Antimicrobial Mechanism of Antimicrobial Peptide from Paenibacillus ehimensis against Penicillium expansum Spores

Abstract

Penicillium expansum, a common spoilage organism in postharvest fruits, can cause fruit decay and deterioration and endanger human health. It is of great significance to investigate the antimicrobial mechanism of the antimicrobial peptide from Paenibacillus ehimensis on P. expansum spores. The antimicrobial activity of the antimicrobial peptide against P. expansum spores was determined by using the two-fold dilution method as well as measuring the time-killing curve. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to evaluate the effect of the antimicrobial peptide on the ultrastructure of P. expansum spores. The effects of the antimicrobial peptide on the cell membrane and reactive oxygen species (ROS) accumulation of P. expansum were analyzed by fluorescence probes. The results showed that the minimum inhibitory concentration (MIC) of the antimicrobial peptide against P. expansum spores was 3.5 AU/mL. The spore germination rate was significantly decreased by 28.30%, 84.57% and 100% by the antimicrobial peptide at concentrations of 0.5 MIC, 1 MIC and 2 MIC compared with the blank control (P < 0.05). After treatment with the antimicrobial peptide, the spores appeared seriously sunken, the intracellular contents were leaked out, and the morphology and structure were changed. The antimicrobial peptide damaged the cell wall of P. expansum, resulting in the leakage of alkaline phosphatase. The antimicrobial peptide depolarized the cell membrane potential in a dose-dependent manner, and increased the cell membrane permeability, leading to K+ leakage. The fluidity of the cell membrane was increased, which in turn resulted in a significant decrease in DPH fluorescence intensity (P < 0.05). The integrity of the cell membrane was damaged by the antimicrobial peptide, so the fluorescence intensity of SYTOX-Green and the contamination rate of PI were increased. Moreover, the antimicrobial peptide at 1 MIC and 2 MIC increased the fluorescence intensity of DCFH-DA significantly (P < 0.05) and resulted in ROS accumulation, which affected the physiology and metabolism of P. expansum spores. This study indicated that the target sites of the antimicrobial peptide against P. expansum spores were mainly the cell membrane and ROS metabolism.

Keywords

• paenibacillus ehimensis; antimicrobial peptide; penicillium expansum; spore; antimicrobial mechanism