectors inside host cells. Our recent research has indicated that the effector protein EspZ plays an important role in controlling the flow of effector proteins into host cells. It appears to do this only once injected, and host cells that have been engineered to express EspZ are resistant to injection of effectors. Bacteria that lack EspZ cause excessive damage to host cells, likely because they inject effectors at elevated levels. These data suggest that EspZ is a novel natural inhibitor of Type III secretion that may arrest the injection process once it has been delivered into host cells. Our pilot data suggest that EspZ may interact with other LEE-encoded proteins to close the pore created by the syringe, and that it may be modified once it enters host cells. The nature and consequences of interactions between EspZ and other proteins, and of modification of EspZ, are not known. We therefore propose to:1. Define when EspZ is injected into host cells, and whether targeting of the protein to the host cell membrane coincides with arrest of Type III secretion.2. Identify proteins that interact with EspZ and determine how such interactions arrest Type III secretion.3. Define the nature and consequences of modification of EspZ inside host cells.4. Determine the role of EspZ in bacterial persistence and disease in animal models.An understanding of how EspZ modulates Type III secretion will aid the rational design of strategies to control EHEC and EPEC infections and carriage by farm animals. Inhibitors of the process may be used to treat infections in humans or reservoir hosts. Moreover, we will explore the possibility of creating transgenic animals that are refractory to infection as a consequence of expression of EspZ in intestinal cells.