Interactions of lipid membranes with chitosan and epsilon-toxin: a biophysical approach

点击翻译按钮获取中文摘要

Entamoeba histolytica is a protozoan parasite responsible for an estimated 100 000 deaths annually and is a major health problem in the developing countries. This parasite has strong cytolytic activity, which has been related to a class of pore-forming toxins, amoebapores, produced by this organism. Amoebapores (and more generally, pore-forming toxins) are capable of perforating the plasma membrane of target cells, thereby killing them. Although the cytotoxicity of amoebapores has been studied previously, there is little understanding of the biophysical processes associated with toxicity and the role of the lipid membrane physical properties. In the past few years, we have developed novel experimental methodologies to investigate the biophysics behind toxin activity, focusing on Clostridium perfringens a-toxin and the pore-forming toxins NetB and Pneumolysin. This project will investigate, using in vitro model systems, the biophysical factors determining cytolytic activity of amoebapores. Initially, we shall seek to establish the main lipid species responsible for recruitment of the toxin to the lipid membrane and its activation, using model membrane systems (Langmuir monolayers of lipids and bilayer lipid vesicles), as well as the effect of the toxin on the lipid organisation in the membranes. Then, we shall investigate the effect of biochemical and biophysical properties of the plasma membrane on the susceptibility of human red blood cells to amoebapores. The properties to be studied will be membrane elasticity, electrical properties and morphology, which are likely determinants of toxin activity. We will also modify the biochemical status of the membrane using oxidative stress, lipid scrambling and cell ageing in order to determine the role of cell surface biochemistry in modifying cell responses to toxins. There are some minor amino acid changes between amoebapores from Entamoeba species with different levels of virulence attributed to these differences but a mechanism explaining these differences in virulence is lacking. We shall study these amoebapores from different Entamoeba species using the above techniques as we expect these differences to affect membrane solubility. These studies will identify the factors responsible for cell lysis under the action of the toxin and likely suggest novel ways of increasing cell resistance to amoebapores.