Structure-function studies on Clostridium difficile large toxins

艰难梭菌感染（CDI）是医院获得性腹泻的最重要原因。艰难梭菌是一种厌氧菌，存在于高达3%的健康成年人和66%的婴儿的肠道中。然而，艰难梭菌很少引起儿童或健康成人的问题，因为它是由肠道正常细菌群控制的。当某些抗生素扰乱肠道内细菌的平衡时，艰难梭菌可以迅速繁殖并产生导致疾病的毒素。CDI的范围从轻微到严重腹泻，到更不寻常的严重肠道炎症（称为假膜性结肠炎）。接受过广谱抗生素（影响多种细菌的抗生素）治疗的人、患有严重潜在疾病的人和老年人的风险最大——报告的cdi中，超过80%发生在65岁以上的人群中。CDI通常通过卫生保健工作人员和其他接触受感染患者或被细菌或其孢子污染的环境表面（如地板、便盆、厕所）的人的手传播。当艰难梭菌遇到不利条件时，例如在体外，就会产生孢子。它们非常耐寒，可以长时间在衣服和环境表面上生存。然而，随着医院护理和卫生意识的提高，2010年健康保护局（确定和应对传染病造成的健康危害和紧急情况的机构）记录的病例数量有所减少，但仍然是国家保健服务的主要威胁和重大经济负担。有证据表明，甲硝唑和万古霉素（两种临床上常用的抗生素）的耐药性正在增加，因此迫切需要开发替代疗法。对于目前治疗方案非常有限的严重CDI病例尤其如此。流行病学和疾病严重程度的变化，特别是在过去十年中出现的菌株（例如027核糖型）方面的变化，突出表明需要更多地了解这种世界性病原体。通过与英国Porton Down健康保护局的Clifford Shone博士的学术合作，我们已经着手阐明与CDI有关的一些关键分子的分子结构，例如艰难梭菌主要毒素（毒素a和b和二元毒素）。目前，我们对艰难梭菌的这些蛋白质分子是如何引起疾病的，以及针对这些毒素产生的抗体如何中和它们的活性的机制，还存在相当大的空白。更好地了解毒素结构和作用的这些方面将大大有助于设计新的治疗方法，包括改进疫苗、基于抗体的治疗方法和基于小分子抑制剂（药物）的治疗方法。

Clostridium difficile infection (CDI) is the most important cause of hospital-acquired diarrhoea. C. difficile is an anaerobic bacterium that is present in the gut of up to 3% of healthy adults and 66% of infants. However, C. difficile rarely causes problems in children or healthy adults, as it is kept in check by the normal bacterial population of the intestine. When certain antibiotics disturb the balance of bacteria in the gut, C. difficile can multiply rapidly and produce toxins which cause illness. CDI ranges from mild to severe diarrhoea and to, more unusually, severe inflammation of the bowel (known as pseudomembranous colitis). People who have been treated with broad spectrum antibiotics (those that affect a wide range of bacteria), people with serious underlying illnesses and the elderly are at greatest risk - over 80% of CDIs reported are in people aged over 65 years. CDI is usually spread on the hands of healthcare staff and other people who come into contact with infected patients or with environmental surfaces (e.g. floors, bedpans, toilets) contaminated with the bacteria or its spores. Spores are produced when C. difficile bacteria encounter unfavourable conditions, such as being outside the body. They are very hardy and can survive on clothes and environmental surfaces for long periods. However, with better hospital care and hygiene awareness, the number of cases recorded in 2010 by the Health Protection Agency (the agency that identifies and responds to health hazards and emergencies caused by infectious disease) has reduced, but still remains a major threat and significant economic burden to the NHS. With evidence of growing antibiotic resistance for metronidazole and vancomycin (two well known antibiotics used in the clinic) there is an urgent need for the development of alternative therapeutics. This is particularly true for cases of severe CDI for which there are currently very limited treatment options. Changes in epidemiology and disease severity, particularly in respect of strains that have emerged over the last ten years (e.g. the 027 ribotype), highlight the need to understand more about this worldwide pathogen. Through an academic collaboration with Dr. Clifford Shone at the Health Protection Agency, Porton Down (UK), we have set about elucidating the molecular structures of some of the key molecules implicated in CDI such as C. difficile major toxins (Toxin-A and-B and the binary toxin). Currently there are considerable gaps in our understanding of how these protein molecules of C. difficile cause disease and the also the mechanism by which antibodies produced against these toxins, neutralise their activity. A greater understanding of these aspects of toxin structure and action would greatly aid the design of new therapeutics including improved vaccines, therapeutics based on antibodies and therapeutic based on small molecule inhibitors (drugs).