Structure and interactions of the Clostridium difficile S-layer with bacteriocins.

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。艰难梭菌感染(CDI)最常发生在医院环境中，影响到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抗生素具有天然抗药性，因此，虽然这些抗生素会杀死人体肠道中的有益细菌，但它们对艰难梭菌没有影响；事实上，它们通过消除竞争而使细菌受益。艰难梭菌表面的蛋白质是它与环境和人类宿主相互作用的附属物。艰难梭菌的表面覆盖着一层蛋白质，形成了包裹在细菌周围的二维晶体。这一表层(S层)起到了盔甲的作用，保护细菌免受我们免疫系统的攻击，是细菌致病所必需的。尽管S层很重要，但我们对它如何在细胞表面组装以及它的样子知之甚少。我们最近与一家生物技术公司合作，该公司开发了一种新的治疗颗粒，这种颗粒附着在S层上，以杀死艰难梭菌。这些颗粒被称为阿维菌素，由弹簧加载的护套内的一根针组成。当阿维菌素结合到细胞表面时，鞘收缩并将针穿过细胞壁和细胞膜，从而杀死细菌。虽然我们知道阿维菌素与S层结合，但我们不知道它们是如何识别S层的，它们在哪里结合，结合时经历了什么变化，如何触发收缩，或者针如何穿透细胞被膜的各个层。在这个项目中，我们将结合艰难梭菌生物学和强大的电子显微镜的互补专业知识：1.了解S层的结构和组织。如果我们想要开发针对S层的新疗法来治疗或预防CDI，这是必不可少的信息。了解阿维菌素颗粒本身的结构以及与艰难梭菌结合并杀死细胞的行为。迫切需要开发新的疗法来对抗CDI，特别是开发既能杀死艰难梭菌又不会对有益的肠道细菌造成损害的疗法。一种新的治疗方法将理想地针对细菌中对其生命周期至关重要的独特部分。S层是这种干预的理想候选者，因为它不像任何其他类型的细菌的表面。阿维菌素本身就是很有希望的治疗药物，也与感染艰难梭菌(噬菌体)的自然病毒密切相关。人们对噬菌体治疗感染的潜力很感兴趣。我们将研究的阿维菌素杀死的许多特征与噬菌体感染直接相关，包括结合到S层，收缩弹簧加载的鞘和穿透细胞被膜。我们对S层的工作将确定如何将这一重要结构作为应对CDI的目标。

Clostridium difficile is an important human pathogen, causing serious illness and even death. C. difficile infection (CDI) occurs most commonly in the 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; indeed they actually benefit the bacterium by removing competition. The proteins on the surface of the C. difficile bacterium are the appendages through which it interacts with its environment and the human host. The C. difficile surface is coated in a single layer of protein that forms a two-dimensional crystal that wraps around the bacterium. This surface layer (S-layer) acts as a coat of armour to protect the bacterium from attack by our immune system and is essential for the bacteria to cause disease. Despite the importance of the S-layer we know very little about how it assembles on the cell surface and what it looks like. We have recently collaborated with a biotech company who have developed new therapeutic particles that attach to the S-layer to kill the C. difficile. These particles, called Avidocins, consist of a needle inside a spring-loaded sheath. When the Avidocin binds to the cell surface the sheath contracts and drives the needle through the cell wall and membrane, killing the bacterium. Although we know that Avidocins bind to the S-layer we do not know exactly how they recognise the S-layer, where they bind, what changes they undergo while binding, how the contraction is triggered or how the needle penetrates the various layers of the cell envelope.In this project we will combine our complementary expertise in C. difficile biology and powerful electron microscopy to:1. Understand the structure and organisation of the S-layer. This is essential information if we want to develop new treatments that target the S-layer to treat or prevent CDI.2. Understand the structure of the Avidocin particle both on its own and in the act of binding to and killing a C. difficile cell.There is an urgent need to develop new therapies to combat CDI 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 bacterium which is essential for its lifecycle. The S-layer is an ideal candidate for this sort of intervention because it doesn't resemble the surface of any other kind of bacteria. Avidocins are promising therapeutic agents in their own right and are also closely related to natural viruses that infect C. difficile (bacteriophage). There is a lot of interest in the potential of bacteriophage to treat infections. Many of the features of Avidocin killing that we will study are directly relevant to bacteriophage infection, including binding to the S-layer, contraction of the spring-loaded sheath and penetration of the cell envelope. Our work on S-layer will identify ways in which this important structure can be targeted to tackle CDI.