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).

Clostridium difficile is a major problem as the aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonises the gut during infection is poorly understood, but undoubtedly involves a set of key toxin molecules. Our aim is to further define roles for the key toxins using a detailed structure-function study. While the sequence of amino acids which make up these proteins can provide a great deal of information about the likely roles of the protein, primarily in relation to (and as a result of) other already characterised proteins, it is the tertiary or 3D structure of the protein that reveals the true interactions between proteins and their ligand/receptor interactions.
Understanding these complex interactions require data at the atomic level obtained by X-ray crystallography with the aid of other complementary biophysical techniques. To facilitate these studies, preliminary work has created a toolbox of reagents including highly purified C. difficile toxins from a variety of strains, recombinant toxin fragments and panels of monoclonal antibodies to the various toxin domains. Currently, there are considerable gaps in our understanding of these toxins both at a detailed mechanistic level and in respect of their roles in the pathogenesis of C. difficile.

Key goals of the proposal are-
- Obtaining a detailed structure of the C. difficile to, in particular the central domains and deciphering their role in receptor binding and intracellular translocation.
- Obtaining further insights into the mechanism of antibody-mediated toxin neutralisation and the structure and location of key epitopes. Key questions include: why some monoclonal antibodies neutralise toxin activity while others do not and the likely impact of sequence variation on antibody binding.
Progress in these areas would improve our understanding of the pathogens of CDI and facilitate the design of improved vaccines, immunotherapeutics and toxin inhibitors.

Clostridium difficile infection (CDI) continues to be a major problem within the healthcare environment. This bacterium mainly infects elderly patients on antibiotics causing a range of symptoms from mild diarrhoea to severe, recurrent diarrhoea which is often life-threatening. There were 23,253 reports in 2010, comprising 20,463 from England, 2,106 from Wales and 684 from Northern Ireland (patients aged 2 and older) and in 2009 over 4000 deaths were directly attributable to CDI. While better management of the disease risk factors together with improved hospital cleaning have reduced case numbers significantly for 2009, there is little doubt that C. difficile will remain a serious problem as a healthcare-associated infection for many years in the future. More virulent strains of the bacterium (ribotype 027 family) emerged in 2001 and they continue to cause disease with increased morbidity and mortality. Recently, other virulent strains (ribotype 078 family) have appeared which are causing increased community-associated CDI. This rapidly changing epidemiology of C. difficile underlines the need for the development of new interventions to manage its associated diseases.
Treatment of C. difficile infection currently relies on antibiotics of which metronidazole and vancomycin constitute the treatments of choice. However, these antibiotics are not effective in all cases and 20-30% of patients suffer relapse of the disease. In addition, there are few treatment options currently available for severe CDI which develops in 3-8% of cases. With evidence of growing antibiotic resistance there is an urgent need for the development of alternative therapeutics.
It is well established that the bacterial cell surface serves two central purposes: protection from and interaction with the environment. Infections by microbes follow a similar pattern: adhesion to host cells followed by complex interactions that often involve secretion of microbe effector proteins e.g. toxins and induction of signaling processes by the host cells. In the case of C. difficile, the toxins and the proteins of the microbe cell surface (the known major players responsible for CDI) are therefore of considerable interest both academically and industrially as they represent a novel interaction site between host and the pathogen. Deciphering these interactions at the molecular level is of tremendous importance. The interruption of such interactions may result in the cessation of potentially harmful infection.
In the proposed programme of research we have tried to highlight the supreme informative power of structural data, which is being sought for some of the key players implicated in CDI. However our interest is not merely to elucidate structures of proteins, but to understand the molecular basis of protein function, their interaction with antibodies and to use this information for the design of therapeutic interventions.The outcome of this research will be of immense interest to many biologists and biomedical researchers where the available structural details are limited. We strongly believe that the functional properties of these proteins can be understood only in terms of their relationship to their three-dimensional structure. The proposed research involves our combined expertise in structural biology (Prof. K. Ravi Acharya, University of Bath, UK) and input from an established key collaborative link with Dr. Clifford Shone at the Health Protection Agency, Porton Down (UK).
We believe that the requested support from the MRC would allow us to accomplish the major aims of this proposal in an area that is extremely important in biomedical research with a long term goal 'To reduce the incidence and consequences of infection' in the priority area of 'Exploiting our assets for the development of new evidence based interventions' as set out by the Health Protection Agency (UK) in line with the MRC policy to support academic research in the UK.