Structure of the assembly platform of the bacterial type II secretion system

细菌II型分泌系统会释放有害蛋白质，对动物、人类和植物都有毁灭性的影响。人类病原体如产肠毒素大肠杆菌、霍乱弧菌和铜绿假单胞菌分别引起儿童腹泻、霍乱和呼吸道感染，每年有许多人死亡。最近的研究表明，II型分泌系统对于已经产生多药耐药的人类院内病原体的生存非常重要。此外，II型分泌系统的损伤已被证明可以使先天免疫系统清除感染。依靠II型分泌系统的植物病原体，如Dickeya、Pectobacterium和Xanthomonascause，在英国和欧洲造成了毁灭性的作物损失。通过这个系统分泌的酶破坏植物组织，威胁粮食安全。细菌II型分泌系统跨越革兰氏阴性细胞壁的两层膜。分泌是由一个内膜复合体组装一个短毛，将招募的效应物从周质（两膜之间的空间）通过外膜的门控孔来实现的。菌毛组装所需的能量由细胞质atp酶提供。我们在II型分泌系统成像方面取得了很好的进展。我们纯化、成像并生成了膜内组装平台和相关atp酶的3D模型。我们制作并成像了整个跨膜分泌系统。在本提案中，我们将扩展这些复合物的稳定性和分辨率，以便我们可以建立准确的膜内组装平台的分子模型。我们还将在外膜分泌素存在的情况下对组装平台进行成像，以捕捉从内膜组装平台延伸出来的质周结构域的排列。我们将使用低温电子显微镜来获得这些结果。为了证明我们可以生产提案中所述的可交付成果，我们收集了细胞膜内组装平台的初步数据，并产生了atp酶的初步结构。我们期望捕获不同状态下的分泌系统，并能够模拟状态之间的结构转换。我们得到了合作者的支持，他可以对细菌中的II型分泌系统进行逆向工程，以确认我们在活细菌实验中看到的相互作用的重要性。作为这项工作的结果，我们将确定细胞膜组装平台的结构及其与细胞质atp酶的相互作用。我们将阐明从显微镜捕获或解剖的不同复合体中可能发生的结构转变，并为揭示atp酶如何驱动菌毛组装提供结构基础。我们还旨在确定外周质中结构域的组织，这将有助于我们了解特定蛋白质底物如何被招募到分泌系统，以及信号如何在外周质和细胞质之间通过内膜组装复合体进行转导。最终，这项工作将有助于开发对抗感染的新抗生素。

The bacterial type II secretion system delivers harmful proteins which can have devastating effects on animals, humans and plants. Human pathogens such as enterotoxigenic Escherichia coli, Vibrio cholerae and Pseudomonas aeruginosa cause children's diarrhea, cholera and respiratory infections, respectively, and there are many deaths annually. It has recenly been demonstrated that the type II secretion system is very important for nosocomial pathogen survival in humans where multi-drug resistance has developed. Moreover, impairment of the type II secretion system has been shown to allow the innate immune system to clear infection. Plant pathogens relying on the type II secretion system such as Dickeya, Pectobacterium and Xanthomonascause are responsible for devastating crop loss in the UK and Europe. The lytic enzymes secreted through this system destroy plant tissues and threaten food security. The bacterial type II secretion system spans the two membranes of the Gram-negative cell wall. Secretion is achieved by an inner-membrane complex assembling a short pilus to push the recruited effector from the periplasm (the space between the two membranes) through the gated pore in the outer-membrane. The energy needed for pilus assembly is provided by the cytoplasmic ATPase. We have made excellent progress in imaging the type II secretion system. We have purified, imaged and generated 3D models of the inner-membrane assembly platform and associated ATPase. We have produced and imaged the entire envelope-spanning secretion system. In this proposal we shall extend the stability and resolution of these complexes so that we can build accurate molecular models of the inner-membrane assembly platform. We shall also image the assembly platform in the presence of the outer-membrane secretin to capture the arrangement of the periplasmic domains extending from the inner-membrane assembly platform. We shall achieve these results using cryo-electron microscopy. To demonstrate we can produce the deliverables stated in the proposal we have collected preliminary data on the inner-membrane assembly platform and produced a preliminary structure of the ATPase. We anticipate capturing the secretion system in different states and to be able to model the structural transitions between states. We are supported by our collaborator who can reverse engineer the type II secretion system in the bacterium to confirm the importance of the interactions we see in our experiments in the live bacterium.As a result of this work, we will determine the structure of the inner-membrane assembly platform and its interaction with the cytoplasmic ATPase. We shall elucidate the structural transitions that can occur from the different complexes trapped or dissected from micrographs and shall provide a structural basis for revealing how the ATPase drives pilus assembly. We also aim to determine the organisation of domains in the periplasm, and this will help us to understand how specific proteins substrates are recruited to the secretion system and how signal is transduced between periplasm and the cytoplasm across the inner membrane assembly complex. Ultimately this work will help underpin the development of new antibiotics to combat infection.