Structural and functional characterisation of cell wall proteins of Clostridium difficile.

The bacterium Clostridium difficile can cause serious intestinal infections, and elderly patients in hospitals are particularly at risk. Rates of infection are increasing in the UK and elsewhere, and infection is linked to the widespread of use antibiotics. The first step in an infection is when the bacteria colonise the gut, where they then grow and cause disease. At present we cannot prevent colonisation, and so the bacteria are free to multiply and produce toxins that cause diarrhoea and other potentially fatal symptoms. There is no vaccine against C. difficile, and treatment with antibiotics is not always effective. Developing new therapies and treatments against C. difficile is essential, but this is very difficult because we know little about the bacterium. We do know that on the outside of the bacteria there are many surface proteins. These proteins are essential for colonisation, and if we can interfere with their functions we will be able to prevent disease. In this project we will study the surface proteins. We will use several biochemical techniques to unravel their molecular structures, discovering exactly what the proteins look like. We will also explore how the proteins fit together on the surface of the bacterium. Not all strains of C. difficile are identical, and in this project we will investigate three strains that are particularly prevalent in the UK. We will also perform a limited vaccination study in animals.The information we will gain will help enormously in the design of new inhibitors of colonisation or in new vaccines against infection.

Clostridium difficile associated disease (CDAD) is the most common cause of antibiotic associated diarrhoea in UK hospitals. The incidence of disease is rising, and management and treatment of disease is problematic. There are no vaccines against CDAD, and the only treatments rely on a limited number of antibiotics. In order to cause disease, C. difficile firstly colonises the intestine and then produces toxins that mediate cell damage and diarrhoea. Colonisation is clearly an essential process, but we know very little about the mechanisms involved.

Over the last 5 ? 6 years, our laboratory has characterised the cell wall proteins of C. difficile. The surface or S-layer is composed of two proteins (SLPs), a low molecular weight SLP (LMW SLP) and a high molecular weight SLP (HMW SLP). These proteins are involved in adherence of the bacterium to host tissues; in particular the LMW SLP binds to cell lines and to human enteric tissues. Extensive variation is seen in the amino acid sequences of the SLPs from different strains, in particular in the LMW SLP which is presumed to be surface exposed. We show here that the HMW and LMW SLPs are held together by non-covalent forces to form a complex, the H/L complex and that specific residues of the LMW SLP are essential for complex formation. We have also determined the X-ray crystal structure of the LMW SLP from strain 630, the genome sequence strain.

In this application our aims are:
(1) to solve the X-ray structures of (a) the HMW SLP from strain 630, (b) the H/L complex from strain 630, (c) the LMW SLP from the three major strains that cause disease in the UK ? strains 001, 106 and 027 and (d) the variable domains of two other adhesins, Cwp66 and CwpV;
(2) to analyse the molecular biology of the assembly of the H/L complex through mutagenesis and by construction of mutants in C. difficile;
(3) to carry out a limited vaccination study in hamsters using structurally designed SLPs as antigens.

This project will provide a substantial amount of structural data on the important cell wall proteins from C. difficile strains most relevant to infection in the UK. It will reveal molecular details of the assembly of the S-layer, a process that could be the target for novel therapeutics. Finally the vaccine study will reveal whether colonisation could potentially be prevented by immunisation with S-layer proteins.