Interactions of lipid membranes with chitosan and epsilon-toxin: a biophysical approach
Lead Research Organisation:
University of Exeter
Department Name: Engineering Computer Science and Maths
Abstract
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.
Organisations
People |
ORCID iD |
| Peter Petrov (Primary Supervisor) | |
| Skye Marshall (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509656/1 | 01/10/2016 | 30/09/2021 | |||
| 1783559 | Studentship | EP/N509656/1 | 01/10/2016 | 26/11/2020 | Skye Marshall |
| EP/R513210/1 | 01/10/2018 | 30/09/2023 | |||
| 1783559 | Studentship | EP/R513210/1 | 01/10/2016 | 26/11/2020 | Skye Marshall |
| Description | Using lipid monolayers and bilayers: Different properties of lipids (e.g. charge, saturation, shape), and different combinations of lipids and cholesterol have varying effect on their affinity to membrane-active, biological molecules (see below). Red cells have varying susceptibilities to the epsilon protein (see below). Amoebapore protein: Producing this protein in the lab was attempted using a standard technique (transfecting E.coli with an amoebapore encoding plasmid) and a more complicated technique (cell-free protein synthesis) however both these techniques did not yield results. If the amoebapore protein is to be produced it would be more appropriate to work with a lab that has already established production of this protein or order in a chemically synthesized version. It is possible that we can work with ISCA biochemicals in Exeter who can produce this protein and we can investigate its effects in our systems. Chitosan: This polysaccharide was investigated due to is link to the amoebapore protein: the link being that they are both produced by the Entamoeba histolytica pathogen which causes disease in humans. Chitosan was found to show preference for lipids that represent red blood cell inner membrane leaflets however it did change the visco-elastic properties of lipids representing both inner and outer-leaflets of the red cell membrane. When the attempt to produce amoebapore protein was ceased, C. perfringens epsilon protein was tested, using biophysical and biological techniques, instead. Different mutations of this protein reacted differently to the systems I tested it with. I used lipid monolayers and bilayers as well as red blood cells. With the red blood cells I used both room temperature and 37oC settings. New research questions arising from this research include: how do the epsilon monomers find each other on the cell membrane in order to form oligomers?, why can a certain mutant pores form in a lipid bilayer at room temperature but not in a red blood cell?, why is there a temperature dependence of activity?, which amino acids are involved in binding to the membrane? The epsilon protein research has been done in collaboration with Monika-Bokori Brown and Catalin Chimeral at the University of Exeter. |
| Exploitation Route | If ISCA biochemicals can create the amoebapore protein we could have an ongoing collaboration with them to investigate this protein. The epsilon protein experiments have opened up several new questions and will continue to be investigated at the University of Exeter |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Security and Diplomacy |