Evaluation of the Pathogenesis of Pseudomonas aeruginosa's Flagellum Before and After Flagellar Gene Knockdown by Small Interfering RNAs (siRNA)

Pseudomonas aeruginosa possesses a polar flagellum made up of flagellar subunits, which are encoded by fliC gene. Flagella have important roles in the motility, chemotaxis, and establishment of P. aeruginosa in the acute phase of infections. The inhibition of flagellar expression may be a promising therapeutic approach to prevent the pathogenesis. The gene-silencing effect of siRNA may be useful for this strategy. The current study investigated the efficacy of siRNA on the expression of flagellin, because it is an important protein in the initial stages of P. aeruginosa infections. The current research designed and synthesized 21 bp siRNA duplexes against P. aeruginosa flagella. Quantitative RT–PCR was performed to determine whether the siRNAs inhibit the expression of the flagellin mRNA in vitro. The efficacy of siRNA was determined by the motility test and in a murine model of hematogenous pulmonary infection. In quantitative RT–PCR,it was shown that the siRNA significantly inhibited the expression of the flagella mRNA. FilC gene knockdown by the siRNA resulted in a significant decrease in the expression of the flagellar mRNA in the siRNA group as compared with that of the control (P < 0.05). In the motility test,the motility was inhibited in the siRNA group more effectively than in the control group. In the murine infection model, a significant decrease in the number of viable bacteria was detected in the siRNA group when compared with the control (7.87 ± 0.54 in the former versus 4.69 ± 0.35 log cfu/mL in the latter mean ± SD, P < 0.05). The development of delivery systems into bacteria with an efficacy compatible to that in human use could be a key for the potential utility of siRNA for the prophylaxis and treatment of P. aeruginosa-induced hematogenous pulmonary infections in humans.