Structure-function analysis of Type IV secretion systems by cryo-electron microscopy

人类、其他动物和植物中的许多疾病都是由细菌引起的。这些疾病种类繁多，包括食物中毒、牙痛、炭疽，甚至某些形式的癌症。细菌发明了许多不同的策略来渗透到不同的生物体中，利用它们的资源导致宿主的疾病。这些策略被称为细菌致病性，包括在细菌和细胞膜上运输不同的重要蛋白质及其复合物。为了将有毒分子和其他蛋白质转移到目标细胞中，细菌发展出了几种特殊的系统，这些系统被称为分泌系统。其中一个特征是IV型分泌（T4S）系统，它对不同的条件具有特殊的适应性，能够运输大型大分子复合物。例如，在消化性溃疡和胃癌的发病机制中起主要作用的人类病原体幽门螺杆菌编码一个T4S系统，即Cag-T4S系统，该系统介导毒素CagA的注射。T4S系统是由12个蛋白（VirB1-11和VirD4）组成的大分子组装体。大多数T4S系统有三个专用的atp酶（VirD4（或偶联蛋白）、VirB11和VirB4），它们在为底物转运和装置组装提供能量方面发挥重要作用。在过去的十年中，我们对T4S系统的理解取得了重大进展：通过晶体学方法研究了几个结构元素，并通过电子显微镜方法揭示了T4S系统核心组织的总体结构。然而，更大的复合物仍有待研究，特别是包含所有12个组分的完整组装T4S系统。此外，T4S系统的动态特性仍有待揭示，包括每个atp酶在分泌过程中所起的确切作用。研究表明，三种atp酶VirD4、VirB11和VirB4相互作用，参与底物转移过程。然而，关于atp酶与其他T4S装置相互作用的细节所知甚少。该项目的总体目标是揭示atp酶和核心之间相互作用和信号传导的结构基础。结合不同的方法，如分子遗传学、生物化学、晶体学和结构电子显微镜，将揭示T4S系统的总体结构及其在这个复杂系统中各组成部分之间的相互作用。了解T4S系统的功能是本课题的主题，也是开发更好的治疗和预防传染病的重要一步。

Numerous diseases in humans, in other animals, and also in plants are caused by bacteria. The diseases are extremely diverse including food poisoning, tooth ache, anthrax, and even certain forms of cancer. Bacteria invented many various strategies to penetrate into different organisms to use their resources leading to illness of the host. These strategies, called bacterial pathogenicity, include transport of different important proteins and their complexes across the bacterial and cell membranes. To transfer toxic molecules and other proteins into target cells bacteria developed several types of special systems that are named as secretion systems. One of them that is characterised as a Type IV secretion (T4S) system is exceptionally adaptable to different conditions and able to transport large macromolecular complexes. For example, the human pathogen Helicobacter pylori, which plays a major role in the pathogenesis of peptic ulcers and gastric cancer, encodes a T4S system, the Cag-T4S system, which mediates the injection of the toxin CagA. T4S systems are macromolecular assemblies usually composed of 12 proteins (VirB1-11 and VirD4). Most T4S systems have three dedicated ATPases (VirD4 (or the coupling protein), VirB11, and VirB4) that play essential roles in supplying the energy for substrate translocation and apparatus assembly. Significant progress has been made in the past decade in our understanding of T4S systems: several structural elements were studied by crystallographic methods and the overall architecture of the T4S system core organisation was revealed by methods of electron microscopy. However, larger complexes remain to be investigated, notably a fully assembled T4S system containing all 12 components. Moreover, the dynamic nature of T4S systems remains to be revealed, including the exact role each ATPase plays during the secretion process. It has been shown that the ATPases VirD4, VirB11 and VirB4 interact with each other, and they are involved into substrate transfer process. However, very little is known concerning the details of the interactions of ATPases with each other and with the rest of the T4S apparatus. The overall aim of the project is to reveal the structural basis of interactions and signalling between ATPases and the central core. Combination of different methods such as molecular genetics, biochemistry, crystallography, and structural electron microscopy will reveal a total architecture of the T4S system and interactions between its components within this intricate system. Understanding how T4S system functions is a subject of this proposal and an important step for development of better treatment, and prevention of infectious diseases.