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The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αβ were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and β were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both Land D-PLNC8 αβ caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. the D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, β1-22, β7-34 and β1-20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αβ substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αβ is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use. Although antibiotics are the most effective treatment against bacteria of the genus Staphylococcus (including the species S. aureus and S. epidermidis), these opportunistic pathogens are one of the leading causes of severe bacterial infections in humans connected to chronic wounds and medical devices, e.g. catheters and prosthetic implants 1. These persistent infections are generally difficult to treat, which increases the risk for bacterial dissemination and development of systemic complications 2,3. Furthermore, considering the gradual increase in antimicrobial resistance, treatment may be even more difficult to achieve as the available options become limited 4. Consequently, there is an urgent need to find new approaches in human medicine against bacterial infections, and bacteriocins represent a promising avenue that requires more consideration 5,6. Bacteriocins are antimicrobial peptides that are produced by most microorganisms that contribute their defence mechanisms. These peptides are divided into class I-V based on their structural characteristics. Class I includes small peptides (<5 kDa) with unusual amino acids, such as lanthionine and β-methyllanthionine that are post-translationally introduced and class II peptides are synthesized in precursor forms and processed (<10 kDa), and includes bacteriocins composed of two peptides (class IIb), such as PLNC8 αβ. Class III bacteriocins are large (>10 kDa) and sensitive to heat, class IV are small (<10 kDa) and circular peptides. Class V are small (<5 kDa), circular or linear peptides that are characterized by containing cross-linkages between cysteine residu...

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Solid-state nanopores provide a highly versatile platform for rapid electrical detection and analysis of single molecules. Lipid bilayer coating of the nanopores can reduce nonspecific analyte adsorption to the nanopore sidewalls and increase the sensing selectivity by providing possibilities for tethering specific ligands in a cell-membrane mimicking environment. However, the mechanism and kinetics of lipid bilayer formation from vesicles remain unclear in the presence of nanopores. In this work, we used a silicon-based, truncated pyramidal nanopore array as the support for lipid bilayer formation. Lipid bilayer formation in the nanopores was monitored in real time by the change in ionic current through the nanopores. Statistical analysis revealed that a lipid bilayer is formed from the instantaneous rupture of individual vesicle upon adsorption in the nanopores, differing from the generally agreed mechanism that lipid bilayer forms at a high vesicle surface coverage on a planar support. The dependence of the lipid bilayer formation process on the applied bias, vesicle size, and concentration was systematically studied. In addition, the nonfouling properties of the lipid bilayer coated nanopores were demonstrated during long single-stranded DNA translocation through the nanopore array. The findings indicate that the lipid bilayer formation process can be modulated by introducing nanocavities intentionally on the planar surface to create active sites or changing the vesicle size and concentration.

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