Antibacterial Effect And Mechanism Of Mannosylerythritol Lipid-A On Listeria Monocytogenes

Listeria monocytogenes is a gram-positive foodborne pathogen with strong stress resistance,which can survive in various food processing environments such as low temperature and dryness,and can prolong the survival period by forming biofilm.Ingestion of food contaminated by L.monocytogenes can cause abdominal pain and diarrhea in mild cases,and severe diseases such as sepsis,meningitis,and fetal abortion in severe cases,with a lethality rate as high as 20-30%.Mannosylerythritol lipids-A（MEL-A）are glycolipid biosurfactants mainly produced by fermentation of yeast and fungi,and are widely used in daily chemical and biomedical fields because of their excellent physical and chemical properties and biological activities.Previous studies have shown that MEL-A have good antibacterial activity against gram-positive bacteria,however,there is a lack of research on the antibacterial mechanism of MEL-A,and its antibacterial activity and mechanism against L.monocytogenes are still unclear.Therefore,in this study,the antibacterial activity and mechanism of MEL-A against the planktonic and biofilm states of L.monocytogenes were confirmed by multi-omics such as Tn-seq,transcriptomics,non-targeted metabolomics,and gut microbiomics.The main research results are as follows:（1）MEL-A had significant inhibitory effects on the planktonic cells of L.monocytogenes with a minimum inhibitory concentration（MIC）of 32μg/mL.The growth curve results showed that MEL-A significantly inhibited the growth of bacteria in the logarithmic growth phase and retarded the proliferation of the bacteria.SEM and TEM as well as flow cytometric analysis revealed that MEL-A had a significant disruptive effect on the cell membrane structure of L.monocytogenes,and the treatment with MEL-A caused the cells to become elongated and irregular,with a significant increase in surface roughness,and led to the leakage of intracellular nucleic acids and proteins.In addition,the synergistic treatment of MEL-A with HHP and LA significantly increased the antibacterial rate against L.monocytogenes.The synergistic treatment increased the antibacterial rate of HHP from 15.3%to 94.86%at 200 MPa,and the antibacterial rate of LA from 50.0%to 99.4%at 1 mg/mL.（2）Five key resistance genes of L.monocytogenes under MEL-A treatment,lmo2360、spl、lmo2754、hemL and lmo1757,were screened at the whole gene level based on Tn-seq.Markerless deletion mutantΔlmo2360 andΔlmo2754 were constructed by homologous recombination and Cre-lox recombination system,and the complementation strainsΔlmo2360+lmo2360 andΔlmo2754+lmo2754 were obtained by recombinant plasmid construction.The results of growth curve and morphological analysis of the markerless deletion mutants indicated that lmo2360 and lmo2754 play an important role in MEL-A resistance in L.monocytogenes.The results of antibiotic resistance assay,molecular docking and D,D-carboxypeptidase activity assay showed that MEL-A affected the activity of D,D-carboxypeptidase through binding to Lmo2754,which in turn affected the biosynthesis of peptidoglycan,leading to blocked cell wall synthesis and bacterial lysis and death,which may be one of the key mechanisms of MEL-A to inhibit the growth of L.monocytogenes.（3）The effect of MEL-A on the transcriptional regulation of L.monocytogenes was revealed by transcriptomic analysis.GO enrichment results showed that MEL-A significantly up-regulated cell membrane-related genes,further demonstrating the impairment of cell membrane structure by MEL-A.KEGG enrichment results showed that MEL-A significantly affected the transmembrane transport of iron,sugar and amino acids by ABC transporters.In addition,MEL-A caused significant up-regulation of the AGR family and significant down-regulation of the PTS system,indicating that MEL-A significantly affected arginine biosynthesis,sugar transport and sugar phosphorylation of the PTS system in L.monocytogenes at the transcriptional level.Transcriptomic analysis ofΔlmo2360 andΔlmo2754 showed that lmo2360 and lmo2754 played an important role in the biosynthesis of valine,leucine and isoleucine and protein synthesis.（4）MEL-A significantly inhibited the biofilm formation of L.monocytogenes with MBIC and MBEC of 64μg/mL and 256μg/mL,respectively.The results of laser confocal microscope observation and intracellular bacterial viability assay showed that MEL-A significantly reduced the thickness of biofilm and the ratio of live/dead cells,and significantly inhibited the early viability of bacteria in the biofilm.In addition,MEL-A significantly reduced the crawling motility and initial adhesion of L.monocytogenes and affected bacterial adhesion by influencing the secretion of extracellular polysaccharides,e DNA and proteins.The results of non-targeted metabolomics analysis indicated that MEL-A affected cell membrane fluidity and permeability by significantly up-regulating unsaturated fatty acids,lipids and glycosidic metabolites,and protein biosynthesis,nucleotide metabolism and DNA synthesis and repair by significantly down-regulating amino acid metabolism and nucleic acid metabolism,which may be the key mechanism of MEL-A to inhibit biofilm formation.（5）MEL-A significantly inhibited L.monocytogenes infection in vivo in an oral infection model,and the MEL-A-treated group showed a significant decrease in bacterial counts in both the spleen and liver compared to the control group.Gut microbiomic analysis revealed that the MEL-A-treated group resulted in significant changes in the intestinal flora during the infection.The results showed that MEL-A at the genus level led to significant decreases in Prevotellaceae＿UCG-001,Escherichia-Shigella and Listeria and significant increases in Lactobacillus.The in vivo antimicrobial activity of MEL-A was found by LEfSe analysis and may be closely linked to the increased abundance of Lactobacillus in the intestine.（6）The safe dosage range of MEL-A was confirmed by in vivo and in vitro safety evaluation.The results of in vitro cell experiments showed that MEL-A did not significantly affect the viability of intestinal epithelial cells when the concentration did not exceed 5 mg/mL.The results of acute oral toxicity test in vivo showed that the LD₅₀of MEL-A on mice was greater than 20 g/kg BW,and the survival rate,body weight,organs and blood of mice would not be significantly affected at a dose of 20 g/kg BW.