Pseudomonas Aeruginosa Mexab-of Oprm Efflux Pump Inhibitor Screening Model As Well As Antisense Rna, Complementary Rna Positive Efflux Pump Mexab-of Oprm Outer Expression And Regulation

It is well known that the emergence of drug-resistance bacteria,especially multidrug resistance(MDR),has become a serious problem in the clinical treatmen and some known antibiotics have become useless to cure diseases caused by the drug-resistance bacteria.Several approaches could induce bacterial drug resistance.One of the approaches is effluxing antibiotics out of the cells of bacteria by efflux pumps to decrease the intracellular concentrations of antibiotics.Besides decreasing the intracellular concentrations to protect bacteria,efflux pumps may also increase the probability of the drug-resistance mutations in bacteria.Therefore, it is important to seek efflux pump inhibitors to relieve the bacterial drug resistance induced by efflux pump and repress the emergence of drug-resistance mutations.Pseudomonas aeruginosa is a familiar conditional pathogen which may lead to nosocomial infection.The drug resistance of P.aeruginosa is complex with multiple mechanisms.However,two main approaches are important in the MDR of P.aeruginosa: one approach is blocking the antibiotics by the outer membrane with low permeability; the other is effluxing antibiotics by MDR efflux pumps.There are seven kinds of RND efflux pumps in P.aeruginosa,Among them, MexAB-OprM efflux pump is the only one that may both be constructively expressed and affect the drug resistance of P.aeruginosa observably.Therefore,MexAB-OprM efflux pump is a ideal target for anti-P.aeruginosa drugs by increasing bacteria sensitivity to antibiotics.In this study,two means were designed to search the efflux pump inhibitors of MexAB-OprM efflux pump.The first one is to establish a screening model targed on the efflux pump and to screen inhibitors to the efflux pump of P.aeruginosa.For the purpose,a model strain was constructed by biotechnological method that MexAB-OprM was cloned and expressed in P.aeruginosa N150 lacking multiple efflux pumps.Then a high-throughput screening model targetd on the MexAB-OprM efflux pump was established after determining antimicrobial susceptibility,examing mexAB-oprM transcription and testing susceptibility of known efflux pump inhibitors.Appling the screening model,we screened inhibitors to MexAB-OprM efflux pump from the pools of compounds and natural products.The second one is to seek oligonucleotide inhibitors of MexAB-OprM efflux pump by means of RNA technology.In theory,the antisense RNA could specificly inhibit the expression of target gene,so that it could be useful in therapies of microorganism infections and cancers.We devised a series of antisense RNAs of the mexAB-oprM opron,and conducted the expression plasmids to express the antisense RNAs in the P.aeruginosa K335 or K854 accordingly.Different effects of the series of antisense RNAs were evaluated by the ciprofloxacin susceptibility testing of the antisense RNA expression strains.Then mexR and mexA gene in the mexAB-oprM opron were determined to be appropriate target genes for antisense RNAs,and the upstream sequences of the genes and the enough complementary length were considered necessary for antisense repression.These fashion of antisense RNA in P.aeruginosa was explored to direct the research of the antisense oligonucleotide inhibitors of MexAB-OprM efflux pump.In the study,we also investigate the gene-specific silencing induced by parallel complementary RNA in P.aeruginosa.The discovery of gene-specific silencing induced by parallel complementary RNA in Escherichia coli revealed a novel route of gene regulation by RNA.If gene-specific silencing induced by pRNA could be proven to work in other prokaryotes besides E.coli,it would be a novel tool for regulating gene expression in prokaryotes,with many potential applications in biotechnology.The parallel complementary RNA also could be developed to be parallel complementary oligonucleotide drugs for antibacterial treatments as the antisense oligonucleotides.To know whether parallel complementary RNA could induce gene-specific silencing in Pseudomonas aeruginosa,the strain expressing parallel complementary RNA of mexA was compared to the control strains,The result showed that for the former the MIC of some antimicrobial agents which are substrates of the MexAB-OprM efflux pump droped about 50%and the initial accumulation rate of ethidium bromide increased twofold. These results suggest that gene-specific silencing was induced by parallel complementary RNA.This is the first time that such a route for gene silencing has been reported in a bacterium other than Escherichia coli.Gene-specific silencing induced by parallel complementary RNA may be useful as a novel biotechnology tool for gene regulation in prokaryotes,and it would direct the the research of the parallel complementary oligonucleotide inhibitors of MexAB-OprM efflux pump.