| Trueperella pyogenes（T.pyogenes）can cause suppurative infection in many domestic animals,wild animals and even human beings,which poses a serious threat to animal husbandry,endangered species protection and human health.However,the resistance of T.pyogenes is not optimistic,and resistant strains of T.pyogenes have been isolated all over the world,including multidrug-resistant strains.Therefore,it is necessary to develop new antibacterial drugs to deal with the increasingly serious resistance of T.pyogenes.People pay more and more attention to finding new non-antibiotic antibacterial drugs from natural products.Luteolin is one of the most representative natural flavonoids,and studies have shown that luteolin has good antibacterial activity.This study will comprehensively analyze the antibacterial activity of luteolin against T.pyogenes in vitro and in vivo,and deeply explore the action mechanism of luteolin against T.pyogenes,in order to provide detailed theoretical basis for the development and utilization of luteolin as a new target antibacterial drug.By measuring the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of luteolin against T.pyogenes,and combining with the growth of T.pyogenes under the action of luteolin,the antibacterial activity of luteolin against T.pyogenes in vitro was evaluated.To evaluate the antibacterial activity of luteolin in vivo by analyzing the effect of luteolin on the survival rate of Galleria mellonella larvaes infected with T.pyogenes.The results showed that the MIC of luteolin against 20 strains of T.pyogenes was 78～156μg/m L,and the MBC was 625～1250μg/m L.The growth of T.pyogenes could be obviously inhibited by luteolin with sub-inhibitory concentrations（1/2 MIC and 1/4 MIC）,which indicated that luteolin had good antibacterial activity against T.pyogenes in vitro.Luteolin treatment could significantly improve the survival rate of Galleria mellonella larvaes infected by T.pyogenes,indicating that luteolin had certain antibacterial activity in vivo.By analyzing the effects of luteolin on cell wall,cell membrane,protein expression and nucleic acid synthesis,the mechanism of luteolin against T.pyogenes was preliminarily explored.After T.pyogenes was treated with luteolin,the effects of luteolin on cell wall and cell membrane were analyzed by detecting the leakage of alkaline phosphatase（AKP）,the penetration of fluorescent probe through cell wall,the leakage ofβ-galactosidase and the entry of extracellular substances into the cell,measuring the cell membrane potential of the strain and observing the ultrastructure of the strain.The effect of luteolin on the expression of protein was analyzed by quantifying the total protein of T.pyogenes and SDS-PAGE.The effect of luteolin on nucleic acid synthesis was analyzed by quantifying the intracellular nucleic acid of T.pyogenes,scanning the ultraviolet-visible spectrum of interaction between luteolin and DNA and detecting the DNA topoisomerase activity of the strain.The results showed that after luteolin treatment,AKP located between the cell wall and the cell membrane leaked out of the cell,and the fluorescent probe could pass through the cell wall and enter the cell membrane;intracellularβ-galactosidase leaked out of the cell,and extracellular substances could also enter the cell through the cell membrane.TEM observation results showed that luteolin made the bacterial surface rough,some substances oozed out of the cell,the cell wall was partially missing and the cells were deformed.Luteolin could also depolarize the cell membrane potential.Luteolin could affect the expression of proteins in T.pyogenes and significantly reduce the intracellular total protein content.After luteolin treatment,the content of nucleic acid in T.pyogenes cells decreased significantly.Luteolin could embed in DNA and inhibit the activities of DNA topoisomerases I and II,thus interfering with nucleic acid metabolism.The above research showed that luteolin could play an anti-T.pyogenes role by destroying the integrity of cell wall,increasing the permeability of cell membrane,affecting the expression of proteins and inhibiting the synthesis of nucleic acid.RNA-seq and TMT-quantitative proteomic techonology were used to analyze the transcriptomics and proteomics data of T.pyogenes treated with luteolin,to explore the action mechanism of luteolin on transcription level and protein expression level,and to clarify its target.According to the analysis results of omics data,q RT-PCR was used to detect the relative expression of energy metabolism-related genes in T.pyogenes after luteolin treatment.The results showed that 967 genes were differentially expressed（675 up-regulated genes and 292down-regulated genes）and 1054 proteins were