An essential channel in Clostridium difficile sporulation: structure and function

艰难梭菌孢子形成的重要通道：结构和功能

• 批准号: MR/M000923/1
• 负责人: Paula S Salgado
• 金额: $ 56.69万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FM000923%2F1
• 关键词: essential; channel; Clostridium; difficile; sporulation

Clostridium difficile is one of the major causes of hospital acquired infections in the UK, a major health concern that puts a significant economic burden on healthcare systems, estimated to cost over 3000 million euros a year in the EU. Recent changes in hospital hygiene reduced the overall number of infections; however, C. difficile infections (CDI), still caused about 1500 deaths in 2012 (about 6 times more than MRSA) in England and Wales alone. C. difficile is resistant to most antibiotics and usually grows when the normal population of gut bacteria is disturbed by these drugs. Recent outbreaks show greater antibiotic resistance, more serious disease and higher risk of repeating infections, posing an increased challenge to healthcare in the coming years. One of the main issues in fighting CDI is the limited understanding of C. difficile. In order to identify new methods to fight this serious health problem, we need to investigate the infection mechanisms at the molecular level.A key mechanism is the bacteria's ability to form dormant cells - spores - which are responsible for CDI transmission and recurrent disease. Spores released from infected patients survive in the environment due to their resistance to common disinfectants, high temperatures and radiation. Despite its clear importance, formation of spores is poorly understood and our work focuses on one of the key aspects in this process.During sporulation, the cell divides asymmetrically, with a smaller "daughter" cell (forespore) becoming surrounded by the mother cell with a double-membrane system, physically isolating it from the outside medium. In order for the full spore to develop, communication between the two cells has to be maintained across this physical barrier. We have identified an essential complex formed by two proteins, one from each cell: SpoIIQ, present in the forespore membrane and SpoIIIAH, a mother-cell membrane protein, that we propose creates the communication channel.Inhibition of channel function is predicted to prevent spore formation, therefore interrupting the C. difficile transmission cycle, making it an attractive target for therapeutic intervention. Our studies will provide the necessary detailed knowledge to make sporulation, and this channel in particular, feasible targets for therapeutic research for C. difficile infections.In this study, we will determine the structure of the channel, its functional details, and identify the molecules transported between the two cells. Our approach is two-fold: in vitro techniques will allow us to dissect the structural and biochemical properties and in vivo studies will unravel the functional details. We will use protein X-ray crystallography to determine the structure of the whole channel formed across two membranes, the first such complex from this type of bacteria to be solved. A new fluorescence microscopy method, recently developed by our collaborators (Portugal), will be used for in vivo co-localisation of the proteins and studies of the channel effect in gene expression control. We will also elucidate the previously unidentified SpoIIQ activity in the degradation of components of the cell wall and investigate its role in correct localisation and function of the complex. Using purified proteins, we will explore a range of biochemical and biophysical techniques, complemented by in vivo studies. to determine the architecture and stability of the complete channel and identify different potential ligands. The spore is the infectious agent of C. difficile and prevention of sporulation is an attractive therapeutic route, however molecular targets remain to be characterised. This work will elucidate key molecular details in C. difficile spore formation, which are crucial to exploit sporulation as an effective therapeutic target for treatment and control of C. difficile infections, that affect over 123,000 patients a year in Europe.

