SiRNA-mediated Gene Silencing Of Mexb From The MexAB-OprM Efflux Pump In Pseudomonas Aeruginosa

Objective:The MexAB-OprM multidrug efflux pump is an important component of the human pathogenic bateria Pseudomonas aeruginosa. We investigated the efficacy of siRN A on the expression of MexB gene and the importance of MexAB-OprM efflux pump system in chronic Pseudomonas aeruginosa lung infection.Methods:We designed and synthesized 21bp siRNA against the MexB gene and electrotransformed the siRNA into the P. aeruginosa strain PAO1. Changes in broad-spectrum antibiotic susceptibility in response to siRNA-mediated MexB gene silencing were measured by determining the minimum inhibitory concentration (MIC) by the E-test method. RT-PCR was performed to determine whether the siRNA inhibit the expression of the MexB mRNA in vitro. The efficacy of siRNA was determined in a murine model of chronic P. aeruginosa lung infection. Macroscopic and histopathological changes of the lungs, bacterial loads, and the levels of cytokines [interleukin(IL)-lb), interleukin(IL)-12) in BALF, myeloperoxidase (MPO) were measured at 3,5and7 days post-infection. Meanwhile the pathogenic effects of the wild-type P.aeruginosa PAO1(K767) and its double mutant nalB andΔmexB were determined by infecting a murine model of chronic P. aeruginosa lung infection in the concentration of 1×107 colony-forming units per milliliter(CFU/ml). The mice were injected with Meropenem (100mg/kg,twice/d) on days 1,2 and 3 after infection with the P.aeruginosa strains. Then the indexs were measured on days 3,7 and 14 after infection with the P.aeruginosa strains.Results:siRNAs caused a remarkable increase in sensitivity of PAO1 to meropenem, ciprofloxacin, and ceftazidime. RT-PCR showed that siRNAs significantly inhibited the expression of the MexB mRNA (p<0.05). In the murine infection model, treatment with siRNAs led to significantly reduced bacteria burden of the day 5 and 7 post-infection. Comparabled with the siRNAnon group and Scrambled siRNA group, the siRNA caused a marginal pathological changes of the lung. In addition, MexB-siRNA treatment enhanced neutrophil recruitment and production of inflammatory cytokines (IL-lb, IL-12) in the early infection stage (day 3) (p<0.05), both of which decreased by day 7. Meanwhile bacterium burden inΔmexB group obviously descended after 7d and 14d infection of mice, meanwhile pathological change in this group was much lighter than K767 group and nalB groups. Contrast with K767 and nalB groups, neutrophil recruitment (MPO) and inflammatory cytokines (IL-lb, IL-12 and TNF-a) ofΔmexB group heightened in early infection stage (3d), and these expression level in this group descended in later infection stage (14d). Serum macrophage inflammatory protein-2(MIP-2) level of three groups (ΔmexB, K767 and nalB) in infective stages (3d,7d,14d) continued to advance, and there were statistical significance among the three groups (P< 0.05)Conclusion:siRNA could inhibit both mRNA expression and the activity of P. aeruginosa in vitro. In vivo, siRNA was effective in reducing the bacterial load in a murine model of chronic lung infection. Targeting of MexB with siRNA appears to be a novel strategy for treating P. aeruginosa infections. This research could provide evidence for studying new antipathogenic drugs targeted to the pathogenicity of Pseudomonas aeruginosa. In addition, the pathopoiesis effects characteristics of the MexAB-OprM efflux pump system knockout strain are obviously milder than that of the MexAB-OprM efflux mutant strain in the animal models. The functional MexB-OprM efflux pump system of Pseudomonas aeruginosa play important roles in the persistence of the chronic infection in lungs.