How do antimicrobial peptides disrupt the lipopolysaccharide membrane leaflet of Gram-negative bacteria?

Haoning Gong; Xuzhi Hu; Lin Zhang; Ke Fa; Mingrui Liao; Huayang Liu; Giovanna Fragneto; Mario Campana; Jian Ren Lu

doi:10.1016/j.jcis.2023.01.051

How do antimicrobial peptides disrupt the lipopolysaccharide membrane leaflet of Gram-negative bacteria?

J Colloid Interface Sci. 2023 May:637:182-192. doi: 10.1016/j.jcis.2023.01.051. Epub 2023 Jan 13.

Authors

Haoning Gong¹, Xuzhi Hu², Lin Zhang², Ke Fa², Mingrui Liao², Huayang Liu², Giovanna Fragneto³, Mario Campana⁴, Jian Ren Lu⁵

Affiliations

¹ Biological Physics Laboratory, Department of Physics and Astronomy, Faculty of Science and Engineering, The University of Manchester, Manchester M13 9PL, UK; State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
² Biological Physics Laboratory, Department of Physics and Astronomy, Faculty of Science and Engineering, The University of Manchester, Manchester M13 9PL, UK.
³ Institut Laue-Langevin, Grenoble 38042, France.
⁴ ISIS Pulsed Neutron & Muon Source, STFC Rutherford Appleton Laboratory, Didcot OX11 0QX, UK.
⁵ Biological Physics Laboratory, Department of Physics and Astronomy, Faculty of Science and Engineering, The University of Manchester, Manchester M13 9PL, UK. Electronic address: j.lu@manchester.ac.uk.

PMID: 36701864
DOI: 10.1016/j.jcis.2023.01.051

Abstract

Hypothesis: It is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains unclear how AMPs disrupt the outer membrane (OM) of Gram-negative bacteria (GNB) and then compromise the inner membrane. We hypothesise that different AMPs impose different structural disruptions, with direct implications to their antimicrobial efficacies.

Experiments: The antimicrobial activities of three typical AMPs, including the designed short AMP, G₃, and two natural AMPs, melittin and LL37, against E. coli and their haemolytic activities were studied. Lipopolysaccharide (LPS) and anionic di-palmitoyl phosphatidyl glycerol (DPPG) monolayer models were constructed to mimic the outer membrane and inner membrane leaflets of Gram-negative bacteria. The binding and penetration of AMPs to the model lipid monolayers were systematically studied by neutron reflection via multiple H/D contrast variations.

Finding: G₃ has relatively high antimicrobial activity, low cytotoxicity, and high proteolytic stability, whilst melittin has significant haemolysis and LL37 has weaker antimicrobial activity. G₃ could rapidly lyse LPS and DPPG monolayers within 10-20 min. In contrast, melittin was highly active against the LPS membrane, but the dynamic process lasted up to 80 min, with excessive stacking in the OM. LL37 caused rather weak destruction to LPS and DPPG monolayers, leading to massive adsorption on the membrane surface without penetrating the lipid tail region. These findings demonstrate that the rationally designed AMP G₃ was well optimised to impose most effective destruction to bacterial membranes, consistent with its highest bactericidal activity. These different interfacial structural features associated with AMP binding shed light on the future development of active and biocompatible AMPs for infection and wound treatments.

Keywords: Anti-infection; Antimicrobial peptide; Lipopolysaccharide; Neutron reflection; Structural disruption.

MeSH terms

Anti-Bacterial Agents / chemistry
Anti-Infective Agents* / chemistry
Antimicrobial Peptides
Bacteria / metabolism
Cell Membrane / metabolism
Escherichia coli / metabolism
Gram-Negative Bacteria / metabolism
Lipopolysaccharides* / chemistry
Lipopolysaccharides* / pharmacology
Melitten / metabolism
Melitten / pharmacology

Substances

Lipopolysaccharides
Antimicrobial Peptides
Melitten
Anti-Infective Agents
Anti-Bacterial Agents