Structure-Function Studies on Clostridium Difficile Large Toxins

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).