Activities of enteropathogenic/enterohemorrhagic E. coli effector proteins

腹泻病是一个主要的人类健康问题，每年可导致约200万人死亡。在引起肠道疾病的细菌因子中有肠致病性E.大肠杆菌（EPEC）和与之密切相关的肠出血性E.大肠杆菌（EHEC）。EPEC和EHEC都是通过与宿主肠道细胞紧密结合，诱导吸收性微绒毛变性而致病的。虽然两者都是细胞外病原体，但它们将过多的效应蛋白注入宿主细胞，从而影响许多细胞功能。这种宿主细胞机制的颠覆的一个广泛研究的例子是细胞肌动蛋白细胞骨架的重组，以在附着的细菌下形成肌动蛋白骨架，这是一个已知对有效定植至关重要的过程。而某些E.尽管EPEC/EHEC对大肠杆菌效应蛋白的作用机制已有所阐明，但仍有许多EPEC/EHEC传递到宿主细胞的蛋白的功能至今尚不清楚。拟议的研究项目的目的是阐明所选EHEC/EPEC效应蛋白在真核细胞中的功能，并进一步阐明这些细菌操纵和感染宿主细胞的不同机制。由于许多细菌效应蛋白具有冗余和/或协同功能，当在EPEC/EHEC中突变时，它们具有很少或没有已知的表型。因此，遗传方法将得到体外研究的补充和支持。这些将需要克隆和纯化选定的细菌效应蛋白，这将允许鉴定新的真核靶点。然后使用基于细胞培养的模型验证体外鉴定的蛋白质-蛋白质相互作用。

Diarrhoeal disease is a major human health problem which can be held accountable for roughly 2 million deaths each year. Among the bacterial agents causing diarrhoeal disease are the enteropathogenic E. coli (EPEC) and the closely related enterohemorrhagic E. coli (EHEC). Both, EPEC and EHEC cause disease by intimately attaching to host intestinal cells, and inducing the degeneration of absorptive microvilli. Although both are extracellular pathogen, they inject a plethora of effector proteins into the host cell, which affect many cellular functions. One extensively studied example of such a subversion of host cell mechanisms is the reorganisation of the cellular actin cytoskeleton to form actin pedestals under the attaching bacteria, a process that is known to be crucial for efficient colonisation. While the mode of action of some E. coli effector proteins has been explained, there are still a number of proteins delivered to the host cell by EPEC/EHEC whose function is unknown to date. The aim of the proposed research project is to elucidate the functions of selected EHEC/EPEC effector proteins in the eukaryotic cell and also to further throw light on the different mechanisms used by these bacteria to manipulate and infect the host cell. As many bacterial effector proteins have redundant and/or cooperative functions, they have little or no known phenotype when mutated in EPEC/EHEC. Thus, the genetic approach will be complemented and backed up by in vitro studies. These will entail the cloning and purification of selected bacterial effector proteins, which will allow for the identification of novel eukaryotic targets. The protein-protein interaction identified in vitro will then be validated using cell culture based models.