differentially expressed（536 up-regulated proteins and 518 proteins）.The results of KEGG enrichment analysis of differentially expressed genes and proteins showed that luteolin could significantly affect the ribosome and oxidative phosphorylation metabolic pathway of T.pyogenes.All the differentially expressed genes and proteins related to ribosome structure were significantly up-regulated,and all the differentially expressed genes and proteins related to oxidative phosphorylation were significantly down-regulated.These two metabolic pathways were the first two pathways with the most differentially expressed genes and proteins,and the KEGG enrichment analysis results of transcriptomics and proteomics data were consistent.In addition,most of the differentially expressed proteins in glycolysis and TCA cycle were also significantly down-regulated.The results of q RT-PCR showed that the genes related to energy metabolism of T.pyogenes were significantly down-regulated after luteolin treatment,which was consistent with the omics data.The above results suggest that targeted energy metabolism is one of the important ways for luteolin to exert its anti-T.pyogenes effect.Based on the analysis results of transcriptomics and proteomics data,this study detected and analyzed the inhibitory effect of luteolin on energy metabolism of T.pyogenes through a series of experiments,and revealed its action mechanism deeply.The energy metabolism level of T.pyogenes after luteolin treatment was detected by bioenergy metabolism analyzer and the ATP content in the cell was determined to analyze the effect of luteolin on the energy metabolism of the strain.The action mechanism of luteolin inhibiting oxidative phosphorylation of T.pyogenes was revealed through the determination of respiratory chain complex I～V activity,the superposition rate of luteolin and respiratory chain complex I～IV inhibitors,the molecular docking and affinity detection of luteolin and respiratory chain complex subunits.The mechanism of luteolin inhibiting glycolysis and TCA cycle was clarified by measuring the activities of enzymes related to glycolysis and TCA cycle of T.pyogenes after luteolin treatment.The results showed that luteolin could reduce the energy metabolism level of T.pyogenes and significantly reduce the ATP content in the cell.Luteolin could significantly inhibit the activity of respiratory chain complexes I,II and V,indicating that the targets of luteolin inhibiting oxidative phosphorylation may be complexes I,II and V.The superposition rates of luteolin and respiratory chain complexes I and II inhibitors were low,which indicateed that luteolin could target complexes I and II to inhibit the electron transfer of respiratory chain.The results of molecular docking showed that luteolin could strongly bind to the subunits of complexes I,II and V through hydrogen bonds and hydrophobic forces,among which the binding ability to the subunit of complex I was the strongest,followed by the subunits of complexes V and II,and the binding sites involve important functional domains of subunits.The affinity constants of luteolin and NADH-ubiquinone oxidoreductase subunit F（complex I subunit）and ATP synthase subunitα（complex V subunit）are 1.717×10-6 M and 1.010×10-5 M,respectively,indicating that luteolin could bind to both,and the binding ability of luteolin and complex I subunit was stronger than that of complex V subunit.In addition,luteolin could significantly inhibit the activities of pyruvate kinase and phosphofructokinase in glycolysis and the activities of citrate synthase and succinate dehydrogenase in TCA cycle.To sum up,luteolin had good antibacterial activity against T.pyogenes in vitro and in vivo,and its antibacterial mechanism was diverse,including destroying cell wall and cell membrane,affecting the expression of proteins,inhibiting nucleic acid synthesis,inhibiting energy metabolism and so on.Inhibition of energy metabolism was one of the important mechanisms of luteolin’s anti-T.pyogenes effect.Luteolin could reduce the level of oxidative phosphorylation by inhibiting the activities of respiratory chain complexes I and II and the electron transfer in respiratory chain,binding with ATP synthase subunitαand inhibiting the activity of complex V in catalyzing ATP synthesis.Luteolin could also inhibit the activities of enzymes related to glycolysis and TCA cycle,which eventually led to the inhibition of energy metabolism and ATP synthesis of T.pyogenes.This study provides an important theoretical basis for the development and utilization of luteolin as a new antibacterial drug,and will also provide new ideas for the development of new target antibacterial drugs. |
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