Identification and expression of a novel antimicrobial peptide Gloverin and its antibacterial effect against Staphylococcus aureus.

Abstract

Background: Antimicrobial peptides (AMPs) have attracted extensive attention in various fields. Gloverin is a group of AMPs derived from Lepidoptera insects. In this study, a novel gloverin CpGlv was identified through sequence alignment. The open reading frame (ORF) of mature CpGlv, devoid of its signal peptide region, was recombinantly expressed in Escherichia coli. The antibacterial activity of the fusion protein CpGlv against Staphylococcus aureus (S. aureus) was then assessed by determining the minimum inhibitory concentration (MIC) and using scanning electron microscopy (SEM) to observe the ultrastructure of S. aureus cells. Furthermore, the effects of CpGlv on the cell wall and membrane permeability of S. aureus were evaluated by detecting the leakage of intracellular nucleic acids, proteins, alkaline phosphatase (ALP), and β-galactosidase, as well as through the propidium iodide (PI) staining experiment. Finally, the influence of CpGlv on the S. aureus biofilm formation was investigated using the crystal violet staining assay.

Results: The results showed that the ORF of CpGlv contained a nucleotide sequence of 591 bp. The expressed fusion protein CpGlv had a molecular weight of approximately 24 kDa. The purified CpGlv exhibited significant antibacterial activity against S. aureus, with a MIC of 40 µg/mL. 2×MIC of CpGlv caused S. aureus cells to exhibit adhesion, swelling, deformation, and even rupture. Further, CpGlv resulted in the leakage of intracellular nucleic acids, proteins, ALP, and β-galactosidase in S. aureus. After PI staining, there was intracellular fluorescence intensity in S. aureus treated by CpGlv. Additionally, 1/4×MIC of CpGlv significantly reduced the biofilm formation of S. aureus.

Conclusion: A novel gloverin CpGlv was identified, expressed, and purified, and the recombinant CpGlv exhibited antimicrobial activity against S. aureus by increasing the cell membrane and wall permeability, and inhibiting the biofilm formation, laying a foundation for in-depth research on the antibacterial mechanism and application of CpGlv.