Structural transitions and cellular remodelling in spore germination

孢子萌发中的结构转变和细胞重塑

• 批准号: BB/W015072/1
• 负责人: Per Bullough
• 金额: $ 113.04万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW015072%2F1
• 关键词: Structural; transitions; cellular; remodelling; spore

Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. However for these spores to cause disease symptoms they must germinate into so-called vegetative cells. Toxins produced by the vegetative cells are most often the main factor in making infected animal or human hosts sick. We wish to understand how this germination takes place and how the active vegetative cell emerges from the dormant spore. This is a remarkable metamorphosis where one intricate type of cell structure is completely transformed into a radically different structure- it is analogous to the metamorphosis of a seed into a seedling and arguably as complex! The process is also interesting because vegetative cells are much more vulnerable to attack, for example by antibiotics or disinfectants, than spores are. Thus if we could work out how to 'germinate to exterminate', we could develop new weapons against a number of diseases and food-spoilage organisms. We will study germination in two selected organisms- Clostridium sporogenes (C. sporogenes) and Clostridioides difficile (C. difficile). C. sporogenes is non-pathogenic but very closely related to C. botulinum which causes botulism, a potentially lethal paralysis. Spores are extremely heat resistant so eradication from food requires costly heat treatment that lowers nutritional value & makes the product less appealing, yet there is increasing demand for minimally processed, safe foods. C. botulinum is also listed as a potential bio-weapon (UK Anti-terrorism, Crime & Security Act 2001). The advantage of using the harmless C. sporogenes is that we can learn a lot about C. botulinum without the need for highly specialised containment facilities. C. difficile is the leading cause of antibiotic-associated diarrhoea, especially in hospital patients and the elderly. Disease occurs when ingested spores germinate in response to bile acids in the gut. The toxins are the major cause of disease & in many cases, death. Ungerminated spores remaining in the gut can be the source of recurrent infections even after treatment.Our multidisciplinary project will apply state of the art techniques of structural and molecular biology to investigate the way these spores germinate. A crucial aspect of this work will involve mapping the proteins that make up spores in 3D molecular detail. To achieve this, we will exploit exciting new developments in microscopy - such as cryo-electron tomography - that allow us to visualise spores and cells in unprecedented detail. We will create 3D images of the spore where we can even identify and locate individual molecules. We will then go on to visualise the structures of spores as they transform into active cells, as might happen when contaminated food is ingested. The project will exploit new developments in imaging methods that allow us to see the structures more clearly. It will involve combinations of molecular genetics, use and development of electron light and scanning probe imaging approaches and computational image processing. Ultimately we aim to make a 3D movie that explains the mechanical and biochemical changes that take place when the active vegetative cell emerges out of the spore.

引起肉毒杆菌中毒和炭疽病的细菌可以在恶劣的条件下生存，并以孢子的形式传播疾病。然而，这些孢子要引起疾病症状，它们必须发芽成所谓的营养细胞。营养细胞产生的毒素通常是导致受感染动物或人类宿主患病的主要因素。我们希望了解这种发芽是如何发生的以及活跃的营养细胞如何从休眠孢子中出现。这是一种非凡的变态，其中一种复杂类型的细胞结构完全转变为一种完全不同的结构——这类似于种子变态为幼苗，并且可以说同样复杂！这个过程也很有趣，因为营养细胞比孢子更容易受到抗生素或消毒剂的攻击。因此，如果我们能够弄清楚如何“发芽消灭”，我们就可以开发出对抗许多疾病和食物腐败生物的新武器。我们将研究两种选定的生物体的发芽——产孢梭菌（C. sporogenes）和艰难梭菌（C. difficile）。孢子梭菌是非致病性的，但与引起肉毒杆菌中毒（一种潜在致命性麻痹）的肉毒梭菌密切相关。孢子具有极强的耐热性，因此从食品中根除孢子需要昂贵的热处理，这会降低营养价值并使产品吸引力降低，但对经过最低限度加工的安全食品的需求却在不断增加。肉毒杆菌也被列为潜在的生物武器（英国《2001 年反恐、犯罪和安全法案》）。使用无害的孢子梭菌的优点是我们可以了解很多关于肉毒杆菌的知识，而不需要高度专业化的收容设施。艰难梭菌是抗生素相关性腹泻的主要原因，尤其是医院患者和老年人。当摄入的孢子响应肠道中的胆汁酸而发芽时，就会发生疾病。毒素是疾病的主要原因，在许多情况下是死亡的主要原因。即使在治疗后，残留在肠道中的未发芽孢子也可能成为复发感染的根源。我们的多学科项目将应用最先进的结构和分子生物学技术来研究这些孢子的发芽方式。这项工作的一个重要方面将涉及以 3D 分子细节绘制构成孢子的蛋白质。为了实现这一目标，我们将利用显微技术令人兴奋的新发展——例如冷冻电子断层扫描——使我们能够以前所未有的细节可视化孢子和细胞。我们将创建孢子的 3D 图像，甚至可以识别和定位单个分子。然后，我们将继续观察孢子转变为活性细胞时的结构，就像摄入受污染的食物时可能发生的情况一样。该项目将利用成像方法的新发展，使我们能够更清楚地看到结构。它将涉及分子遗传学、电子光和扫描探针成像方法的使用和开发以及计算图像处理的结合。最终，我们的目标是制作一部 3D 电影，解释当活跃的营养细胞从孢子中出现时所发生的机械和生化变化。