Objective To identify the efficacy small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps.Methods Four siRNA ( siRNA1,siRNA2,siRNA3 and siRNA4) against mexB gene were designed and prepared by electricity transference in vitro.MICs of antibiotic combined with efflux pump inhibitors against multiple resistant strain PAO1 and PAO3 were determined by E-test method.The mRNA expression levels of efflux pump gene (mexB) were quantified by real time fluorescent quantitative PCR.Results siRNA expression vectors were constructed success by enzyme cut method.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA4,the sensibilities to antibiotic were enhanced.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNAl,siRNA2 and siRNA3,the sensibilities to antibiotic didn't change obviously.48 after PAO1 and multiple drug resistant PAO3 ttransfected with siRNA4,the expression level of mexB was decreased obviously (P ＜ 0.05 ).48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA1,siRNA2 and siRNA3,the expression level of mexB didn't change obviously.Conclusion siRNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps enhanced the sensibility to antibiotic and inhibited the expression of mexB gene.Our results demonstrate the using RNAi may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

Objective To investigate the effect of pvdQ(PA2385)gene on Pseudomonas aeruginosa swarming motility.Methods The plasmid pME6032 with pvdQ gene was constructed and identified,then transformed into Pseudomonas aeruginosa PAO1 using the eleetroporation to build pvdQ-overexpression strain.The pME6032-PAO1 strain was constructed with the same method.The cloning plasmid pEX18Gm containing sacB was successfully used to construct unmarked deletion mutant of pvdQ gene and pvdQ mutant strain. Bacteria were inoculated in LB and were cultured overnight.The clones were measured for the diameter of the swarming zone.The statistical analysis was done using one-factor ANOVA.Results Strains of pvdQ-overexpression and pvdQ-mutant were successfully constructed and confirmed by polymerase chain reaction(PCR).The four strains were compared for the swarming motility by changes in diameter:PAO1(20.52±1.80)mm,pME6032-PAO1 strain(19.39±2.10)mm,pvdQ-overexpression strain(51.20±2.16)mm,pvdQ-mutant strain(3.30±0.55)mm.The diameter of pME6032-PAO1 strain was not significantly different from that of wild strain PAO1(t=-0.1493,P>0.05).However,the diameter of pvdQ(Q-mutant strain was significantly shorter than that of wild strain PAO1(t=2.8525,P<0.05).while the diameter of pvdQ-overexpression strain was longer than that of the wild strain PAO1(t=1.4230,P<0.05).Conclusions pvdQ gene may be involved in regulating the swarming motility of Pseudomonas aeruginosa,which can promote Pseudomonas aeruginosa swarming motility.

Objective To investigate the efficacy of small interfering RNA against Pseudomonos aeruginosa expressing MexA-MexB-OprM multidrug efflux pump in vivo.Methods Two short hairpin (sh)RNA expression vectors targeting the MexB gene,and negative controls,were designed,synthesized,and electrotransformed into the P.aeruginosa strain PAO1.The in vivo therapeutic efficacy of the MexB small interfering (si)RNAs was determined by infecting a murine model of chronic P.aeruginosa lung infection (1 × 107 CFU/ml).The mice were killed on day 3,5 and 7 after infection with the Pseudomonas aeruginosa strains.Results In the murine infection model,treatment with MexB-siRNAs led to significantly reduced bacteria burden of the bellows by day 5 and 7 post-infection,and reduced the P.aeruginosa-induced pathological changes.In addition,MexB-siRNA2 treatment enhanced neutrophil recruitment and production of inflammatory cytokines (IL-1β,IL-12) in the early infection stage (day 3) (P＜0.05),both of which decreased by day 7.Conclusion MexB-siRNA could inhibit both mRNA expression and the activity of P.aeruginosa in vitro.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.

Objective To investigate the effects of NOD2 on inflammatory responses of macrophages to Staphylococcus aureus. Methods Real-time RT-PCR detected NOD2 gene expression of macrophages infected by S. aureus. Synthesis of siRNA against NOD2 and interfere with macrophages, observed the effects of NOD2 gene silencing to phagocytosis of 5. aureus, cytokine secretion, activation of nuclear transcription factors, cell apoptosis of the macrophages infected by S. aureus using F.I .IS A, flow cytometry etc. Results S. aureus infection of macrophages can cause increased expression of intracellular NOD2. NOD2 gene silencing of macrophage lead to the decreased ability of phagocytosis with S. aureus, the lower levels of cytokines secretion, deficiencies of NF-κB activation. S. aureus can cause macrophage apoptosis, with the apoptosis rate increased with time. Conclusion The intracellular pattern recognition receptor NOD2 play a key role in pathogen recognition, signal transduction, activation of nuclear transcription factors in the process of macrophages infected by S. aureus.

