Browsing by Author "Holroyd, Dale"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemAtomic force microscopy : a novel tool for the analysis of the mechanism of action of antimicrobial peptides on target membranes(Stellenbosch : Stellenbosch University, 2003-03) Holroyd, Dale; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry .ENGLISH ABSTRACT: Nanoscale visualisation of live cells and cellular components under physiological conditions has long been a goal in microscopy. The objective of this study was to validate the use of Atomic Force Microscopy (AFM) as a new tool in unravelling the mysteries of antimicrobial peptide mechanism of action. Using the simplest AFM imaging technique, we were able to analyse the influence of haemolytic melittin and anti-bacterial magainin 2 on different target membranes at nanometer resolution, without using fixing agents. First, magainin 2 was synthesised and purified by gel permeation chromatography and high performance liquid chromatography (HPLC). The purity of magainin 2 and melittin, isolated from bee venom (Sigma-Aldrich), was verified with electro spray ionisation mass spectrometry (ESI-MS). Second, dose-response experiments were used to determine the optimum peptide/target cell ratio that would allow interaction with the membrane without causing lysis. Third, peptide/target-cell samples were placed on silica plates and visualised using contact mode AFM. Images obtained of the cells before and after peptide treatment, showed distinct changes in cell membrane surface topology. We observed grooves, lesions, membrane collapse and vesiculation depending on the concentration, type of peptide and target-cell used, allowing us to make conclusions regarding the mechanism of action of melittin and magainin 2. In comparison with model membrane studies, our AFM results show that a peptide can function by more than one mechanism of action depending on the structural composition of the membrane, which appears to have specific segregated lateral organisation. Magainin 2 (non-toxic) selectively targets cell membranes using different mechanisms of action. In this way it can lyse bacterial membranes (anti-bacterial agent) using one mechanism, while using another mechanism to interact with mammalian cells at physiological concentrations, without destroying them. In contrast, melittin (toxic) is non-selective, and uses the same mechanism of interaction with bacterial and mammalian cells. In conclusion, we propose a new holistic model for the mechanism of action of antimicrobial peptides.