Elucidating the interactions between antimicrobial peptides and periplasmic osmolytes in Escherichia coli

Thesis

Antimicrobial resistance poses a major threat to global health, and Gram-negative bacteria remain particularly difficult to combat due to their complex cell envelopes. Antimicrobial peptides (AMPs) are promising therapeutic candidates, but their precise mechanisms of action remain debated, and only a few have been successfully translated into effective drugs. A critical but underexplored factor is how interactions with the crowded, chemically heterogeneous periplasm influence AMP behaviour during their envelope translocation. In particular, the role of periplasmic osmolytes in modulating peptide aggregation, membrane interactions, and pore formation has received little attention.

This thesis investigates how osmolytes affect AMP behaviour in both solution and membrane environments. Three representative α-helical amphipathic cationic peptides were selected to capture differences in charge density. Atomistic molecular dynamics simulations were used to investigate peptide aggregation in crowded solutions representing the E. coli periplasm. Complementary coarse-grained simulations of magainin 2 with realistic E. coli inner membranes under an induced transmembrane potential were performed to study peptide-mediated pore formation. Both membrane protein-independent and -dependent translocation mechanisms were observed.

In solution, all AMPs aggregated into micelle-like structures, with sequence, initial conformation, and solute phase composition strongly influencing aggregation. Osmolytes stabilised aggregates through residue-specific electrostatic shielding of repulsive same-charge interactions and through more promiscuous polar interactions, providing the first mechanistic insight into how periplasmic solutes shape AMP organisation before membrane encounter.

In membranes, four pore-formation pathways were identified, with two predominating: single-peptide “snorkelling” along membrane proteins and AMP aggregate-induced bulk membrane rupture. Osmolytes did not alter the overall pore formation frequency but shifted the mechanism distributions, favouring aggregate-driven rupture over snorkelling, by promoting larger aggregates.Overall, this work demonstrates that realistic periplasmic environments fundamentally shape AMP behaviour in ways not captured by simplified models, offering mechanistic insights to guide the rational design of AMPs that retain their activity within the complex cell envelope of Gram-negative bacteria.

Authors: Schroeder, C

• DOI: 10.5287/ora-0nrparyn7
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: coarse-grained; computational electrophysiology; osmolytes; CompEL; molecular dynamics; AMP; MD simulations; periplasmic osmolytes; cupiennin; gram-negative; antimicrobial peptides; AMPs; latarcin; atomistic; cell envelope; gram-negative cell envelope; magainin; periplasm
• Subjects: Computational Microbiology