An essential channel in Clostridium difficile sporulation: structure and function

艰难梭菌是英国医院获得性感染的主要原因之一，这是一个主要的健康问题，给医疗保健系统带来了重大的经济负担，据估计，欧盟每年的成本超过30亿欧元。最近医院卫生方面的变化减少了感染的总人数；然而，艰难梭菌感染（CDI）在2012年仅在英格兰和威尔士就造成了大约1500人死亡（大约是MRSA的6倍）。艰难梭菌对大多数抗生素具有耐药性，通常在正常肠道细菌群受到这些药物干扰时生长。最近爆发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，对未来几年的医疗保健构成更大的挑战。抗击CDI的主要问题之一是对艰难梭菌的有限了解。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。一个关键的机制是细菌能够形成休眠细胞——孢子——这是CDI传播和疾病复发的原因。从受感染患者身上释放出来的孢子能够在环境中存活，因为它们对普通消毒剂、高温和辐射具有抵抗力。尽管孢子的形成具有明显的重要性，但人们对其了解甚少，我们的工作主要集中在这个过程中的一个关键方面。在产孢过程中，细胞不对称分裂，较小的“子”细胞（前孢子）被具有双膜系统的母细胞包围，物理上将其与外界培养基隔离。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。我们已经确定了一个由两个蛋白质组成的基本复合物，一个来自每个细胞：存在于前孢子膜的SpoIIQ和我们提出的创建通信通道的母细胞膜蛋白SpoIIIAH。据预测，通道功能的抑制可以阻止孢子的形成，从而中断艰难梭菌的传播周期，使其成为治疗干预的一个有吸引力的靶点。我们的研究将为制造孢子提供必要的详细知识，特别是这个通道，为艰难梭菌感染的治疗研究提供可行的靶点。在这项研究中，我们将确定通道的结构，其功能细节，并识别在两个细胞之间运输的分子。我们的方法是双重的：体外技术将使我们能够解剖结构和生化特性，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越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合体。我们的合作者（葡萄牙）最近开发了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的通道效应研究。我们还将阐明先前未确定的SpoIIQ在细胞壁成分降解中的活性，并研究其在复合物的正确定位和功能中的作用。使用纯化的蛋白质，我们将探索一系列生化和生物物理技术，并辅以体内研究。确定整个通道的结构和稳定性，并确定不同的潜在配体。孢子是艰难梭菌的传染因子，预防产孢是一种有吸引力的治疗途径，但分子靶点仍有待确定。这项工作将阐明艰难梭菌孢子形成的关键分子细节，这对于利用孢子形成作为治疗和控制艰难梭菌感染的有效治疗靶点至关重要。艰难梭菌感染每年影响欧洲超过123,000例患者。

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.