The Mechanism Of Escherichia Coli Membrane Proteins Promoting Its Tolerance To Nalidixic Acid

With the widespread antibiotic tolerance,how to completely and effectively kill drug-resistant pathogens has become one of the primary problems which every medical research institutes around the world have to face.As the "armor" to protect bacteria and the "scout" to sense the external environment,membrane proteins play a very important role in protecting bacteria and adapting to the environment.However,how membrane proteins respond to antibiotic stress and coordinate with other cellular factors to protect cells needs further study.In order to study the mechanisms of membrane proteins protecting bacteria against drug treatment,transcriptome sequencing was first performed to examine the changes of gene expression in the wild-type Escherichia coli treated with and withoutnalidixic acid.The significantly differentially expressed genes were screened and the functions of the proteins encoded by these genes were annotated.A total of 16 genes encoding membrane proteins were selected for further study.The changes of the expression levels of these 16 genes under drug treatment were confirmed by using real-time quantitative PCR.The susceptibility of the selected gene mutants to nalidixic acid was then measured,and seven of them（△din F、△pho U、△sul A、△fhu B、△fiu、△cyb B、△pst A）were more sensitive to nalidixic acid than the wild type strain,while mutants △omp G and △ omp F that encode pore proteins showed resistance to drugs at certain concentrations when compared with the wild type strain.This suggested that the membrane proteins encoded by these genes are involved in their susceptibility to antibiotic stress.The proteins encoded by these nine genes can be divided into two categories: two kinds of pore proteins and seven kinds of membrane proteins.Pore proteins include Omp G and Omp F.The survival rate of the two gene mutants（especially the △omp F mutant）is higher than that of the wild type strain at certain concentrations of nalidixic acid（40-60 μg/m L）,which we speculate is related to their cellular functions,that is,the decreased expression or lack of these two proteins leads to the decrease in the number of pore proteins and the decrease in membrane permeability that finally leads to the decreased penetration of quinolones into the cells and the development of drug tolerance.Ultra-performance liquid chromatography（UPLC）was used to verify this hypothesis,and the results showed that fewer drugs were detected in the mutant strains than in the wild-type strains when treated with 40 μg/m L of nalidixic acid.Meanwhile,two strains over-expressing Omp G and Omp F were constructed,and the sensitivity of the two strains to drugs was tested.It was found that the membrane protein overexpressing strains were indeed more sensitive to drugs than the wild-type strain.Therefore,the pore proteins Omp G and Omp F could protect cells by reducing their expression to resist drug entry under drug pressure.To further understand the protective mechanism of other seven membrane proteinencoding genes,the efflux pump activity of the mutant strains of these genes was measured to see whether mutations in these membrane proteins could alter theirefflux pump activity.The results showed that the activity of the efflux pumps of four mutant strains △pho U,△fiu,△sul A,and △din F was significantly lower than that of the wild type strain.Therefore,these membrane proteins might protect bacterial cells through elevating efflux pump activity.We then examined the reactive oxygen species（ROS）levels of the mutants during drug treatment to see whether these membrane proteins could protect the bacteria by affecting intracellular ROS levels.The results showed that the intracellular ROS level of four of the mutant strains（△sul A,△din F,△ fhu B and △cyb B）was significantly higher than that in the wild-type strain after drug treatment,while the intracellular ROS level of the △fiu mutant decreased.Thus,the deletion of these five genes affected the ROS homeostasis in bacteria under antibiotic treatment.However,it is still unclear how these membrane proteins affect the intracellular ROS levels to protect cells.The study on the mechanism of E.coli membrane proteins promoting its drug resistance would help us to further reveal the molecular mechanism of bacterial resistance,and lay a theoretical foundation for the development of drugs targeting bacterial membrane proteins.