Interference with gene expression in twitching motility of Peudomonas aeruginosa by RNAi

RNAi干扰铜绿假单胞菌抽搐运动基因表达

• 批准号: 18590860
• 负责人: KADOTA Junichi
• 金额: $ 2.46万
• 依托单位: Oita University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590860/
• 关键词: Pseudomonasaerurinnasa; Twitching motility; RNAi; Biofilm; Th1; Th2

Twitching motility of Pseudomonas aeruginosa ha s potent virulence in chronic respiratory infection. We estimated whether the siRNA targeted twitching motility of P aeruginosa was useful in the treatment. We designed the siRNA targeted piIH piII, pig piIK and pilA using the software in the web site (http: //design.rnai.ip) and ordered their synthesis from SIGMA Genosys siRNA service. 2mM of each siRNA was exposured to 2.5×10^5 of. P. aeruginosa PAO-1 in LB broth for 4h at 37℃. The ability of twitching motility were estimated by stabbing the cells with toothpick to the bottom of the 2mm thick agar plate (1.5%) and incubating for 48h at 37℃. The maximum radius of gray-hazy zone around of the colony (twitching zone)was measured as twitching motility. Only the siRNA targeted pilK had suppressive effect (79±9% to control) significantly. For more active exposure of siRNA targeted pilK to P aeruginasa PAO-1, we proposed to use the electroporation method. 2mM of siRNA targeted pilK was mixed with 40μl of the concentrate of P aeruginosa PAO-1 (2×10^<10>cfu/ml)in PBS with 10% glycerol in a cubet and pulsed 2.5kV electricity using Gene Pulser (Bio Rad). Immediately the cells were cultured in SOC medium for 1h at 37℃ and estimated the twitching motility as above. Only 60% of resulted cells were suppressed the twitching motility. We attribute the failure of introducing the siRNA to the bacterial cytoplasm to the thicker cell wall of Paerugiaosa was than that of Escherirhie coli.

铜绿假单胞菌的抽搐运动在慢性呼吸道感染中具有很强的毒力。我们评估了靶向铜绿假单胞菌抽搐运动的siRNA是否在治疗中有用。我们使用网站（http：//design.rnai.ip）中的软件设计了靶向piIH piII、猪piIK和pilA的siRNA，并从SIGMA Genosys siRNA服务订购了它们的合成。将2 mM的每种siRNA稀释至2.5×10^5。铜绿假单胞菌PAO-1在LB肉汤中于37℃下培养4 h。用牙签刺入2 mm厚的琼脂平板底部（1.5%），37℃孵育48 h，测定抽搐运动能力。测量殖民地周围灰色模糊区（抽搐区）的最大半径作为抽搐运动性。只有靶向pilK的siRNA具有显著的抑制效果（相对于对照的79±9%）。为了使siRNA靶向的pilK更主动地暴露于铜绿假单胞菌PAO-1，我们提出使用电穿孔方法。将2 mM的siRNA靶向的pilK与40μ 1的铜绿假单胞菌PAO-1的浓缩物（2× 104 <10>cfu/ml）在含有10%甘油的PBS中在立方体中混合，并使用Gene Pulser（Bio Rad）脉冲2.5kV电。立即将细胞在SOC培养基中在37℃下培养1小时，并如上所述估计抽搐运动。只有60%的细胞的抽搐运动受到抑制。我们将siRNA导入细菌细胞质的失败归因于Paerugiaosa的细胞壁较厚。