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.

像肉毒杆菌中毒和炭疽病这样的细菌能在恶劣的条件下生存，并以孢子的形式传播疾病。然而，为了使这些孢子产生疾病症状，它们必须萌发成所谓的营养细胞。营养细胞产生的毒素通常是导致受感染的动物或人类宿主生病的主要因素。我们希望了解萌发是如何发生的，以及活跃的营养细胞是如何从休眠的孢子中出现的。这是一种非凡的蜕变，一种错综复杂的细胞结构完全转变为完全不同的结构--它类似于种子蜕变成幼苗，可以说是同样复杂！这个过程也很有趣，因为营养细胞比孢子更容易受到攻击，例如抗生素或消毒剂。因此，如果我们能研究出如何“萌发到灭绝”，我们就可以开发出新的武器来对抗一些疾病和食物腐败的有机体。我们将研究两种选定的生物--产孢梭菌和艰难梭状芽孢杆菌的萌发。产孢子杆菌是非致病性的，但与肉毒杆菌密切相关，肉毒杆菌中毒是一种潜在的致命麻痹。孢子具有极强的耐热性，因此从食物中根除需要昂贵的热处理，这会降低营养价值，降低产品的吸引力，但对加工最少的安全食品的需求却在不断增加。C.肉毒杆菌也被列为潜在的生物武器(英国2001年反恐怖主义、犯罪和安全法)。使用无害的产孢子杆菌的好处是，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遏制设施。艰难梭菌是抗生素相关性腹泻的主要原因，尤其是在医院患者和老年人中。当摄入的孢子对肠道中的胆汁酸做出反应而萌发时，疾病就会发生。毒素是疾病的主要原因，在许多情况下是死亡。留在肠道中的未萌发的孢子即使在治疗后也可能是反复感染的来源。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萌发的方式。这项工作的一个关键方面将涉及绘制3D分子细节的组成孢子的蛋白质图。为了实现这一目标，我们将利用显微镜领域令人兴奋的新发展--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，在那里我们甚至可以识别和定位单个分子。然后，我们将继续可视化孢子转化为活跃细胞的结构，就像摄入受污染的食物时可能发生的那样。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发展以及计算机图像处理的组合。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发生的机械和生化变化。