Rational Design of Antimicrobial Peptides and the Investigation of Membrane Disruption Mechanisms

Abstract

Multi-drug resistance (MDR) is a global threat because of extensive misuse and overuse of antibiotics. To fight against MDR infections, some membrane targeting antimicrobial peptides (AMPs) have been developed. Unlike traditional antibiotics, AMPs kill bacteria by physically disrupting cytoplasmic membranes. Because the membrane disruption mechanism is fast, bacteria are less likely to mutate or select a countering mechanism to become resistant to AMPs. Based on the previous studies, G(IIKK)3I-NH2 (G3) was chosen as a model AMP because of its potent antimicrobial activity and high selective index (SI). In this thesis, a series of surfactant-like AMPs were designed derived from G3. The peptide hydrophobicity was modified by substitution and deletion of key amino acid residues. It was demonstrated that AMPs with enhanced hydrophobicity had improved antimicrobial activity, but possessed drawbacks in terms of worse biocompatibility and selectivity. AMPs’ interactions with small unilamellar vesicle (SUV) models were studied by circular dichroism (CD), fluorescein dye leakage, Zeta potential and small-angle neutron scattering (SANS) experiments. AMPs’ interactions with planar lipid monolayer models were investigated by neutron reflectivity (NR), external reflection Fourier transform infrared (ER-FTIR) spectroscopy and Brewster angle microscopy (BAM). Together with molecular dynamics (MD) simulations using lipid bilayers and direct stochastic optical reconstruction microscopy (dSTORM) on real bacteria, all these techniques were combined to demonstrate the membrane disruption processes. All results point to the conclusion that a potent AMP must firstly transform its structure from random-coil monomers to α-helical nanoaggregates and then co-assemble with anionic bacterial membranes. Formation of AMP nanoaggregates is a crucial step before further generating cytoplasmic leakage channels inside the bacterial cell membranes. To gain insights into the self-assembling effect of AMPs during the antimicrobial process, a series of self-assembling antimicrobial lipopeptides (AMLPs) were developed based on the general formula of Cx-G(IIKK)2I-NH2 (acyl chain length x = 4-12, denoted as CxG2). With the increase of acyl chain length, the lipopeptides showed enhanced antimicrobial activity and self-assembling propensity. These lipopeptides can self-assemble into supramolecular nanofibres and form hydrogels. The self-assembly of C12G2 nanofibres involved quick disassembly into small α-helical aggregates upon association with negatively charged bacterial membranes, whilst remaining inactive against non-charged human cells. Thus, self-assembling AMPs provide a promising strategy to further improve their biocompatibility whilst achieve faster and more efficient bactericidal activity. The knowledge gained in this thesis will be helpful for the future development of clinically applied AMPs.

Date of Award	31 Dec 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jian Lu (Supervisor), Andrew Mcbain (Supervisor) & Thomas Waigh (Supervisor)