Evolution Of Metallo-β-lactamase SMB-1 From Serratia Marcescens Under The Selection Pressure Of Meropenem

With the widespread application of β-lactam antibiotics,Metallo-β-lactamases(MβLs)super bacteria possesse resistant to almost all kinds of antibiotics.MβLs have become a major threat to public health,due to they can evolve in a rapidly changing selective environment and have the ability to quickly adapt their substrate preference.In order to explore the evolution process of resistance and drug resistance of them in response to antibiotic selection pressure.The phenotypic and genotypic changes that occurr during the evolution process.This study takes the subclass B3 MβL SMB-1 from Serratia marcescens as the research object,through microbial evolutionary culture experiments of in it evolution were carried out in order to simulate bacteria are evolutionary dynamics response towards increasing concentrations of an antibiotic.It explores the pathway SMB-1 acquires drug-resistant mutations and how to resist antibiotic selection pressure via changes in molecular structure.The main contents of this paper are as follows:1.The minimum inhibitory concentration(MIC)of the E.coli BL21(DE3)containing p ET-28b/SMB-1 was measured to be 8 μg/m L,and investigated its growth,migration and evolutionary status in the apparatus containing discontinuous concentration gradient meropenem(MEM).After the culture,the bacterial samples were randomly collected for sequencing,and seven base mutations were found,all located at 650 bp downstream of the gene,which were divided into missense mutations and synonymous mutations.2.The MβL SMB-1 and the mutant enzymes A242 G,M5 obtained on the microbial evolution plate were expressed and purified,and their MIC and catalytic activity against MEM were determined.Meanwhile,the secondary structures of wild-type and mutant enzymes were detected by circular dichroism spectroscopy.The results showed that the mutations caused A242 G and M5 to increase the MIC of MEM by at least 120-fold.The catalytic efficiency of A242 G and M5 was 1.7-fold and 2.5-fold higher compared with that of WT SMB-1,respectively.The conformation of the mutant enzyme also changed,with a decrease in the amount of ordered structure α-helix and the increased content of the random structure.3.Differences in molecular recognition and interactions between wild-type and mutant enzymes and MEM were revealed at the molecular level by homology modeling,molecular docking,and molecular dynamics simulations.It results indicated that the mutation may not influence the overall structure of SMB-1,but it was shown herein to changed from an ordered to a disordered structure near the active site and increase the flexibility of loop1,which endowed the enzyme with broaden the binding pocket,more conducive to the exposure of active sites and less steric hindrance.Thus,it is beneficial to better binding of antibiotics and the hydrolyzed product exit from the binding groove faster.In the binding process of wild-type and mutant enzymes with MEM,leading to several changes such as decrease in hydrogen bonding,increase in the metal acceptor interaction and the non-bond interactions.In summary,E.coli BL21(DE3)harboring SMB-1 gradually accumulated resistance or drug resistance mutations through the establishment of adaptive evolution as a strong and survival sustainable strategy to evade the selection pressure of antibiotics,and evolved a more refined and efficient mutant enzymes in the process of resistance and drug-resistance.