Design and Evaluation of Short Bovine Lactoferrin-Derived Antimicrobial Peptides against Multidrug-Resistant Enterococcus faecium

Abstract

Enterococcus faecium has become an important drug-resistant nosocomial pathogen because of widespread antibiotic abuse. We developed short and chemically simple antimicrobial peptides (AMPs) with a selective amino acid composition, fixed charge, and hydrophobicity ratio based on the core antimicrobial motif of bovine lactoferrin (LfcinB6). Among these peptides, 5L and 6L (both 12 residues long) demonstrated a narrow spectrum and high antibacterial activity against drug-resistant E. faecium isolates with a minimal inhibitory concentration (MIC) that ranged from 4–16 µg/mL. At 32 µg/mL, peptides 5L and 6L inhibited E. faecium strain C68 biofilm formation by 90% and disrupted established biofilms by 75%. At 40 µg/mL, 5L reduced 1 × 107E. faecium persister cells by 3 logs within 120 min of exposure, whereas 6L eliminated all persister cells within 60 min. At 0.5× MIC, 5L and 6L significantly downregulated the expression of a crucial biofilm gene ace by 8 folds (p = 0.02) and 4 folds (p = 0.01), respectively. At 32 µg/mL, peptides 5L and 6L both depolarized the E. faecium membrane, increased fluidity, and eventually ruptured the membrane. Physiologically, 5L (at 8 µg/mL) altered the tricarboxylic acid cycle, glutathione, and purine metabolism. Interestingly, in an ex vivo model of porcine skin infection, compared to no treatment, 5L (at 10× MIC) effectively eliminated all 1 × 106 exponential (p = 0.0045) and persister E. faecium cells (p = 0.0002). In conclusion, the study outlines a roadmap for developing narrow-spectrum selective AMPs and presents peptide 5L as a potential therapeutic candidate to be explored against E. faecium.

A frog‐derived antimicrobial peptide as a potential anti‐biofilm agent in combating Staphylococcus aureus skin infection

“…The antibacterial efficacy of OG9 and OG9c‐De‐NH 2 was further evaluated in an ex vivo porcine skin model, as reported. 27 The porcine skin was shaved and cleaned with 70% ethanol and dd H 2 O. The polyethylene tubing was glued to the cleaned skin surface with cyanoacrylate glue.…”

Molecular Dynamics Simulations Help Determine the Molecular Mechanisms of Lasioglossin-III and Its Variant Peptides’ Membrane Interfacial Interactions

“…In the second phase, frames corresponding to the important events during the 500 ns simulation runs, particularly the initiation of membrane–peptide interaction and the fully membrane-bound peptides (i.e., at the end of the 500 ns simulation), were converted into the corresponding AA models , (Figures S1–S3) and further simulated for at least 1 ns using the CHARMm-36 force field. All membrane simulations were done using 128 lipid molecules with an equal number of lipid moieties on the upper and lower membrane leaflets . Gram +ve bacterial membranes were modeled using DOPC (1,2-dioleoyl- sn -glycero-3-phosphocholine)/DOPG (dioleoylphosphatidylglycerol) in a 7:3 ratio, , while gram −ve membranes were modeled using POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine)/POPG (palmitoyl-oleoyl-phosphatidylglycerol) in a 3:1 ratio. − Mammalian membranes were modeled using POPC (1-palmitoyl-2-oleoylphosphatidylcholine) with 30% cholesterol .…”

“…Gram +ve bacterial membranes were modeled using DOPC (1,2-dioleoyl- sn -glycero-3-phosphocholine)/DOPG (dioleoylphosphatidylglycerol) in a 7:3 ratio, , while gram −ve membranes were modeled using POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine)/POPG (palmitoyl-oleoyl-phosphatidylglycerol) in a 3:1 ratio. − Mammalian membranes were modeled using POPC (1-palmitoyl-2-oleoylphosphatidylcholine) with 30% cholesterol . The simulation studies were initiated by using the alphafold-derived peptide structure at least 1.5 nm in parallel to the upper membrane leaflet solvated by 3 nm thick TIP3P water model above and below the membrane–peptide system . Energy minimization and equilibration of the membrane–peptide systems were performed at 310 K and a pressure of 1.0 atm as per the Charmm-GUI protocol.…”

“…4 However, with the advent of coarse−grained molecular dynamics (CG-MD) simulation strategies, such studies have become more feasible and use minimal computational resources. 5 The caveat in such cases is that CG-MD only provides low-resolution data, which we address by converting the CG models into all-atom (AA) models and continuing the simulation for a much shorter timescale 6 for the desired event under consideration. This is done using the Charmm-GUI interface, recently designed to address the challenges associated with the interconversion of CG and AA models.…”

“…All membrane simulations were done using 128 lipid molecules with an equal number of lipid moieties on the upper and lower membrane leaflets. 6 Gram +ve bacterial membranes were modeled using DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DOPG (dioleoylphosphatidylglycerol) in a 7:3 ratio, 13,14 while gram −ve membranes were modeled using POPE (1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/POPG (palmitoyl-oleoyl-phosphatidylglycerol) in a 3:1 ratio. 15−17 Mammalian membranes were modeled using POPC (1-palmitoyl-2oleoylphosphatidylcholine) with 30% cholesterol.…”