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Title: The development of computer-designed and naturally occurring antimicrobial peptides to target methicillin-resistant Staphylococcus aureus (MRSA) using SPOT peptide synthesis
Author: Ashby, Martin
ISNI:       0000 0004 7655 1423
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2018
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The aim of this thesis is the identification and development of computer-designed and naturally occurring antimicrobial peptides (AMPs) which demonstrate potential as clinically viable compounds in the treatment of MRSA infections. To achieve this aim the method of SPOT peptide synthesis was employed, this allows for hundreds of unique peptide sequences to be synthesised rapidly and relatively cheaply. Using this technique, peptide libraries were constructed based upon two general strategies: 1. Synthesis of novel 9-mer peptide sequences predicted in silico to possess a high therapeutic index. 2. Synthesis of short naturally occurring AMPs sequences ranging in length from 5-17 residues along with truncated forms of longer peptides. In addition, two potent novel peptides were optimised by designing additional libraries containing hundreds of derivatives based upon their sequences. Each library was screened for both antimicrobial and hemolytic activity and several peptides were identified that demonstrate potent in vitro antistaphylococcal activity (MIC 0.5-4 pg/ml) and minimal hemolytic activity (HCso 111- >250 pg/ml). To assess the activity of the peptides in vivo the model organism Galleria mellonella was used. Neither artificial or natural peptides demonstrated substantial toxicity towards this organism, however no peptide could rescue 6. mellonella from a systemic MRSA infection. When applied topically to G. mellonella, several of the peptides could significantly reduce the bacterial burden on the skin. Mode of action studies revealed that all peptides cause rapid depolarisation of the bacterial membrane and differing degrees of membrane permeabilisation. Using small angle X-ray scattering morphological changes that take place in bacteria exposed to different concentrations of peptides for different lengths of time could be tracked. Visualisation of bacteria treated with one novel peptide revealed changes to the intracellular environment. In conclusion, the usefulness of SPOT synthesis in identifying several peptides with potent antimicrobial activity towards MRSA and minimal toxicity towards mammalian cells in vitro was demonstrated. Analysis ofthe screening data revealed peptide features which could be correlated with their level of activity, thus improving our ability to predict active peptides in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available