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

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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.