Clone, Expression And Evolution Of Active Efflux Pump Gene EmrE From Shigella

Shigella is the main pathogen of shigellosis. It is an epidemic contagion throughout the world, especially in the devoloping countries.Since antimicrobial agents were widely used to treat shigellosis, antimcrobial resistance in Shigella has been widely dispreaded. Now increasing numbers of Shigella exhibit resistance to multiple antibacterials, which constitutes great difficulty to the prevention and treatment of shigellosis. Active efflux is a major mechanism of resistance to drugs in pathogenic microorganisms. Many investigations have revealed the presence of efflux transporters in Shigella.To study the function and evolution of the active efflux pump gene emrE in clinical isolates of Shigella, antibiotics susceptibility of the ten clinical strains of Shigella was tested by the two fold agar dilution technique. The results showed that the strains were resistant to many kinds of antiobiotics. Finally, the strain H₂₄ was selected for its significant antiobiotic resistance.A pair of primers were designed and PCR was performed using genome DNA of the drug resistant-strain H₂₄ as the template. The PCR product, including coding sequences of emrE and its native promoter, was ligated into the vector pMD18-T to construct the recombinant pMD-emrE plamid which was subsequently transformed into E.coli DH5a. Then positive bacterial clones were identified by restriction enzyme digestion and the gene was sequenced and analyzed.The result showed that the cloned emrE gene fragment was 1100bp , 100% homogenous to the sequence of emrE of S. sonnei Ss046. Phylogenetic tree was constructed using the data of the gene sequences from Genebank. Phylogenetic tree analysis indicated that horizontal gene transferring was scarce for emrE among different bacterial genomes.Drug susceptibility of the recombinant strain was determined by the two fold agar dilution technique,with and without the membrane deenergizer carbonyl yanidem-chlorophenyl hydrazone (CCCP) to clarify the emrE gene function. The results showed that the pMD-emrE (DH5α) exhibited increased resistance to tetracycline(1-fold) and erythromycin(2-fold). The MICs of the seven antimicrobial agents to the pMD-emrE (DH5α) and the control strain pMD (DH5α) decreased to almost the same level with carbonyl cyanidem-chlorophenyl hydrazone being present.The total RNA was extracted from the pMD-emrE (DH5α) strain as well as the pMD (DH5α) strain. The disparity of mRNA level of emrE eflux genes between the pMD-emrE (DH5α) and the control strain pMD ( DH5α) was measured by RT-PCR using gapA gene as inter reference. It was indicated that the level of expression of emrE in the recombinant strain was significantly enhanced compared with that in control strain.In conclusion, emrE contributed to the resistance of tetracycline and erythromycin in the drug-resistant strain H₂₄ and horizontal gene transferring for emrE was scarce among different bacterial genomes.