The Study On Antisense Peptide Nucieic Acid Targeting MOTA Gene Inhibiting Biofilm Formation Of Pseudomonas Aeruginosa PAO1 In Vitro

PART1 SCREENING OF ANTISENSE OLIGONUCLEOTIDES TARGETING THE MOTA MESSAGE RNA OF PAO1 BY COMPUTER PROGRAMME PREDICTION AND DOT BLOT HYBRIDIZATIONObjective: To screen the antisense oligonucleotides targeting the motA message RNA (mRNA) of PAO1 by full length gene targeting, which bind to the mRNA strongly. Methods: Sequences of antisense oligonucleotide binding closely to the key gene motA for the early biofilm formation of Pseudomonas aeruginosa PAO1 were designed using Mfold software/RNA structure4.6 and dot blot hybridization. The second structure of motA mRNA was predicted by computer programme Mfold and RNA Structure 4.6. Six antisense oligonucleotides were designed based on the minimal free energy theory, and another two negtive antisense oligonucleotides were designed. The motA gene was cloned into T vector and then transcribed in vitro. In the process of in vitro transcription, digoxigenin-11-uridine-5'-triphosphate was incorporated to label the mRNA. And then dot blot hybiridization was used to screen the motA mRNA accessible sites which showed strong binding affinity to the antisense oligonucleotides. Results: The full motA gene was obtained by PCR, and four antisense oligonucleotides showed binding affinity to the motA mRNA in vitro. Conclusion: The antisense oligonucleotides showed strong binding affinity were obtained. And the further studies will focus on the mechanism of biofilm formation accomdated by antisense technology targeting the motA mRNA of PAO1. PART 2 THE INHIBITION EFFECT OF PEPTIDE NUCLEIC ACID ON BIOFILM FORMATION OF PSEUDOMINAS AERUGINOSA PAO1 IN VITROObjective: To study the inhibitory effect of peptide nucleic acid (PNA) on biofilm formation of Pseudomonas aeruginosa PAO1in vitro. Methods: PNA was synthesized according to the sequence of antisense oligo- nucleotide which was chosen in the part 1.The PNA was linked to the cell penetrating peptide (CCPs) (KFF)3K to form CCPs-PNA. Pseudomonas aeruginosa PAO1 was treated with CCPs, PNA and CCPs-PNA at different concentrations, and the inhibitory effect of PNA on biofilm formation of Pseudomonas aeruginosa PAO1 was quantitatively detected and observed under a microscope. The inhibitory effect of PNA on motA gene expression and motility was observed simutaneously. Results: PNA and CCPs-PNA inhibited the biofilm formation of Pseudomonas aeruginosa PAO1in a dose-dependent manner. The inhibitory effect of CCPs-PNA was better than that of PNA on biofilm formation of Pseudomonas aeruginosa PAO1. The inhibitory rate of (KFF)3K-PNA was 30%, 50% and 70%, respectively, for the biofilm formation of Pseudomonas aeruginosa PAO1 at the concentration of 1,5 and 10μmol/ L. The inhibitory rate of PNA was 3% and 10%, respectively, for the biofilm formation of Pseudomonas aeruginosa PAO1 at the concentration of 5μmol/L and 10μmol/L. No effect of CCPs at different concentration was observed on the biofilm formation of Pseudomonas aeruginosa PAO1. The expression of motA was sharply reduced when treated with (KFF)3K-PNA, but reduced slightly when treated with PNA, and had no reduction when treated with CCPs. The motility of Pseudomonas aeruginosa PAO1 was significantly decreased after treated with CCPs-PNA. The PAO1 radial diameter ratio was 3:5 between (KFF)3K-PNA treatment group and control group. Conclusion: The PNA targeting motA gene can inhibit the biofilm formation of Pseudomonas aeruginosa PAO1 at the initial stage by suppressing its motility and adsorption, which can be greatly increased by (KFF)3K , suggesting that the motA gene or its encoded product can be used as a good target to prevent biofilm formation of Pseudomonas aeruginosa.