Bacterial toxins

Supervisors: Professor David Moss and Dr. Ajit Basak

1. Structural studies of gram +ve clostridial phospholipase Cs

The genus Clostridium is diverse and heterogeneous group of gram-positive, bacteria, which are ubiquitous and widely distributed and responsible for many serious human and animal diseases. We have successfully cloned, over expressed and purified this enzyme from various strains of C. perfringens, C. bifermentans, C. absonum, C. barati and C. sordellii. We are interested in determining the 3D-structure of clostridial phospholipase C proteins (PLC) from these bacteria. We have already solved the X-ray structure of C perfringens PLC, which attacks cell membranes and is a major cause of gas gangrene. We now wish to determine the 3D-structures of PLCs from other species that exhibit a range of toxicities and their complex with potential inhibitors and their analogues. These studies will further our understanding of the molecular processes involved in protein-membrane interactions involved in gangrenous disease in man and enterotoxemia in sheep and cattle. The project will appeal to students who wish to study protein-membrane interactions in the propagation of disease.

2. Structure analysis of Clostridium perfringens enterotoxin

The C. perfringens enterotoxin (CPE), a 35kDa protein, is the primary virulence factor in type-A clostridial food poisoning that causes approximately 1 million cases of food poisoning per annum in the UK. We have initiated crystallographic studies of this important toxin, in collaboration with the School of Medicine at the University of Pittsburgh, USA. We have crystallised the full-length protein (proto-toxin) and collected a native X-ray dataset at a resolution of 2.7Å. Recently we have also cloned and purified the activated toxin. The research student would determine the crystal structure of the toxin in order to understand the mechanism of interaction of CPE with its receptors that have already been identified and expressed in intestinal cells. The project would appeal to students interested in structure-function studies in a medically important area.

3. Structural studies LT-toxin and its enzymatic domain

The LT-toxin of C. sordellii belongs to the family of large clostridial cytotoxins and causes diarrhoea and enterotoxaemia in domestic animals and in gas gangrene in man. The toxin is a glucosyltransferase that modifies and inhibits small G-proteins of the Ras family, Ras and Rap as well as Rac proteins. The gene encoding for LT-toxin has been cloned and has been sequenced, with molecular mass of 254kDa. In order to understand the mechanism of function we have targeted this project from two different perspectives. We have purified the protein and produced small crystals of the full toxin. We have also cloned and expressed the enzymatic domain of this protein. The student would carry out X-ray structural studies on the LT-toxin in order to throw light on its mode of action.

References:

1. Naylor, CE., Eaton, JT., Howells, A., Justin, N., Moss, DS., Titball, RW. and Basak, AK. (1998) Structure of the key toxin in gas-gangrene. Nature Structural Biology 5:738-746.

2. Eaton, JT., Naylor, CE., Howells A., Moss, DS., Titball, RW. and Basak, AK. (2002) Crystal structure of C. perfringens a -toxin with the active site closed by a flexible loop region. J. Mol. Biol. 319:275-281.

3. Cole, AR., Gilbert, M., Popoff, M., Moss, DS., Titball, RW. And Basak, AK. (2004) Clostridium perfringens e-toxin shows structural similarity to the pore-forming toxin Aerolysin. Nature Structural and Molecular Biology. 11, 797-798.

BBSRC EARMARKED STUDENTSHIP

4. Understanding the role of Clostridium perfringens epsilon-toxin in enterotoxaemia

The e-toxin is produced by Clostridium perfringens types B and D as an inactive prototoxin (Mw 32.7kDa), which is activated by proteolytic cleavage peptides at the N- and C-terminal, to yield a 29.5kDa mature toxin. The e-toxin is a major pathogen in the farming industry and symptoms are usually quickly followed by death. Recently we have determined the 3D-structure of the e-prototoxin in studies supported by a BBSRC project grant. We now need to determine the X-ray structure of the active toxin. We also need to study the heptameric form of the toxin by electron microscopy, which may form membrane channels. These structural studies will give us more insight into the molecular basis of serious animal disease.

5. Structural studies of Botulinum Neurotoxin (Type-C and Type-F)

Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and is one of the more potent toxins known being the cause of botulism. There are seven serotypes of this protein (A-G) and they share significant sequence homology and possibly structural similarity. However, each BoNT has unique substrate specificity and the peptide bond cleavage selectivities of their catalytic domains are different. The reason for this unique specificity of these neurotoxins is not clear. In the interest of inhibitor design leading to the therapeutic use to all serotypes, it is essential to use X-ray crystallography to elucidate the differences in their 3D-structures that are responsible for their unique characteristics. We have already grown small crystals of BoNT (type-C) full-length protein and cloned and purified its catalytic domain. Crystallisation trials of type-F protein are in progress. This project would appeal to students who wish to study the structure function relationships and understand more about neurotoxins.