Study On The Enhancement Of Susceptibility Of MRSA To A Novel Cephalosporin Compound WYBQ-4 By D-amino Acid

Staphylococcus aureus(S.aureus),is a common gram-positive bacterial pathogen,which is an important cause of skin and soft tissue infection,intravascular infection,pneumonia,infectious arthritis,endocarditis,osteomyelitis and sepsis.With the emergence and spread of Methicillin-resistant Staphylococcus aureus(MRSA),it has become one of the main causes of hospital infection.MRSA can produce a new Penicillin binding proteins(PBP2a)2a,which is related to β-The low affinity of lactam antibiotics makes it one of the main reasons for MRSA resistance.In addition to the infection caused by plankton,Staphylococcus aureus can also secrete extracellular polysaccharide and other substances,which attach to the surface of the host and form biofilm.The existence of biofilm also increases the difficulty of clinical treatment.WYBQ-4(C22H22N605S3),as a new type of cephalosporin compound,has trans-methoxy group in the 7α site of its β-lactam ring,which can effectively form steric hindrance,so that it has strong stability against β-lactam enzyme.However,after putting it into clinical use,Staphylococcus aureus will also gradually develop resistance to it.Therefore,new therapeutic strategies to enhance the anti-MRSA effect of WYBQ-4 are urgently needed.As natural small molecules,amino acids have high safety.Studies have shown that different amino acids combined with various antibiotics can improve the ability of antibiotics to fight bacterial infection,and different amino acids can also inhibit the formation of a variety of bacterial biofilms.However,whether amino acids can improve the sensitivity of MRSA to cephalosporins WYBQ-4 in the way of combined drug use and its mechanism have not been thoroughly studied.Objective:In this study,amino acids which can improve the sensitivity of MRSA USA300 to WYBQ-4 were screened from L-amino acids and D-amino acids.Furthermore,the possible key binding sites of the screened D-serine and USA300,and whether the combination of D-serine and WYBQ-4 has synergistic inhibitory effect on the formation of USA300 biofilm,and explore its possible mechanism.After that,the actual therapeutic effect of D-serine combined with WYBQ-4 in vivo was studied by constructing a mouse MRSA pneumonia model.This study provides a new experimental basis for the further development of drugs that can treat MRSA infection.Methods:1.Amino acids that have synergistic effect on USA300 combined with WYBQ-4 were screened from L-amino acids and D-amino acids by checkerboard method.The Time-kill curve was used to confirm the synergistic effect again.Further study was conducted to determine whether the selected D-amino acids had a co-effect on Methicillin-Sensitive Staphylococcus aureus(MSSA)ATCC29213.2.PBP2a protein was induced by the vector pET28a,and was isolated and purified by His Trap FF affinity chromatography.Affinity of PBP2a protein with D-serine screened by checkerboard method was investigated using Bocillin experiment.3.The binding pattern of D-Serine and PBP2a protein was detected by molecular docking experiment,and the key site of PBP2a protein and D-Serine binding was estimated.4.The multi-step induction of drug resistance was used to make USA300 more resistant to WYBQ-4,and to study whether D-serine can improve the sensitivity of drug-resistant strain UW20 to WYBQ-4.5.The inhibition of D-serine on USA300 and UW20 biofilm formation was confirmed by crystal violet staining when used alone or in combination with WYBQ-4.6.Through enzymolysis and autolysis experiments,the mechanism of D-serine inhibiting biofilm formation was discussed.7.A mouse model of MRS A pneumonia was established,and the therapeutic effect of D-serine combined with WYBQ-4 on MRSA pneumonia in mice was investigated by survival rate,bacterial load in lung tissue,and lung histopathological changes in turn.Results:1.The effect and mechanism of amino acid combination with WYBQ-4 to increase the sensitivity of MRSA to itBy checkerboard method,the sensitivity of USA300 to WYBQ-4 was improved by adding D-serine,and the synergistic effect was confirmed again by the Time-kill curve.There was no synergistic effect on ATCC29213.PBP2a protein with the size of 73kDa was successfully induced and expressed by SDS-PAGE.Subsequent Bocillin experiments showed that as D-serine concentration increased,it was able to bind competitively to PBP2a.Molecular docking also indicated that Glu-523,Asn-505,Lys-506 and Asn-507 in PBP2a may be the key binding sites of D-serine.Then,the MIC value of USA300 against WYBQ-4 was increased from 8μg/ml to 256μg/ml by a multi-step induction of resistance.After the use of D-serine,the MIC value can still be significantly reduced.2.Inhibitory effect of D-serine on MRSA biofilm and its mechanismCrystal violet staining proved that D-serine alone could inhibit the initial adhesion stage of USA300 biofilm,and D-serine could enhance the inhibition effect of WYBQ-4 on USA300 biofilm.It indicated that D-serine combined with WYBQ-4 had synergistic effect on USA300 biofilm.The results of enzymolysis experiment showed that the extracellular matrix of USA300 biofilm was mainly composed of protein,followed by eDNA and less extracellular polysaccharide.The autolysis experiment showed that D-serine could inhibit bacterial autolysis and affect the release of eDNA.3.Therapeutic effect of D-serine combined with WYBQ-4 on MRSA-infected mouse pneumonia modelIn vivo experiments showed that the survival rate of MRSA-infected mice was significantly improved by 80%when treated with D-serine combined with WYBQ-4 compared with the monotherapy group.Moreover,the number of colonies in lung tissue was significantly reduced,and the pathological changes of lung tissue were significantly reduced,which was closer to the blank control group.Conclusions:1.D-serine can improve the sensitivity of MRSA bacteria USA300 to cephalosporin WYBQ-4,and also has a synergistic effect on inducing drug-resistant bacteria.2.D-serine can act on PBP2a specific to MRSA,and Glu-523,Asn-505,Lys-506 and Asn-507 of PBP2a protein may be the key residues of D-Serine.3.D-serine can inhibit the biofilm formation of drug-resistant bacteria UW20 induced by USA300 and WYBQ-4,and D-serine can also enhance the inhibition effect of WYBQ-4 on biofilm formation.4.The combined application of D-serine and WYBQ-4 has a good therapeutic effect on mouse USA300 pneumonia model.