艰难梭菌是英国医院获得性感染的主要原因之一，这是一个重大的健康问题，给医疗保健系统带来了巨大的经济负担，估计在欧盟每年花费超过30亿欧元。最近医院卫生的改变减少了感染的总数，然而，C。艰难梭菌感染（CDI）在2012年仅在英格兰和威尔士就造成约1500人死亡（约为MRSA的6倍）。C.艰难梭菌对大多数抗生素具有耐药性，通常在肠道细菌的正常种群受到这些药物干扰时生长。最近的疫情显示出更大的抗生素耐药性，更严重的疾病和更高的重复感染风险，在未来几年对医疗保健构成了更大的挑战。反对CDI的主要问题之一是对C语言的理解有限。很难为了找到对抗这一严重健康问题的新方法，我们需要在分子水平上研究感染机制。一个关键机制是细菌形成休眠细胞-孢子-的能力，这是CDI传播和复发疾病的原因。从受感染的病人身上释放出来的孢子由于对普通消毒剂、高温和辐射的抵抗力而在环境中存活。尽管孢子的形成非常重要，但人们对它的了解却很少，我们的工作集中在这个过程中的一个关键方面。在孢子形成过程中，细胞不对称分裂，一个较小的“子”细胞（前孢子）被母细胞包围，形成一个双膜系统，将其与外部介质物理隔离。为了使完整的孢子发育，两个细胞之间的交流必须跨越这个物理屏障。我们已经确定了由两种蛋白质形成的基本复合物，一种来自每个细胞：SpoIIQ，存在于前孢子膜中，SpoIIIAH，一种母细胞膜蛋白，我们提出创建通讯通道。艰难的传播周期，使其成为治疗干预的有吸引力的目标。我们的研究将提供必要的详细知识，使孢子形成，特别是这一渠道，可行的目标，为治疗研究的C。在这项研究中，我们将确定通道的结构，其功能细节，并确定两个细胞之间转运的分子。我们的方法是双重的：体外技术将使我们能够剖析结构和生化特性，体内研究将揭示功能细节。我们将使用蛋白质X射线晶体学来确定跨两个膜形成的整个通道的结构，这是第一个从这种类型的细菌中得到解决的复杂结构。我们的合作者（葡萄牙）最近开发的一种新的荧光显微镜方法将用于蛋白质的体内共定位和基因表达控制中通道效应的研究。我们还将阐明以前未确定的SpoIIQ活性在细胞壁成分的降解，并研究其在正确的定位和功能的复杂的作用。使用纯化的蛋白质，我们将探索一系列的生物化学和生物物理技术，辅以体内研究。以确定完整通道的结构和稳定性并鉴定不同的潜在配体。孢子是C.艰难梭菌和孢子形成的预防是一种有吸引力的治疗途径，然而分子靶点仍有待表征。这项工作将阐明C.艰难梭菌孢子形成，这对于利用孢子形成作为治疗和控制艰难梭菌的有效治疗靶点至关重要。艰难感染，在欧洲每年影响超过123，000名患者。

项目成果

Inducible Expression of spo0A as a Universal Tool for Studying Sporulation in Clostridium difficile.

• DOI: 10.3389/fmicb.2017.01793
• 发表时间: 2017
• 期刊: Frontiers in microbiology
• 影响因子: 5.2
• 作者: Dembek M;Willing SE;Hong HA;Hosseini S;Salgado PS;Cutting SM
• 通讯作者: Cutting SM

The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis.

• DOI: 10.1111/mmi.13311
• 发表时间: 2016-04
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Serrano M;Crawshaw AD;Dembek M;Monteiro JM;Pereira FC;Pinho MG;Fairweather NF;Salgado PS;Henriques AO
• 通讯作者: Henriques AO

The engulfasome in C. difficile: Variations on protein machineries.

艰难梭菌中的吞噬体：蛋白质机器的变化。

• DOI: 10.1016/j.anaerobe.2019.102091
• 发表时间: 2019
• 期刊: Anaerobe
• 影响因子: 2.3
• 作者: Kelly A

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs.

• DOI: 10.1128/mbio.02229-17
• 发表时间: 2018-03-27
• 期刊: mBio
• 影响因子: 6.4
• 作者: Herrero-de-Dios C;Day AM;Tillmann AT;Kastora SL;Stead D;Salgado PS;Quinn J;Brown AJP
• 通讯作者: Brown AJP

Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment.

• DOI: 10.1111/mmi.14091
• 发表时间: 2018-11
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Dembek M;Kelly A;Barwinska-Sendra A;Tarrant E;Stanley WA;Vollmer D;Biboy J;Gray J;Vollmer W;Salgado PS
• 通讯作者: Salgado PS