ABSTRACT
To investigate whether parallel complementary RNA (pRNA) could induce gene-specific silencing in Pseudomonas aeruginosa, pRNA of the mexA gene was expressed in it. Compared to the control strains, the strain expressing pRNA of mexA showed a 50% decrease in minimum inhibitory concentrations (MICs) of several antimicrobial agents and a twofold increase in the initial accumulation rate of ethidium bromide, all of which are substrates of the MexAB-OprM efflux pump. These results suggest that gene-specific silencing was induced by pRNA. 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 pRNA may be useful as a novel biotechnology tool for gene regulation in prokaryotes.
Subject(s)
Gene Knockdown Techniques/methods , Gene Silencing , Pseudomonas aeruginosa/genetics , RNA, Complementary/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Membrane Transport Proteins , Microbial Sensitivity TestsABSTRACT
Drug-resistant (including multidrug-resistant) bacteria increase continuously with the wide use of antibiotics, which have seriously threatened the human health. It is an important way to fight against drug-resistance by screening and developing novel drugs based on the various mechanisms of the bacterial drug tolerances. Meanwhile, the basic research related to the new drug R. & D. and studies on the new screening methods for the antimicrobial agents should be taken seriously and strengthened, so as to accelerate the process of finding new drugs and meet the challenge of new pathogens and new drug-resistant strains.
Subject(s)
Anti-Bacterial Agents , Bacteria/drug effects , Drug Design , Drug Resistance, Multiple, Bacterial , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Bacteria/genetics , Bacterial Physiological Phenomena/drug effects , Drug Evaluation, Preclinical , Drug Resistance, Multiple, Bacterial/genetics , Drug Resistance, Multiple, Bacterial/physiology , Humans , PeptidesABSTRACT
OBJECTIVE: To establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point. METHODS: Efflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain. Expression of OprM protein was induced in E. coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector. In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly. RESULTS: OprM protein was highly expressed. Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM. A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5%. CONCLUSION: OprM plays an important role in drug efflux. The established model has good specificity and maneuverability.
