Thesis (MSc)--Stellenbosch University, 2003. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53306 |
| Date | 03 1900 |
| Creators | Holroyd, Dale |
| Contributors | Rautenbach, M., Stellenbosch University. Faculty of Science. Dept. of Biochemistry . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | [116] p. : ill. |
| Rights | Stellenbosch University |
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