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.

Objective To investigate the effect and mechanism of siHybrids technique on inhibiring the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro. MethodsTargeting the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 ,we designed and synthesized three siHybrids molecule and one negative scamble siHybrids molecule. Pseudomonas aeruginosa PAO1 were intervened by the siHybrids molecules in 50 nmol/L, respectively. And the experiments were made of control groups[blank and scamble (sc) -001]and intervened groups[siHybrids( si ) -001, siHybrids( si ) -002 and siHybrids(si) -003]of Pseudomonas aeruginosa PAO1. The targeting efflux pump gene mexB mRNA expressions of Pseudomonas aeruginosa PAO1, including all groups, were measured by real-time PCR in 12 h and 24 h after interference in vitro. Further, the minimal inhibitory concentration (MIC) of chlormycetinCP, erythrocin( EM ), levofloxacin ( L-OFLX), ceftazidime ( CAZ), meropenem (MER) to those groups were detected by using Mueller-Hinton broth dilution before and after interference. ResultsThe relative mexB mRNA amounts of Pseudomonas aeruginosa PAO1 intervened by different siHybrids were not much more different from each other after 12 h,but the expression of mexB mRNA of the intervened group ( si-001 ,si-002 ,si-003 ) was much lower than control groups after interference for 24 h. The relative mexB mRNA amounts, comparing 12 h with 24 h, we would find the blank control and negative control submit escalating trend. And the intervened control ,three different siHybrids were all with a downward tendency. However, in presence of 50 nmol/L siHybrids, the minimal inhibitory concentration(MIC) of CP, EM, L-OFLX, CAZ, MER to those controls were not much more different before and after interference. Conclusion On level of the mRNA expressions, siHybrids could inhibit the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro, and within 24 h would be able to function effectively. Further, the effect was time-dependent.

ObjectiveTo investigate the alteration of MexB protein expression by plasmid containing siRNA template strand was transformed into Pseudomonas aeruginosa.Methods siRNA molecules specifically against mexB were designed and ligated into pGPU6/GFP/Neo vector to construct the recombinant plasmid pGPU6/GFP/Neo-siRNA.MexB gene was cloned into expression vector pET22b+ to construct plasmid pET22b+/mexB,the recombinant expression plasmid was transformed into E.coli BL21 (DE3)plysS and protein MexB was induced to express,then purified protein MexB was used to prepare specific antibodies in rabbits.The plasmids with siRNA molecules specifically against mexB were transformed into wild type strain,clinical multiresistant strain and mexB overexpression strain of Pseudomonas aeruginosa by electroporation respectively,and the changes of the expression of MexB were detected in 8,12,24 h respectively by Western blot.ResultspGPU6/GFP/Neo-siRNA was constructed successfully.Protein MexB was expressed successfully and the rabbit polyclonal antibodies against MexB was prepared well.The gene silence of mexB by siRNA molecules was effective in the three strains of Pseudomonas aeruginosa,but it depended on the silencing time.ConclusionThe expression of MexB was reduced in 8 h and 12 h,but in 24 h,the expression was unchanged.

Objective To investigate whether Pseudomonas aeruginosa pvdQ( PA2385 ) gene reveals altered antibiotic susceptibility under swarming conditions. Methods The plasmid pME6032 with pvdQ gene was constructed and identified, then transformed into Pseudomonas aeruginosa PAO1 by the electroporation, building pvdQ overexpression strain. Using the same method building pME6032-PAO1 strain.Bacteria were inoculated in LB overnight , measuring the colony diameter of the swarming zone . Results Strains of pvdQ overexpression was successfully constructed by real-time PCR. Comparison of two strains of the swarming motility of change in diameter: The result showed that PAO1 and pvdQ overexpression strains can both improve the antibiotics resistance. Swarmer cells of pvdQ overexpression strain exhibited a 2- to 4-fold increase in antibiotic resistance toward ceftazidime,ciprofloxacin, meropenem and polymyxin B compared to PAO1 on BM2-swarming agar plates. Conclusion pvdQ gene played an important role in elevating the antibiotics resistance, which through prarticipated in the swarmer cell differentiation.

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.
Animals ; Cell Line ; Cytokines ; immunology ; Inflammation Mediators ; immunology ; Mast Cells ; immunology ; microbiology ; Mice ; NF-kappa B ; immunology ; Nod2 Signaling Adaptor Protein ; immunology ; Staphylococcus aureus ; physiology