Structure-function studies on Clostridium difficile large toxins

艰难梭菌感染(CDI)是引起医院获得性腹泻的最重要原因。艰难梭菌是一种厌氧细菌，存在于多达3%的健康成年人和66%的婴儿的肠道中。然而，艰难梭菌很少对儿童或健康的成年人造成问题，因为它受到肠道正常细菌群的控制。当某些抗生素扰乱肠道细菌的平衡时，艰难梭菌会迅速繁殖并产生导致疾病的毒素。CDI的范围从轻度到重度腹泻，以及更不寻常的严重肠炎(称为伪膜性结肠炎)。接受过广谱抗生素治疗的人(那些影响多种细菌的人)、有严重潜在疾病的人和老年人风险最大--报告的CDI中超过80%是65岁以上的人。CDI通常传播到医护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)的人的手中。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，可以在衣服和环境表面上长时间存活。然而，随着更好的医院护理和卫生意识，2010年卫生保护局(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NHS的主要威胁和重大经济负担。随着甲硝唑和万古霉素(临床上使用的两种广为人知的抗生素)对抗生素耐药性的增加，迫切需要开发替代疗法。对于目前治疗选择非常有限的严重CDI病例来说，情况尤其如此。流行病学和疾病严重性的变化，特别是关于过去十年出现的毒株(例如027核糖型)，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 Slight博士的学术合作，我们已经着手阐明CDI中涉及的一些关键分子的分子结构，如艰难梭菌主要毒素(毒素-A和-B以及二元毒素)。目前，对于艰难梭菌的这些蛋白质分子如何致病，以及产生抗这些毒素的抗体中和它们的活性的机制，我们的理解存在相当大的差距。更好地了解毒素结构和作用的这些方面将极大地帮助设计新的疗法，包括改进的疫苗、基于抗体的疗法和基于小分子抑制剂(药物)的疗法。

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).