Characterisation of the essential C. difficile S-layer secretion system

艰难梭菌是一种重要的人类病原体，可引起严重疾病甚至死亡。艰难梭菌相关疾病（CDAD）最常发生在医院环境中，影响已经健康状况不佳的患者，但社区感染是一个日益严重的问题。艰难梭菌对许多常见的抗生素有天然的抵抗力，所以当这些抗生素杀死人类肠道中的有益细菌时，它们对艰难梭菌没有影响，实际上通过消除竞争对细菌有益。迫切需要开发新的治疗方法来对抗CDAD，特别是开发能够杀死艰难梭菌但不会对有益肠道细菌造成损害的治疗方法。理想情况下，一种新的治疗方法将针对细菌的一个独特部分，这个部分对细菌的生命周期至关重要。包裹在艰难梭菌表面的蛋白质是细菌与人类宿主相互作用的附属物。我们的研究重点是这些表面结构是如何组装的。这种组装的早期关键步骤是蛋白质从细胞内部运输，穿过脂质膜到达细胞表面。我们发现这一步骤需要一种特殊的分泌系统，称为辅助分泌系统。这种分泌系统及其运输的蛋白质对生物体的生命及其引起疾病的能力至关重要，使其成为未来开发治疗CDAD的新疗法的理想靶点。然而，我们对这个系统如何运作的了解还有许多重要的空白，包括系统如何识别它分泌的蛋白质，以及系统如何与细胞中的其他蛋白质协同工作以实现分泌。我们必须详细了解它的运作，以便开发出阻断分泌和杀死细菌的药物。我们将采用多学科的方法来回答四个关键问题：1。辅助Sec系统位于单元的哪个位置？我们将使用先进的显微镜技术来观察细胞表面新蛋白出现的位置，以确定辅助Sec系统是否定位于细胞的特定部分。我们的初步数据表明，细胞内可能存在少量不同的分泌通道。通过辅助Sec系统有效分泌还需要哪些额外的蛋白质？辅助Sec系统的核心是一个分子马达SecA2，它为系统提供动力，还有三种蛋白质SecYEG，它们在膜上形成一个通道，分泌的蛋白质通过这个通道运输。迄今为止，我们的工作主要集中在这四个方面。然而，其他类似的系统需要更多的蛋白质才能充分发挥效率。我们将使用马达蛋白SecA2分离和鉴定它在细胞中与之相互作用的其他蛋白。辅助Sec系统如何识别它所运输的蛋白质？辅助Sec系统最重要的特征是能够以惊人的准确性识别和运输一些特定的蛋白质。我们将结合生物化学和遗传学来确定该系统如何识别其目标。这一信息对于开发特异性抑制该系统的药物至关重要。从长远来看，我们将在这个项目中获得的见解的基础上开发干扰艰难梭菌附属Sec系统的化学物质。这可能会导致CDAD新疗法的发展。

Clostridium difficile is an important human pathogen, causing serious illness and even death. C. difficile associated disease (CDAD) occurs most commonly in a hospital setting, affecting patients who are already suffering ill health, but infections in the community are an increasing problem. C. difficile is naturally resistant to many common antibiotics so whilst these antibiotics kill the beneficial bacteria in the human gut they have no effect on C. difficile and actually benefit the bacterium by removing competition. There is an urgent need to develop new therapies to combat CDAD and, in particular, to develop therapies which kill C. difficile but do not cause damage to the beneficial gut bacteria. A new therapy would ideally target a distinctive part of the bacteria which is essential for the bacteria's lifecycle.The proteins which coat the C. difficile surface are the appendages through which the bacterium interacts with its human host. Our research focuses on how these surface structures are assembled. A critical early step in this assembly is the transport of the proteins from the interior of the cell, where they are made, across a lipid membrane and onto the cell surface. We have discovered that this step requires a special secretion system, called the accessory Sec system. This secretion system, and the proteins it transports, are essential to the life of the organism and its ability to cause disease, making it an ideal target for the future development of new therapies for the treatment of CDAD. However there are many important gaps in our knowledge of how this system operates, including how the system recognises the proteins it secretes and how the system works with other proteins in the cell to enable secretion. It is essential that we develop a detailed understanding of its operation in order to allow the development of drugs that will block secretion and kill the bacteria.We will adopt a multidisciplinary approach to answer four key questions:1. Where in the cell is the accessory Sec system located?We will use advanced microscopy techniques to visualise where on the surface of the cell new protein appears, to determine if the accessory Sec system is localised to a particular part of the cell. Our preliminary data indicates that the there may be a small number of distinct secretion channels in the cell.2. What additional proteins are required for efficient secretion via the accessory Sec system?At the core of the accessory Sec system is a molecular motor, SecA2, which powers the system and three proteins, SecYEG, which form a channel in the membrane through which the secreted protein is transported. To date our work has focussed on these four components. However other similar systems require several more proteins for full efficiency. We will use the motor protein SecA2 to isolate and identify other proteins it interacts with in the cell.3. How does the accessory Sec system recognise the proteins it transports?The most important characteristic of the accessory Sec system is the ability to recognise and transport a few specific proteins with astonishing accuracy. We will use a combination of biochemistry and genetics to determine how the system recognises its targets. This information will be crucial to the development of drugs which inhibit this system specifically.In the longer term we will build on insights gained from this project to develop chemicals that interfere with the accessory Sec system in C. difficile. This could lead to the development of new treatments for CDAD.