Membrane Disruption Mechanisms of Antimicrobial Peptides and Their Combinations with Antibiotics: An Insight from Experimental Studies and MD Simulations

Abstract

Antimicrobial peptides (AMPs) offer great potential in the fight against multi-drug resistant (MDR) microbes via the mode of membrane targeting. However, the potency and selectivity of AMPs must be substantially improved. By combining experiments and molecular dynamics (MD) simulations, this thesis work focuses on exploring how representative cationic AMPs interact with the bacterial outer and inner membranes. Antimicrobial efficacy, dynamic killing, neutron reflection (NR) and small angle neutron scattering (SANS) together with MD simulations have been used to investigate the membrane-lytic actions of four AMPs, G(IIKK)3I-NH2 (G3), G(IIKK)4I-NH2 (G4), previously-studied GLLDLLKLLLKAAG-NH2 (LDKA, biomimetic) and GIGAVLKVLTTGLPALISWIKRKR-NH2 (Melittin, natural). Distinctly different intramembrane nanoaggregates were formed when the four AMPs were bound to the inner and outer membranes. G3 and G4 formed smaller but further inserted intramembrane nanoaggregates into bacterial membranes which were well correlated to their greater antimicrobial efficacy and faster dynamic killing. Substitutions of hydrophobic or cationic amino acids in G3 (also denoted as GIK) led to AMPs of G(WWKK)3W-NH2 (GWK), G(FFKK)3F-NH2 (GFK), and G(IIRR)3I-NH2 (GIR), with varying antimicrobial activities. Electronic microscopy imaging and fluorescence assays revealed structural disruptions by AMP binding to bacterial cell walls, anionic lipoteichoic acids (LTA) and cytoplasmic membrane. GWK and GIR can rigidify the cytoplasmic membrane and decrease the diffusive efficiency of the anionic lipid membrane more significantly than GIK and GFK, associated with intramembrane peptide nanoaggregates. Synergistic combination of an antibiotic and an aiding agent provides an important but largely unexploited option to ‘repurpose’ existing biomaterial’s space while addressing issues of potency, spectrum, toxicity and drug-resistance. Antibiotic tetracycline/minocycline (TC/MC) combined with the broad-spectrum antimicrobial lipopeptides (C8GIK and C8GIR) has been shown to improve the efficiency of membrane targeting and intramembrane accumulation. Binary antibiotic-lipopeptide combinations displayed synergistic effects against both Gram-positive and Gram-negative bacterial strains including 3 MDR strains, featured by fast time-killing and high TC/MC uptake. Finally, GIIKDIIKDIIKDI and GIIKKIIDDIIKKI (denoted as 3D and 2D, respectively), designed by selective substitutions of cationic residues of Lys (K) in the extensively studied peptide G(IIKK)3I with anionic residue Asp (D), interacted differently with the inner and outer membranes of Gram-negative bacteria in a pH-responsive manner. Different antimicrobial efficacies of 2D and 3D were underlined by the interplay between their ability to bind to the outer membrane lipid LPS (lipopolysaccharide), outer membrane permeability change and inner membrane depolarization and leakage. This thesis study has thus provided useful data to present AMPs as a promising alternative to combat antimicrobial resistance. Rational structural design together with intramembrane structural analysis offers an effective route for optimizing AMP performance.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jian Lu (Supervisor) & Thomas Waigh (Supervisor)