Evolution Of CphA From Aeromonas Hydrophila Under Imipenem Selection Pressure

Today,the global spread of metal-producing?-lactamase-producing super bacteria has seriously threatened public health.The increase in multidrug resistance has led to the evolution of many superbugs.The synthesis of highly efficient,broad-spectrum selective metal beta-lactamase inhibitors has been a competition between chemists and bacteria.To date,all clinically effective metal?-lactamase inhibitors have been rapidly hydrolyzed by metal?-lactamase.In order to explore the ways in which superbugs acquire resistance and resistance,and the interaction between the two,this article takes the Cph A enzyme of the B2subclass as the research object and cultivates it through an improved experimental device for microbial evolution and cultivation.It shows how the resistance of super bacteria is obtained and how to cope with the pressure of antibiotic selection through the change of molecular conformation.This paper mainly researches from the following three aspects:1.By measuring the minimum inhibitory concentration?MIC?of p ET-28b/Cph A engineering bacteria,the measured MIC was 12?g/m L.Taking this recombinant bacterium as the research object,the growth,migration and evolution of the imipenem?IMP?evolution device with discontinuous concentration gradient were investigated.After the end of the culture,the sequencing results of the bacteria samples were randomly collected and analyzed,which showed that the bases 650 bp downstream of the gene were susceptible to mutation,and all were missens mutation.2.Two of the mutated enzymes,K249T and M1,were purified,expressed and characterized,and their antibacterial concentration and catalytic activity against IMP were determined.The results showed that the enzyme activities of K249T and M1 were increased by 32%and37%compared to the wild type,respectively.The mutations caused K249T to increase the bacteriostatic concentration of IMP by at least 100 times,and K218R to the bacteriostatic concentration of IMP by 85 times.It indicates that it contributes a lot to IMP resistance.3.Based on the study of how these modifications affect the catalytic efficiency of Cph A enzymes,the molecular mechanism of the interaction between mutant enzymes and antibiotic substrates was revealed by means of calculation methods of homology modeling,molecular docking,and molecular dynamics simulation.The results showed that the mutation caused by drug resistance caused the migration of the enzyme carbon skeleton,changed the coordination ability of Cys221 and Zn²⁺,and increased the cavity volume of the enzyme active site,so that sufficient Zn²⁺could play a role in the proton shuttle.In addition to stabilizing the local conformational structure,Zn²⁺also plays an active role in catalysis,which is more conducive for IMP to enter the active pocket of the enzyme and then undergo a hydrolysis reaction.At the same time,the change in the geometry of the active site is a direct cause of high K_catand low K_mvalues.In short,the mutation of Cph A enzyme under IMP selection pressure,and the results of MIC,enzyme activity,and kinetic simulation of mutant samples show that bacteria have completed the evolution of Cph A enzyme in the process of drug resistance and resistance,and due to different evolution pressure creates a more refined structure.