Study The Effect And Mechanism Of Noval Coumarin Derivatives Aginst MRSA Biofilm

ObjectiveBacterial biofilm is a self-organized structure of polymer matrix formed by bacteria that attach to surfaces.Biofilm can protect bacteria against the attact from antimicrobial agents,and reduce the host immune function and cell phagocytosis,leading antibiotics resistance significantly.The data from the Centers for Disease Control and Prevention shows that 65% to 80% of human bacterial infections are associated with biofilm and 50% of nosocomial infections are associated with biofilms on medical devices.Staphylococcus aureus is an important clinical pathogen,and also a common pathogen in biofilm infections.It often causes skin infections and a variety of tissues and organs suppurative inflammation,and is one of the important pathogens that causes the hospital cross-infection.As an important virulence factor,biofilm formation plays a significant role in the medical device infections caused by Staphylococcus aureus,and biofilm on the implant medical devices causes major morbidity and mortality.Thus it is urgent to develop the new drugs to treat the biofilm induced infections.Recently,coumarin compounds atract widespread concern due to the various of pharmacological properties.We synthesed series of new coumarin derivatives previously,and screened out several compounds that have significant antibacterial activity against staphylococcus aureus,but whether they can inhibit the bacterial biofilm is unclear.In this study,the inhibitory activity of the new coumarin derivative against the biofilm formation was evaluated on the clinical common methicillin-resistant Staphylococcus aureus(MRSA),and we further explore the molecular mechanism of the coumarin compound aganist MRSA biofilm,and provide new targets for treat clinical biofilm infections caused by staphylococcus aureus.Method1.Screening for staphylococcus aureus stains those have better ability of biofilm formation: The strains capable of forming biofilm were screened using Congo red agar plates,and those on the plate were able to form dry and shiny colonies are biofilm formation positive strains.The strains were then cultured in 96-well plates,and the film-forming ability of positive strains was measured by crystal violet semi-quantitative method.The fluorescence staining biofilm formation process was observed under optical microscope.2.Determination of coumarin derivative DC H anti-MRSA(USA300)biofilm activity in vitro: using micro-dilution method and plate count to measure the MIC and MBC of DCH against MRSAUSA300,the inhibitory effect of DCH to biofilm formation was observed by crystal violet staining method.3.Effects of DCH on the biofilm formation in vivo:The catheter was placed into the bladder of SD rats,and then injected with 1 × 107 CFU USA300 bacteria to establish urinary tract infection model.The rats at 1h,24 h and 48 h after infection were treated with different concentrations DC H,after 3 days the rats were killed to and the catheter was obtained and were supersonic shaken after washed and then plated on agar plates for bacterial counts,the urine,bladders and kidneys were also obtained and plated on agar plates after homogenized.4.Impacts of DCH on the expression of biofilm-associated genes: USA300 was incubated with different concentrations of DCH for 3 h,then the bacteria were collected.After centrifugation,the bacteria were treated with lysozyme and lucosporin,and the total RNA was extracted and the genomic DN A was removed,RT-PCR was used to detect the gene expression of icaA and atlA.5.Exploring possible targets of DCH by proteomics analysis : to explore the possible targets,the protein expression differences of MRSA USA300 before and after DCH treatment were compared by isobaric tags for relative and absolute quantitation(iTRAQ)technology.6.The affinity of DCH to ArgR protein: prepared chips through the PlexArray HT(V3 version)biomolecular was used to study the affinity of DC H to ArgR,and SPRi(Surface plasmon resonance imaging)was applied to analysize DC H and argR protein binding kinetics and affinity parameters.7.The molecular docking of DCH and ArgR protein: prepared DC H single crystal by interfacial diffusion,using Modeller in Discovery Studio 3.5 software for multiple sequence alignment,screening out the Escherichia coli argR protein structure as the best similarity as Staphylococcus aureus homologous protein using SWISS-MODEL automatic mode for homology modeling.Result1.DCH inhibited the biofilm formation of MRSA USA300 and can eliminate the mature biofilm at some levelin vitro: Four strains of Staphylococcus aureus were screened by Congo red agar experiment then the MRSA USA300 strain was selected for its strongest biofilm-forming ability by crystal violet semi-quantitative method.Compound DCH can inhibit the biofilm formation(P <0.001)even under MIC concentration,and can erase mature biofilm at a certain degree.2.DCH inhibited the biofilm formation of MRSA USA300 in vivo: DCH could significantly inhibite USA300 biofilm formation on catheter in infection rats,the number of bacteria on the catheters that treated with DCH was also significantly decreased compared to the control group(P <0.001),the number of bacteria from unrine,bladders and kidneys were also significantly decreased compared to the control group.3.DCH reduced the expression of biofilm-related genes: During the biofilm formation,the PIA protein encoded by ica locus and AtlA protein encoded by atlA play important roles.RT-PCR results showed that DCH could significantly inhibit the expression of atlA and icaA(P <0.01)at 1/8MIC and 1/4MIC respectively.4.Arginine metabolic pathway was involved in the anti-biofilm effect of DCH: iTRAQ Quantitative analysis showed that 1647 proteins were identified and 244 protein expression differences occured,of which 97 proteins were upregulated and 147 proteins were downregulated.In the down-regulated proteins,the first five proteins with the largest difference were the ornithine carbamyltransferase,arginine deiminease,immuno globulin G binding protein A,glycine cleavage system protein H,carbamate Kinase.Pathway analysis showed that ornithine carbamyltransferase,arginine deiminease and carbamate kinase were the key enzymes of the MRSA arginine metabolic pathway,encoded by the arc gene cluster,whose expression was inhibited by arginine repressor(argR),the anti-biofilm effect of DCH was assumed to interact with argR.6.DCH has high affinity to argR: according to GenBank published bacteria argR coding gene sequence,the primers were designed to construct argR expression cloning,then the argR protein was purified.The results of SPRi analysis showed that the binding signal of DC H and argR was strong,and the affinity was enhanced with the increasing concentration of argR.The affinity constant of compound DCH and argR was 5.66×10-7 mol/L.7.The molecular docking of compound DCH and ArgR protein: use cultured compound DC H crystal and homologous argR protein model 3D structure,docking results was grouped in the 0.1 nm RMSD standard,and then the lowest docking and binding energy and the most repeated docking operations was selected,combined with all the docking results to determine the active site of DC H binding at the 185-position glycine residues,the molecular docking value was-5.07.ConclusionThe new coumarin compound DC H can significantly inhibit the MRSA biofilm in vitro,reduce the number of bacteria in the infected animals and the biofilm formation.The mechanism is mainly through the combination with the arginine inhibitory factor,thereby reduce the expression of arginine metabolic pathway enzymes(arginine deiminease,ornithine carbamyltransferase and carbamate kinase).The results suggest that blocking the arginine metabolic pathway will be a new mechanism against bacterial biofilm,and also provide a new idea for developing new anti-biofilm drugs.