The Gas-Gangrene Causing Alpha-Toxin: Membrane Interaction and Toxicity

As is frequently documented in the press, people are getting fatter all the time. In fact, by 2010 one in three of us will be clinically obese. Being overweight carries with it a range of health risks, including an increased likelyhood of developing type II diabetes. Diabetes, in turn, has a number of complications, including increased susceptibility to gengrene of the legs and feet. Gangrene is treated by the amputation of the infected leg, while if left untreated it is invariably fatal. This project aims to increase our understanding of what happens in our bodies once we have gangrene, and thus to increase the effectiveness of the treatment.

Gas-gangrene is caused predominantly by a single toxin released by a bacterium that has infected an injury. This toxin attachs to and attacks human or animal cell walls. The result of this attack is the release of certain signals that cause changes in the limb and the rest of your body. We are going to study the interaction between the toxin and human cell walls by using light-emitting markers attached to the toxin or the cell wall and by examining the molecular structure of the toxin.

We will identify which parts of the toxin make it interact strongly with cell walls. And, conversely, what features of the cell wall allow the toxin to attach to it. We will find out how we can change the behaviour of the toxin by changing the features of the cell wall and/or the toxin. This will allow us to devise strategies for mitagating the effects of the toxin in real cases.

Clostridium perfringens phospholipase C (alpha-toxin) is the key determinant of gas gangrene, a major complication of diabetes. We have solved the 3D crystal structure of this toxin in different chemical environments and also the structures of related enzymes, and have consequently developed a model for toxin-membrane interaction. As a result, we hypothesise that specific features of the enzyme and phospholipid membranes promote this interaction, and that understanding the specificity of the toxin-membrane interaction will allow new approaches to the treatment of disease to be devised. It is this hypothesis which we plan to explore with the work described in this application. We will use fluorescence imaging to investigate the factors that influence membrane binding. An assay for membrane binding will be developed, and used to study how changes to both the toxin (via genetic modification and by comparing the toxin to related enzymes) and the membrane structure (by varying phospholipid composition and cell type) affect the membrane-toxin interaction. Where these studies lead to a substrate analogue with promising characteristics for study by X-ray crystallography, we will determine the atomic resolution structure of the enzyme-substrate complex, in order to identify residues essential for catalytic activity. The structures of related enzymes (from C. bifermentans and C. novyii) will be solved using X-ray crystallographic techniques. In combination with the fluorescence results, this atomic resolution information will allow us to identify which residues confer specificty on the toxin-membrane interaction. Understanding this interaction will enable us to understand how the toxin causes tissue necrosis and systemic disease and thus we will be able to alleviate symptoms in a clinical setting.