Multiple Antibiotic Tolerance Mediated By Mutation In The Acid Resistance Gene Of Escherichia Coli

The discovery of antibiotics is an important milestone in the history of human medicine.How antibiotics induce bacterial cell death is centered on the essential cellular function inhibited by the primary drug-target interaction.Recent studies have shown that gallium nitrate,a drug approved by the US FDA for the treatment of hypercalcemia,has a good antibacterial activity.Unlike other antibiotics,the pharmacological properties of plutonium depend on chemical mimicry,which can replace iron in target enzyme molecules,thereby interfering with and disrupting the function of bacterial ferritin and bacterial metabolism,both in vitro and in vivo.Many pathogens,including multi-drug resistant pathogens,exhibit bactericidal activity.However,the specific mechanisms and targets are still unclear.We originally wanted to use E.coli to enrich and screen out the gallium nitrate resistant mutants.We wanted to look for the mutated genes to help us get to know the bactericidal mechanism of gallium.Only the tolerant mutants were screened out as a result.But we unexpectedly discovered that these tolerant mutants were tolerant to three traditional types of antibiotics and industrial bactericide.This indicates that there is a generally tolerant to bactericidal drugs pathway.Combined with whole genome sequencing,we find the corresponding mutation sites,and use phage transduction,CRISPR-Cas9 and other gene editing methods to construct the mutants,and then verify the phenotype.It is found that these mutants share a common characteristic that there are point mutations in the domain of the acid resistance protein.Mutations in this domain can make the protein in a state of sustained activation,resulting in a tolerant phenotype.These mutations are not only tolerant to the three major classes of traditional antibiotics such as ampicillin,kanamycin and ciprofloxacin,but also resistant to fungicides such as hydrogen peroxide and phenol.After drug treatment,intracellular ROS levels significantly reduce compared to the wild type.Later,we find that the tolerant phenotype correlates with the level of phosphorylation of the response regulatory protein.Decreasing the phosphorylation level of the response regulatory protein results in decreased tolerance levels.By looking for genes that are regulated by regulatory protein,the regulatory pathways are further defined.