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Hybridizing two known antimicrobial peptides (AMPs) is a simple and effective strategy for designing antimicrobial agents with enhanced cell selectivity against bacterial cells. Here, we generated a hybrid peptide Lf-KR in which LfcinB6 and KR-12-a4 were linked with a Pro hinge to obtain a novel AMP with potent antimicrobial, anti-inflammatory, and anti-biofilm activities. Lf-KR exerted superior cell selectivity for bacterial cells over sheep red blood cells. Lf-KR showed broad-spectrum antimicrobial activities (MIC: 4–8 μM) against tested 12 bacterial strains and retained its antimicrobial activity in the presence of salts at physiological concentrations. Membrane depolarization and dye leakage assays showed that the enhanced antimicrobial activity of Lf-KR was due to increased permeabilization and depolarization of microbial membranes. Lf-KR significantly inhibited the expression and production of pro-inflammatory cytokines (nitric oxide and tumor necrosis factor‐α) in LPS-stimulated mouse macrophage RAW264.7 cells. In addition, Lf-KR showed a powerful eradication effect on preformed multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms. We confirmed using confocal laser scanning microscopy that a large portion of the preformed MDRPA biofilm structure was perturbed by the addition of Lf-KR. Collectively, our results suggest that Lf-KR can be an antimicrobial, anti-inflammatory, and anti-biofilm candidate as a pharmaceutical agent.

Here we describe the three-dimensional structure and antimicrobial mechanism of mBjAMP1, an antimicrobial peptide (AMP) isolated from Branchiostoma japonicum. The structure of mBjAMP1 was determined by 2D solution NMR spectroscopy and revealed a novel α-hairpinin-like scaffold stabilized by an intramolecular disulfide bond. mBjAMP1 showed effective growth inhibition and bactericidal activities against pathogenic bacteria but was not cytotoxic to mammalian cells. Antimicrobial mechanism studies using fluorescence-based experiments demonstrated that mBjAMP1 did not disrupt membrane integrity. Laser-scanning confocal microscopy indicated that mBjAMP1 is able to penetrate the bacterial cell membrane without causing membrane disruption. Moreover, gel retardation assay suggested that mBjAMP1 directly binds to bacterial DNA as an intracellular target. Collectively, mBjAMP1 may inhibit biological functions by binding to DNA or RNA after penetrating the bacterial cell membrane, thereby causing cell death. These results suggest that mBjAMP1 may present a promising template for the development of peptide-based antibiotics.

Bacillus velezensis GH1‐13 was recently isolated and showed to promote plant growth and strong antagonistic effects against phytopathogenic fungi including Gibberella moniliformis, Fusarium fujikuroi, Bipolaris oryzae, and Colletotrichum gloeosporioides. In this study, the antifungal substances produced by the GH1‐13 strain were purified by ethyl acetate solvent partitioning and solid phase extraction using Sep‐Pak C18 cartridge. The pure antifungal substances were finally purified by preparative reversed phase‐high performance liquid chromatography. We investigated the structure of the purified antifungal compounds using liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. These compounds were identified as surfactin‐type cyclic lipopeptides having an ELLVDLL amino acid sequence with different lengths of lipid chains. The surfactin lipopeptides effectively inhibited the growth of fungal mycelium, suggesting that the GH1‐13 strain will be a potential biocontrol agent against plant pathogenic fungi.

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