No abstract

Many antimicrobial peptides (AMPs) target bacterial membranes and they kill bacteria by causing structural disruptions. One of the fundamental issues however lies in the selective responses of AMPs to different cell membranes as a lack of selectivity can elicit toxic side effects to mammalian host cells. A key difference between the outer surfaces of bacterial and mammalian cells is the charge characteristics. We report a careful study of the binding of one of the representative AMPs, with the general sequence G(IIKK)I-NH (G), to the spread lipid monolayers of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) mimicking the charge difference between them, using the combined measurements from Langmuir trough, Brewster angle microscopy (BAM) and neutron reflection (NR). The difference in pressure rise upon peptide addition into the subphase clearly demonstrated the different interactions arising from different lipid charge features. Morphological changes from the BAM imaging confirmed the association of the peptide into the lipid monolayers, but there was little difference between them. However, NR studies revealed that the peptide bound 4 times more onto the DPPG monolayer than onto the DPPC monolayer. Importantly, whilst the peptide could only be associated with the head groups of DPPC it was well penetrated into the entire DPPG monolayer, showing that the electrostatic interaction strengthened the hydrophobic interaction and that the combined molecular interactive processes increased the power of G in disrupting the charged membranes. The results are discussed in the context of general antibacterial actions as observed from other AMPs and membrane lytic actions.

Molecular dynamics (MD) simulations, stochastic optical reconstruction microscopy (STORM) and neutron reflection (NR) are combined to explore how antimicrobial peptides (AMPs) can be designed to promote the formation of nanoaggregates into bacterial membranes and impose effective bactericidal actions. Changes in the hydrophobicity of the designed AMPs were found to have strong influence on their bactericidal potency and cytotoxicity. G(IIKK) 3 I-NH 2 (G 3 ) achieved low minimum inhibition concentrations (MICs) and effective dynamic kills against both antibiotic resistant and susceptible bacteria. However, a G 3 derivative with weaker hydrophobicity, KI(KKII) 2 I-NH 2 (KI), exhibited considerably lower membrane-lytic activity. In contrast, the more hydrophobic G(ILKK) 3 L-NH 2 (GL) peptide achieved MICs similar to those observed for G 3 , but with worsened haemolysis. Both the model membranes studied by Brewster angle microscopy, Zeta-potential measurements and NR, and the real bacterial membranes examined with direct STORM, contained membrane inserted peptide aggregates upon AMP exposure. These structural features were well supported by MD simulations. By revealing how AMPs self-assemble in microbial membranes, this work provides important insights into their mechanistic actions and allows further fine-tuning of antimicrobial potency and cytotoxicity.

Different bacterial types and their living environments can lead to different saturations in the chains of their membrane lipids. Such structural differences may influence the efficacy of antibiotics that target bacterial membranes. In this work, the effects of acyl chain saturation on the binding of an antimicrobial peptide G4 have been examined as a function of the packing density of lipid monolayers by combining external reflection Fourier transform infrared (ER-FTIR) spectroscopy and neutron reflection (NR) measurements. Langmuir monolayers were formed from 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), respectively, with the initial surface pressures controlled at 8 and 28 mN/m. A reduction in the order of the acyl chains associated with the increase in the layer thickness upon G4 binding was revealed from ER-FTIR spectroscopy, with peptide binding reaching equilibration faster in POPG than in DPPG monolayers. Whereas the dynamic DPPG-binding process displayed a steady increase in the amide I band area, the POPG-binding process showed little change in the amide area after the initial period. The peptide amide I area from ER-FTIR spectroscopy could be linearly correlated with the adsorbed G4 amount from NR, irrespective of time, initial pressure, or chain saturation, with clearly more peptide incorporated into the DPPG monolayer. Furthermore, NR revealed that although the peptide was associated with both POPG and DPPG lipid monolayers, it was more extensively distributed in the latter, showing that acyl chain saturation clearly promoted peptide binding and structural disruption.

Gram-negative bacteria are covered by both an inner cytoplasmic membrane (IM) and an outer membrane (OM). Antimicrobial peptides (AMPs) must first permeate through the OM and cell wall before attacking the IM to cause cytoplasmic leakage and kill the bacteria. The bacterial OM is an asymmetric bilayer with the outer leaflet primarily composed of lipopolysaccharides (LPSs) and the inner leaflet composed of phospholipids (PLs). Two cationic α-helical AMPs were designed to target Gram-negative bacteria, a full peptide G­(IIKK)3I-NH2 (G3), and a hydrophobic lipopeptide C8-G­(IIKK)2I-NH2 (C8G2, with C8 denoting the octanoyl chain). LPS dominates OM functions as the first line of defense against antibiotics, thereby reducing drug susceptibility. This work explores how the two AMPs interact with LPS through several carefully chosen OM models that facilitated measurements from solid-state nuclear magnetic resonance (ss-NMR), small-angle neutron scattering (SANS), and neutron reflectivity (NR). The results revealed that G3 molecules bound preferably to the LPS head region and functioned as bridge molecules to reassemble the dislocated lipids into bilayer stacks. In contrast, C8G2 lipopeptides could quickly penetrate into the central region of the OM to cause direct removal of some membrane lipids. Different structural disruptions implicated different antimicrobial efficacies from these AMPs. The demonstration of the structural features underlying different susceptibilities of the OM to AMPs offers a useful route for the future development of strain-specific AMPs against antimicrobial-resistant pathogens.