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

溶组织内阿米巴是一种原生动物寄生虫，估计每年造成10万人死亡，是发展中国家的一个主要健康问题。这种寄生虫具有很强的细胞溶解活性，这与这种生物产生的一类成孔毒素变形虫有关。变形虫（以及更普遍的，成孔毒素）能够穿透目标细胞的质膜，从而杀死它们。虽然变形虫的细胞毒性已经被研究过，但对与毒性相关的生物物理过程和脂膜物理性质的作用了解甚少。在过去的几年中，我们开发了新的实验方法来研究毒素活性背后的生物物理学，重点是产气荚膜梭菌a-毒素和成孔毒素NetB和溶血素。本项目将利用体外模型系统研究决定变形虫细胞溶解活性的生物物理因素。首先，我们将使用模型膜系统（Langmuir单层脂质和双层脂质囊泡），以及毒素对膜中脂质组织的影响，寻求确定负责将毒素招募到脂质膜及其激活的主要脂质物种。然后，我们将研究质膜的生化和生物物理特性对人红细胞对变形虫敏感性的影响。要研究的性质将是膜弹性，电学性质和形态，这可能是毒素活性的决定因素。我们还将使用氧化应激、脂质混乱和细胞老化来改变膜的生化状态，以确定细胞表面生物化学在改变细胞对毒素的反应中的作用。不同毒力水平的内阿米巴原虫的变形虫之间存在一些微小的氨基酸变化，这些差异归因于这些差异，但缺乏解释这些毒力差异的机制。我们将使用上述技术研究来自不同内阿米巴物种的这些变形虫，因为我们预计这些差异会影响膜溶解度。这些研究将确定在毒素作用下导致细胞裂解的因素，并可能提出增加细胞对变形虫抗性的新方法。

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.