Antimicrobial Properties And Transcriptome Analysis Of Ilex Kudingcha C.J.Tseng Polysaccharides In Guangxi

In the past 30 years,the irrational use or abuse of antibiotics has made more and more pathogenic bacteria resistant to drugs.The replacement speed of antibiotics can not even catch up with the replacement speed of drug-resistant strains,and the high cost of antibiotics on the market and certain drug-induced side effects,so it is urgent to develop new bacteriostatic agents.Ilex kudingcha C.J.Tseng(IKCJT)is a medicinal and edible plant clearly announced by the National Health Commission.It is non-toxic and has obvious antibacterial,antioxidant,hypoglycemic,anti-inflammatory and other effects.It has significant antibacterial activity against drug-resistant bacteria,among which the polysaccharide of IKCJT(Po K)is the main component of its antibacterial effect.To explore the inhibitory effect of Po K on methicillin-resistant Staphylococcus aureus(MRSA USA300)and its bacteriostatic mechanism,USA300 was taken as the research object.The crude polysaccharide extracted from IKCJT by hot water was purified by alcohol precipitation and sevage method.The antibacterial activity of IKCJT against USA300 was investigated.The antibacterial mechanism of polysaccharide from IKCJ against drug-resistant bacteria USA300 was analyzed by transcriptional technology.To speculate the targets of USA300 inhibited by Po K and elucidate the effect of polysaccharides from IKCJT on the pathogenicity of USA300.The main research results are as follows:1.The water extraction crude extract of IKCJT(hereinafter referred to as water extract)has significant effects on Escherichia coli(E.coli)and drug-resistant and drug-resistant(Staphylococcus aureus,S.aureus atcc 6538)and(MRSA USA300)were 4.167,2.083 and2.083 mg/m L,respectively.Compared with IKCJT from Wuming and Tian of Guangxi,the water extract of IKCJT from Daxin County of Guangxi had the largest bacteriostatic zone and a more obvious inhibitory effect on the above three strains of pathogenic bacteria.The content of crude polysaccharide of IKCJT contained in water extract was positively correlated with the size of the bacteriostatic zone.However,penicillin had no significant inhibitory effect on drug-resistant strain USA300,and the crude polysaccharide of IKCJT significantly inhibited the growth of drug-resistant strain USA300.It can be seen that the crude polysaccharide of IKCJT has broad-spectrum antibacterial activity and is the main antibacterial active component of IKCJT.2.The crude polysaccharide of IKCJT was further graded and purified,and three purified components(KDPL1,KDPL2,KDPL3)were obtained.The antibacterial activity of the three purified components were lower than that of the crude Po K.It is speculated that during the purification process of crude polysaccharides,components related to antibacterial activity such as related proteins,uronic acid and sulfuric acid groups were lost,or that several components of crude polysaccharides had synergistic antibacterial effects or each component had different targets,and the activity of each component was not as good as that of multiple components and multiple targets when acting on bacteria alone.3.Transcriptome technology was applied to analyze the inhibitory mechanism of IKCJT polysaccharide on drug-resistant Staphylococcus aureus strain USA300.The findings were as follows:(1)By downregulating fmt C and mva S genes of USA300,the polysaccharide of IKCJT and Ilcan inhibited the synthesis of peptidoglycan,affected the thickness and permeability of cell wall,and successfully invaded the cells,and accelerated the dissolution of USA300 cells by downregulating fmt C gene;(2)The polysaccharide of IKCJT and Ilexin down-regulates the gene vra F,gene vra E and gene sdh C,inhibits the uptake of solute necessary for the growth and metabolism of USA300,and inhibits the efflux of the drug by the cell’s external exhaust pump;(3)By inhibiting the expression of rib A,rib B,rib D,rib H genes and their enzyme activities or the content of flavin mononucleotide(FMN)of their products,the polysaccharide from S.aureus disrupted riboflavin homeostasis;By inhibiting the expression of por A,por B and other genes,the activities of SHD(succinate dehydrogenase)and other enzymes were reduced,and then the energy metabolism and nutrient uptake of USA300 were inhibited.4.The polysaccharide of IKCJT greatly affected the pathogenicity of USA300:(1)By inhibiting the synthesis of Clfb and Spa protein,the polysaccharide from IKCJT reduced the adhesion and virulence of USA300;(2)By down-regulating the expression of sae S and sae R genes,the polysaccharide affected the Sae RS TCS signaling pathway,inhibited the synthesis of multiple downstream virulence factors,inhibited the hemolysis function after hlg A expression and reduced its adhesion,and then reduced the pathogenicity of USA300;(3)The polysaccharide also inhibited several synthase activities of pur K,pur Q,pur L,pur F,pur M,pur H and pur D gene expression,and inhibited the biosynthesis of USA300 source purine,thus reducing the pathogenicity to the host;(4)The treatment of IKCJT and Ilexin polysaccharide also down-regulated several respiratory and substance transformation-related enzyme activities of sdh C,gap B,por A,por B and pck A gene expressions,inhibited glycolysis and gluconogenesis of USA300,and inhibited the biosynthetic ability of amino acids required for protein synthesis on biofilm,and reduces the pathogenicity of USA300.In conclusion,by down-regulating the expression of fmt C and mva S gene of USA300,the polysaccharide of IKCJT Ilicaria reduced the thickness of the cell wall,successfully infiltrated into the cells of the strain,and accelerated the dissolution of USA300.The energy metabolism and nutrient uptake of USA300 were also inhibited by inhibiting the enzyme activities of rib A and rib B.By down-regulating vra F,vra E and other genes,drug efflux by efflux pump was inhibited.In addition,by down-regulating the synthesis of Clfb and Spa protein expression proteins and down-regulating sae S and sae R genes,the adhesion of USA300 can be reduced to reduce its virulence.Down-regulating the expression of hlg A,pur K,gap B,gpm A,sdh C,por A and other genes and inhibiting the enzyme activity of their expression,disrupting the riboflavin homeostasis of USA300,inhibiting the biosynthesis of source purine,glycolysis and gluconeogenesis,thus reducing the pathogenicity of USA300.It can be seen that the polysaccharide of IKCJT has a significant antibacterial function against drug-resistant Staphylococcus aureus.