| Vibrio alginolyticus is a class of marine foodborne pathogenic bacterium that can cause disease even to death in aquatic animals such as fish,shrimp,and crab.And some strains are the main reason for causing spoilage deterioration of food and triggering foodborne diseases in humans,which cause serious hazards and loss to human health and economic.At present,most of antibiotics,bacteriocins are applied to control V.alginolyticus,but these control measures can induce drugresistant strains outbreak,environmental pollution and other new problems,with the disadvantages of high cost and drug selectivity.Therefore,prevention of highfrequency infection of V.alginolyticus,has become one of the important research directions in the field of food and medicine at this stage.In recent years,fatty acids have been found to be important in regulating cell membrane composition,regulating virulence factor expression and inhibiting signaling.As one of the significant fatty acids,palmitic acid has good pharmaco-logical characteristics such as anti-inflammatory and analgesic,which has the advantages of being naturally pollution-free and not producing drug-resistant strains.So,it can be used as a novel natural antibiotic alternative for the prevention project of V.alginolyticus.However,at present,the study of the inhibitory effect and mechanism of palmitic acid on bioactive and virulence factors of V.alginolyticus is few.In this study,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of palmitic acid against V.alginolyticus were determined,the growth,cell membrane and morphology of V.alginolyticus after palmitic acid treatment were monitored.And the lipidomics method based on UHPLC-MS was applied to explore the inhibitory mechanism of palmitic acid against V.alginolyticus.Additionally,the major virulence factors including motility,biofilm,and alkaline serine protease(Asp)of V.algino-lyticus were examined under sub-MIC palmitic acid.Then,the effect of palmitic acid on V.alginolyticus pathogenic and lethal ability was also evaluated by in vivo infection assay.Further,the molecular mechanisms of palmitic acid regulating about V.alginolyticus virulence were investigated using metabolomics(including lipidomics),transcriptomics.The main conclusions are as follows:(1)The antibacterial activity of palmitic acid against V.alginolyticus and the mechanism of inhibition were investigated.It was found that palmitic acid significantly inhibited the growth of V.alginolyticus,and 1MIC palmitic acid increased the extracellular conductivity,extracellular glucose,extracellular nucleic acid,extracellular protein,intracellular reactive oxygen species,and malondia-ldehyde by 4.36-,4.17-,2.18-,1.91-,1.19-,and 1.08-fold,respectively.Found that palmitic acid severely disrupted the integrity,permeability of the cell membrane in V.alginolyticus,and by scanning electron microscopy,palmitic acid caused the cells to highly collapse and forced the life activity till to damage or even apoptosis.The regulatory mechanism of its lipid level was further explored by lipidomic technique method.It was found that palmitic acid acts on the cell membrane to affect processes such as phospholipid metabolism,glycerolipid metabolism,and sphingolipid metabolism.Palmitic acid increases the fluidity and permeability of V.alginolyticus cell membrane,leakage of intracellular substances,and induces cell death;and after palmitic acid treatment,the synthesis process of Cers was significantly enhanced,which implied that palmitic acid promoted the process of cell apoptosis;at the same time,palmitic acid makes many energy of V.alginolyticus stored in the form of TGs,and sugar metabolism is largely used as the main energy supply platform.(2)The effects and molecular regulatory mechanisms of subinhibitory concentrations of palmitic acid on the virulence of V.alginolyticus were investigated.Palmitic acid was found to inhibit motility,biofilm,and alkaline serine proteases of V.alginolyticus in a dose-dependent manner;And exploring the infection of zebrafish with V.alginolyticus after 1/2MIC palmitic acid treatment,we found that the infection symptom of fish body was significantly attenuated,and the lethality rate was reduced to 15%.Further,we explored the mechanisms of virulence regulation at the molecular level based on the combined multi omics technologies of metabolome,lipidome and transcriptome.It was found that palmitic acid significantly acted on the cell membrane,and fatty acid metabolism,phospholipid metabolism,glycerolipid metabolism,and sphingolipid metabolism were significantly regulated,resulting in increased cell membrane fluidity and permeability,and disrupted intracellular biological homeostatic processes,implying a disturbance of the normal toxigenic system.In addition,the amino acid metabolism of V.alginolyticus was seriously disturbed after being treated with palmitic acid,which may lead to the inhibition of the protein synthesis process of V.alginolyticus;Palmitic acid significantly regulates the gene expression of membrane transport related functions,resulting in the weakening of the transport function of V alginolyticus to amino acids,ions,virulence factors and other substances,which may be the key determinant of palmitic acid weakening the infection and pathogenicity of V.alginolyticus.In summary,palmitic acid has a good bacteriostatic activity and virulence regulation on V.alginolyticus,which widely affects the bacterial energy supply,nucleotide metabolism,sugar metabolism,amino acid metabolism,fatty acid metabolism and other pathways,and the cell membrane is used as a target to regulate the structural function of the membrane,in order to affect the biological activity and virulence of the bacteria and achieve the bacteriostatic and virulence regulation effect.So,palmitic acid can be used as a potential novel antibiotic alternative to become the preferred control means for V.alginolyticus and has a promising application as a naturally innocuous bacteriostatic agent in the engineering of Vibrio disease control. |
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