Atomic force microscopy : a novel tool for the analysis of the mechanism of action of antimicrobial peptides on target membranes

Holroyd, Dale (2003-03)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

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.

AFRIKAANSE OPSOMMING: Nanoskaal visualiseering van lewende selle en sellulêre komponente onder fisiologiese toestande is al 'n geruime tyd 'n mikpunt in mikroskopie. Die doel van hierdie studie was om antimikrobiese peptiede se meganisme van werking op teikenselle op nanoskaalvlak met AFM te visualiseer. Sonder om fikseermiddels by te voeg, het ons die eenvoudigste AFM tegniek gebruik om die effek van hemolitiese melittien en anti-bakteriële magainin 2 op verskillende teikenselle, in nanometer resolusie, waar te neem. Eerstens is Magainin 2 gesintesiseer en gesuiwer met behulp van gelpermeasie chromatografie en hoë doeltreffenheid vloeistof chromatografie (HPLC). Die suiwerheid van magainin 2 en kommersiële bye gif melittien, is bevestig met behulp van elektrosproei-ionisasie massaspektrometrie (ESI-MS). Tweedens, is dosis-respons eksperimente gebruik om die optimale peptied/teikensel verhouding te bepaal voordat membraanliese plaasvind. Derdens, is peptied/teikensel monsters op silika plate gevisualiseer met gebruik van kontak AFM. Die beelde van die selle, voor en na peptied behandeling, het duidelike veranderinge in seltopologie getoon. Ons het groewe, letsels, membraaninstorting en vesikulasie, afhangende van die konsentrasie peptied en teikensel gebruik, waargeneem. Dit het ons toegelaat om tot gevolgtrekkings te kom aangaande die meganisme van werking van melittien en magainin 2. In ooreenstemming met model membraan studies, het ons AFM resultate gewys dat 'n peptied veelvoudige meganismes van werking kan hê, afhangend van die strukturele samestelling van die membraan, wat klaarblyklik laterale segregasie toon. Magainin 2 (nie-giftig) is selektief ten opsigte van teikenselle omdat dit gebruik maak van verskillende meganismes van werking op bakteriële en soogdier selle. In teenstelling is melittien (giftig) nie-selektief, en gebruik dieselfde meganisme van werking op bakteriële en soogdierselle. Ten slotte, stel ons 'n nuwe model vir die meganisme van werking voor.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53306
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