| Staphylococcus aureus(S.aureus)is a common foodborne pathogen that poses a serious threat to human health and has become a global public health concern.In recent years,small peptide molecules have been discovered to possess antimicrobial activity and have become a hot topic of research.In the previous stage of research,a novel antimicrobial peptide,MOp2,was identified from the hydrolysate of Moringa oleifera seeds protein,which showed good inhibitory effects against S.aureus.However,the specific mechanism of its antimicrobial action remains unclear.This study focuses on S.aureus as the target strain and investigates the potential molecular mechanisms of MOp2 against S.aureus at 3 levels: transcriptome,proteome,and biofilm.Multiple techniques were employed,including RT-q PCR,enzyme activity assays,flow cytometry,fluorescence microscopy,and molecular docking.The main research findings are as follows:(1)After treatment with MOp2,a total of 579 genes were up-regulated and 564 genes were down-regulated in S.aureus.These differentially expressed genes(DEGs)were closely related to cell membrane permeability,intracellular p H homeostasis,energy metabolism,membrane transport,cell wall synthesis,and protein folding processes.Further confirmation was carried out through activity assays of lactate dehydrogenase,succinate dehydrogenase,and ATPase,and 6 DEGs were validated using RT-q PCR.Ultimately,it was discovered that MOp2 can inhibit S.aureus by altering cell membrane permeability,acidifying the intracellular environment,inhibiting glycolysis,TCA cycle,and respiratory chain processes,as well as hindering cell wall synthesis.(2)Following treatment with MOp2,256 proteins were up-regulated and 285 proteins were down-regulated in S.aureus.These DEPs were mainly related to membrane damage,pantothenate and coenzyme A(Co A)biosynthesis,cell wall synthesis,energy metabolism,protein synthesis,and oxidative stress.Molecular docking results indicated that MOp2 can interact with 8 DEPs,including Pro C,Qox B,SOD2,Dna K,Gro EL,Rpl Y,Acp S,and Fab G.Further research found that MOp2 can inhibit lipid-II synthesis and peptidoglycan biosynthesis,suppress endogenous antioxidant activity,inhibit Co A biosynthesis,inhibit molecular chaperone proteins and ribosomal subunits to hinder protein synthesis,thereby damaging the cell wall,leading to the accumulation of reactive oxygen species within the cell,and causing oxidative damage,DNA fragmentation,changes in nuclear morphology,and phosphatidylserine externalization.Additionally,MOp2 also hindered carbohydrate metabolism and phosphorylation of sugar generation,arginine synthesis,and glutamate transport,affecting the energy and nutritional supply of S.aureus,inhibiting cell protein synthesis,and reducing the development of MOp2 resistance in S.aureus.(3)The minimum biofilm inhibitory concentration(MBIC)and minimum biofilm eradication concentration(MBEC)of MOp2 were found to be 4 mg/m L and 8 mg/m L,respectively.At a concentration of 1×MBIC,MOp2 showed a biofilm clearance rate of63.28% against S.aureus.Fluorescence microscopy observations revealed a decrease in biofilm adhesion and bacterial count after treatment with MOp2,and the initial bacterial adhesion rate was reduced by 44.19% at a concentration of 1×MBIC.Additionally,the surface hydrophobicity of the biofilm decreased after treatment with MOp2.Enzymatic assays showed that extracellular proteins were the most abundant component(49.01%)of the extracellular polymeric substances(EPS)of S.aureus biofilm,followed by extracellular polysaccharides and extracellular DNA.MOp2 was found to inhibit the synthesis of these components in EPS.Molecular docking results indicated that MOp2 can bind to key proteins Agr A,Csh A,Lux S,and Sar A involved in biofilm formation,which may affect the quorum sensing system of S.aureus,thereby inhibiting biofilm formation and reducing the infectivity and virulence of S.aureus. |
