Study On The Pharmacodynamics And Mechanism Of Antibacterial Effects Of Chlorogenic Acid Metabolites

Staphylococcus aureus(SA)is one of the pathogens causing human infectious diseases.SA has caused great concern in clinic due to its wide distribution,strong pathogenicity,high drug resistance,and the production of biofilm and virulence factors.At present,the heat-clearing and detoxicating traditional Chinese medicines(TCM)have been widely used to assist in the anti-infection therapy.It has poor direct antibacterial effect and hardly reaches the concentration for inhibiting bacteria in vivo.However,it has a good effect on inhibiting the release of bacterial virulence and is beneficial to the clearance of bacteria by the immune system of host.In addition,it may cause weaker adverse reactions and less resistance to antibiotics.Therefore,searching for new antibacterial agents from TCM has become a hot topic of research.In this study of the metabolites of chlorogenic acid including Gallic Acid(GA)and Dihydrocaffeic Acid(DCA),we investigated the antimicrobial,anti-biofilm,anti-virulence effects of chlorogenic acid metabolites,and the related mechanisms as well against the MRSA strains isolated from clinic in recent years.Through transcriptomic analysis,we screened the differentially expressed genes,analyzed the biological processes and pathways related to the inhibitory effects of GA and DCA against MRSA.We also carried out quantitative PCR and molecular docking validation on those genes which may be affected in the pathways.It is expected to provide ideas for discovering new antibacterial agents from the heatclearing and detoxicating traditional Chinese medicines and the modernization of TCM.The study included three parts as follows:Part Ⅰ: The inhibitory effect of GA and DCA as chlorogenic acid metabolites on MRSA.From the strains collected clinically in recent years,MRSA2106 strains with MIC values of 1 mg/mL for GA and DCA were selected for experiments.The growth curve experiment showed that both of GA and DCA had inhibitory effects on the growth of the strain at high subMIC.The anti-biofilm experiment showed that DCA presented significant inhibitory effect on the biofilm formation at sub-MIC.Both GA and DCA could eliminate the formed biofilm,and the effect of DCA was stronger than that of GA.As the results observed by scanning electron microscopy,GA and DCA could significantly affect the morphology of biofilm produced by the strain,which could reduce the adhesion and bacterial aggregation,and facilitate the penetration of antibacterial drugs into the biofilm.GA and DCA were used together with cefixime,vancomycin,clarithromycin,gentamicin and levofloxacin,respectively.The results showed that most effects were additive,and nosynergistic and antagonistic effects were observed.The effects of GA and DCA on the release of virulence factor α-hemolysin were also studied.The results showed that both GA and DCA could significantly inhibit the release of α-hemolysin at sub-MIC.Part Ⅱ: the transcriptomic analysis of the effect of chlorogenic acid metabolites GA and DCA on MRSA.A transcriptomic analysis was performed to investigate anti-bacterial,anti-biofilm and anti-virulence effects and underlying mechanisms of GA and DCA on MRSA strains.The results showed that both GA and DCA significantly down-regulated the infection pathway of Staphylococcus aureus.GA down-regulated the expression of spa,clfB and isdA genes in this pathway,up-regulated the expression of agrB and sspA genes in the Agrsystem,and resulted in the production of extracellular secretory proteins that can degrade Spa adhesion proteins.DCA down-regulated the expression of genes associated with infection,including vra G,spa and dlt D,indicating that both GA and DCA could affect the infectivity and virulence factors release of MRSA strains.Transcriptomics analysis also showed that GA and DCA had significant up-regulation or down-regulation effects on multiple biosynthetic and metabolic pathways such as amino acid synthesis,histidine metabolism in MRSA strains,which indicated that GA and DCA could also affect the metabolic process of bacteria.Meanwhile,for icaADBC,the coding gene of PIA,an important component of biofilm,icaA and icaD in GA group showed a slightly down-regulation,but no significant difference,which may be related to the principle and detection sensitivity of the two methods.In summary,the results of transcriptome showed that GA and DCA can affect the related genes in several pathways in MRSA,such as the Staphylococcus aureus infection pathway,and have inhibitory effects on the infectivity of MRSA,such as virulence factor release and biofilm production and elimination.Part Ⅲ: PCR and molecular docking of GA and DCA as chlorogenic acid metabolites on related genes.Based on the previous experimentss,the agrA gene,the main pathogenic gene in the agrsystem,and the icaADBC gene,a biofilm-encoding gene associated with drug resistance,were selected for quantitative PCR analysis.The results showed that GA down-regulated the expressions of agrA icaA,icaD and icaB genes,and DCA down-regulated the expression of agrA,icaA,and icaB genes,indicating that they could not only reduce the release of virulence factors of MRSA,but also inhibit biofilm formation at sub-MIC.Molecular docking results showed that both of them could bind to the site of interaction between DNA and AgrA protein,thus interfering with the binding of DNA to AgrA,affecting the function of AgrA and reducing the pathogenic ability of SA.In addition,DCA has a stronger affinity for AgrA protein than GA.To sum up,based on the previous study on the anti-infective effect of chlorogenic acid,the key metabolites of chlorogenic acid in vivo,GA and DCA,were further investigated to study on the inhibitory effects of GA and DCA(especially DCA)on the biofilm production and virulence factor release of clinically resistant strain MRSA at sub-MIC.Through pharmacological experiments,transcriptome,PCR and molecular docking analysis,we have conducted in-depth research on the regulatory mechanisms related to the infection of SA.Our research reveals the application potential of DCA widely exists in natural plants,it may provide reference for the study of antibacterial effect of TCM and clinical anti-infection treatment.