The Study On Mode Of Action And Target Of Two-peptide Bacteriocin Plantaricin JK

Lactic acid bacteria are identified by FDA as food grade microorganisms.One of its metabolites,lactobacillus bacteriocin,is an antibacterial peptide or protein synthesized by ribosome in its metabolic process with properties of safe,efficient,pollution-free,drug-resistant and so on.It has great potential in food and medicine application.Plantaricin JK(PlnJK)is a Class ?b LAB bacteriocin,which consists of two peptides Plantaricin J(PlnJ)and Plantaricin K(PlnK)and two oligopeptides have obvious synergistic effects.In this study,PlnJK was synthesized by solid-phase synthesis.We achieved its antimicrobial spectrum and studied its mode of action to sensitive bacteria,and finally tried to find the specific targets of PlnJK.The main contents are as follows:1.Determination of inhibition spectrum and Minimum Inhibitory Concentration(MIC)of Plantaricin JK.PlnJ and PlnK were synthesized by solid-phase synthsis.The results of inhibition activity showed that PlnJK could inhibit many Gram-positive bacteria,including Lactobacillus plantarum,Lactobacillus casei and Lactobacillus fermentum.It also had a good inhibitory effect on Staphylococcus.It showed the highest activity against Lactobacillus plantarum ZFM811 and the MIC was 400 nM.2.Effects of pH,temperature and enzyme on the stability of Plantaricin JK.It was found that bacteriocin PlnJK was well pH tolerated,the inhibition activety remained more than 80%after treated with pH 2-12 and was better under acidic condition than that under alkaline condition.PlnJK remained active at 60-100? for 30 min and with the increasing of temperature,the residual activity still remained 91.63%at 100?,indicating that was bacteriocin PlnJK was thermostable.PlnJK was sensitive to protease K,papain,alpha-chymotrypsin,pepsin and trypsin and the activity was almost completely lost after treated with 30 min,but was not sensitive to lysozyme and RNase.3.Study on the mode of action of Plantaricin JK on sensitive strains.Lactobacillus plantarum ZFM811 and Micrococcus luteus GIM 1.226 were used as indicator bacteria.It was found that the transmembrane electrical potential and pH gradient dissipated rapidly after treated with PlnJK,and the intracellular ATP synthesis was also inhibited.The dissipation of transmembrane electrical potential on Micrococcus luteus GIM1.226 was faster than that of Lactobacillus plantarum ZFM811,and the other two aspects were not obvious than that of Lactobacillus plantarum ZFM811.Scanning electron microscopy and transmission electron microscopy of Lactobacillus plantarum ZFM81 1 and Micrococcus luteus GIM 1.226 treated with PlnJK showed that the cell surface and cross-section structure and morphology had changed after treated with PlnJK.In conclusion,the result showed that PlnJK caused the cell membrane of sensitive bacteria damage and eventually lead to cell death.4.Study on the binding of Plantaricin JK to Lipid ?.The results of the agar-well diffusion test and fluorescence leaking test showed that Lipid ? had no effect on the antibacterial activity of PlnJK,it's different with the positive control(Nisin).Thus Lipid ? is not the specific target of PlnJK.5.Find the specific target of Plantaricin JK by the technology of GST pull-down.The cell membrane of Lactobacillus plantarum ZFM811 and Micrococcus luteus GIM1.226 was extracted and the binding of PlnJK to cell membrane was analyzed by the agar-well diffusion test.In order to explore whether there is a target of PlnJK in cell membrane protein,the genes of PlnJ and PlnK were cloned into the expression vector pGEX-6P-1 and the recombinant plasmids pGEX-6P-1-plnJ and pGEX-6P-1-plnK were successfully constructed and transformed into E.coli Rosetta-gami 2(DE3)pLysS.The fusion proteins GST-PlnJ and GST-PlnK were expressed successfully.The fusion proteins GST-PlnJ and GST-PlnK were immobilized on glutathione-agarose resin and acted as bait proteins to capture the potential targets of membrane proteins.Firstly,four membrane proteins with transmembrane helix belonging to Lactobacillus plantarum and one belonging to Micrococcus luteus were captured by iTRAQ technique that may bind to bacteriocin PlnJ;three transmembrane helix membrane proteins belonging to Lactobacillus plantarum and two Micrococcus luteus membrane proteins may bind to bacteriocin PlnK.In addition,there are two membrane proteins belonging to Micrococcus luteus are co-products of GST-PlnJ and GST-PlnK.Finally,by further domain analysis,it was found that 1 possible membrane protein in each cell membrane of Lactobacillus plantarum and Micrococcus luteus with transmembrane domain that specifically binds to PlnJ and 1 possible membrane protein that specifically binds to PlnK as well.