Modeling Design And Digging Out And Functional Analysis Of Key Resistance Gene Of Listeria Monocytogenes Against OLE

Listeria monocytogenes（L.m.）is a kind of common foodborne gram-positive pathogen,which is often found in the contamination ready-to-eat foods such as dairy products,fruits and vegetables and whose clinical symptoms include septicemia,meningitis febrile enteritis,abortion and stillbirth with a fatality rate of 15～30%.Listeria monocytogenes needs low nutritional requirements and can tolerant with high salt environments and wide growth temperature range.Listeria monocytogenes is difficult to prevent and control in food production and storage.Olive Leaf Extract（OLE）has the characteristics of natural antibacterial and antioxidant activity,no biological toxicity and high yield.Its main active substance is oleuropein,which can effectively inhibit L.m.Biofilm formation.Using OLE to gradually replace preservatives and antibiotics as food-borne pathogens L.m.Bacteriostatic agent is always the research hotspot in the field of food safety.Because OLE antibacterial potency is not high,and OLE material calculation method is not so accurate,resulting in OLE industrial production with a large amount of material,limiting the commercial production of OLE as a bacteriostatic agent.In this study,the tolerance of Listeria monocytogenes（L.m.）to olive leaf extract（OLE）was studied.Using Logistic function to model OLE tolerance process of L.m.and lightweight calculation of OLE inhibitory amount;using genetic（transcriptome detection,gene knockout,phenotype analysis）and biochemical（in vitro expression and purification of target protein,BLI affinity analysis）and other ways to analyze L.m.key OLE tolerance gene with excavating and functional analysis.The main research results are as following:（1）The in vitro inhibitory rate of OLE against Listeria monocytogenes F2365 was used to construct the Logistic logic growth model,i.e.,LM（R²=0.88957）of inhibitory rate and OLE concentration,y=1/（1+exp（1.9366-0.11413 x））.In this paper,four critical points including inflection point（PI）,maximum acceleration point（PAM）,maximum deceleration point（PDM）and progressive deceleration point（PDA）in LM were used to describe the inhibitory process of OLE against Listeria monocytogenes F2365.The MIC value obtained by LM-PDA algorithm was verified by in vitro bacteriostasis experiment,which proved that the MIC value from LM-PDA algorithm had the same bacteriostasis effect（OLE inhibited Listeria monocytogenes F2365）as that from 2-fold algorithm with a reduced usage amount of 42.188%for OLE,providing theoretical support for the industrialization of OLE antibacterial products.（2）Transcriptome analysis of differentially expressed genes in L.m.-F2365 transcripts under OLE stress showed that L.m.-F2365-2330（mto）gene was significantly upregulated under OLE induction(FC_3.5=4.10,FC₂₄=10.72).L.m.usually ingests OLE as a nutrient through endocytosis and when L.m.found that OLE is harmful,OLE can be pumped out of the cell by an efflux pump（some membrane transporter）to reduce the concentration of OLE inside the cell,so as to prevent OLE from harmin.L.m.has different membrane transporters according to different antibacterial substances.According to KEGG database analysis,mto gene is membrane protein gene.Therefore,mto was selected as the key response gene of L.m.against OLE,and this gene was studied and verified.（3）Electro-competent Listeria monocytogenes F2365 cells with cell wall mutilation were successfully prepared by using penicillin G+lysozyme method（via SEM verified）.The DNA transcription template of g RNA was successfully assembled and synthesized with a concentration of 39.5 ng/m L and a fragment size of 118 bp.A total yield of 35.7μg g RNA was obtained with a fragment size of 100 nt.The primers for specific amplification of target fragments were successfully designed and the amplification rate reached 82.5%.A total of 99target DNA sequencing samples with concentration between 20 ng/μL～40 ng/μL were prepared with high quality and each sample was detected by Agilent 2100 with a single band of472 bp.Three Listeria monocytogenes F2365-2330 silencing strains of Listeria monocytogenes were obtained by CRISPR/Cas9 gene editing method.The mutation type of Listeria monocytogenes F2365-2330 was nonsense frame-shift mutation for the 28^thsample and the mutation type of the 58^thand 89^thsamples was missense frame-shift mutation.The incidence of frame-shift mutation in this study was 3.03%.（4）The C-terminal mto protein（L.m.-F2365-2330 gene translation protein）with His tag was successfully synthesized in vitro.The molecular weight was 283.57 k Da,and a clear band was detected at the corresponding position by SDS-PAGE.The concentration of mto protein was 38.25±1.03 mg/m L after purification by Ni-NTA.The proportion of oleuropein in OLE detected by HPLC was 18%.Oleuropein was the main active functional component of OLE as inhibitory factor for L.m.against OLE.That is,the affinity degree of OLE and mto protein can be statemented by the affinity degree of oleuropein and mto protein.（5）The MIC values of OLE for the three mutation strains for Listeria monocytogenes F2365-2330 gene were 6.4 mg/m L by 2-fold method with micro-plate reader,while the MIC value for the normal Listeria monocytogenes F2365 was 64 mg/m L.The silencing of Listeria monocytogenes F2365-2330 gene resulted in a significant down-regulation of the MIC value of OLE against Listeria monocytogenes F2365,and the inhibitory rate of OLE on Listeria monocytogenes F2365 decreased by 1 order of magnitude（64 mg/m L→6.4mg/m L）,indicating that the deletion of mto gene caused the OLE efflux pump（OLE membrane transporter）of L.m.cannot be formed to transport excess OLE out of the cell.（6）The mean affinity constant（K_D）of mto protein and oleuropein was detected by BLI,which was 4.46×10^-9M（P mole grade）,and the binding force was strong.The mean affinity constant（K_D）between His protein and oleuropein was 5.31×10^-1M（mole level）,and the binding force was weak.So the interference of His label on the affinity result between mto protein and oleuropein could be deducted.That is,mto protein has a strong affinity with oleuropein,and mto protein can specifically bind OLE（oleuropein）.（7）The OLE tolerance mechanism and mto gene functions of L.m.are as following:L.m.ingested OLE as a nutrient intracellular through endocytosis.When OLE reached a certain concentration and was found to harm L.m.,OLE was specifically bound to oleuropein（OLE）through mto gene product（OLE membrane transport protein）,and OLE was pumped out of the cell to reduce the intracellular OLE concentration,so as to avoid OLE’s effect on L.m..The loss of mto gene caused L.m.to be unable to form OLE efflux pump（OLE membrane transport protein）to transport excess OLE out of the cell,which was manifested as OLE’s effect on L.m.when mto gene was lost.And the mto gene was confirmed to be OLE transmembrane transport protein（OLE efflux pump）gene,which was confirmed to be the key gene of L.m.for OLE tolerance.In this study,the amount of OLE bacteriostatic was reduced by 42.188%through LM-PDA model calculation and experimental verification,which provided theoretical basis for the lightweight of OLE bacteriostatic materials in industrial production.The mto gene was silenced by CRISPR/Cas9 gene editing technology.The function of the target gene was studied by MIC phenotypic verification and BLI affinity detection,and the mto gene was confirmed to be OLE transmembrane transporter protein（OLE efflux pump）gene of L.m.,providing potential research target for enhancing antibacterial efficiency of OLE.