Inhibition Of Gene Expression And Growth Of Multidrug-resistant Acinetobacter Baumannii By Antisense Peptide Nucleic Acids In Vitro

PART1SCREENING OF EFFECTIVE ANTISENSEOLIGONUCLEOTIDES TARGETING THE GYRA MRNAOF MULTIDRUG-RESISTANT ACINETOBACTERBAUMANNIIObjective: To screen the effective antisense oligonucleotides targetingthe gyrA mRNA of multidrug-resistant Acinetobacter baumannii. Methods:Two RNA folding computer programs, Mfold and RNA structure4.6wereused to predict the secondary structure of gyrA mRNA, ten antisenseoligonucleotides were designed based on free energy theory. The full lengthgyrA mRNA was transcribed in vitro and labeled bydigoxigenin-11-uridine-5’-triphosphate. Dot blot hybridization was used toscreen the gyrA mRNA accessible sites which showed strong bindingaffinity to the antisense oligonucleotides. Results: Of the ten antisenseoligonucleotides,five showed binding affinity to gyrA mRNA and one of them showed strong binding affinity. Conclusion: Computer programprediction can rapidly screen antisense sequence of target mRNA, dot blothybridization can be applied to evaluate the efficiency of the antisensesequence in vitro by simulating vivo environment. Combination ofcomputer prediction with dot blot hybridization is a high-flux,rapid wayfor screening of effective antisense oligonucleotides in vitro. PART2INHIBITION OF GENE EXPRESSION ANDGROWTH BY ANTISENSEPEPTIDE NUCLEIC ACIDSTARGETING GYRA IN A MULTIDRUG-RESISTANTACINETOBACTER BAUMANNIIObjective：To investigate the inhibition effect of the peptide nucleicacids targeting gyrA on gene expression and growth of multidrug-resistantAcinetobacter baumannii in vitro. Methods：The synthesis of PNA wasbased on the sequence of the selected oligonucleotides which described inPart1(PNA1). Meanwhile, we designed another PNA targeting the startcodon region of gyrA mRNA, including Shine-Dalgarno (SD) sequenceand AUG (PNA2). Both of the PNAs were synthesized conjugated to the (KFF)3K peptide formed peptide-PNA1(PPNA1) andpeptide-PNA2(PPNA2). CY-623cell cultures were treated by differentconcentrations of PPNA1and PPNA2respectively, compared to treatmentwith PNA without peptide (PNA1) or with the mismatched antisense PPNA(PPNA3). After various hours of incubation, the absorbance of OD600andviable cell counts were measured and total RNA was extracted to evaluatethe gyrA mRNA expression level by reverse transcript (RT)-PCR.Results：Both of the two PPNAs showed significantly inhibition effect ongyrA gene expression and cell growth in a dose-dependent manner. Thelowest inhibitory and bactericidal concentrations of PPNA1and PPNA2were5μmol/L,10μmol/L, respectively. Combination use of the two PPNAsshowed superimposed effect other than synergistic effect. PNA1andPPNA3which used as controls had no growth inhibition effects.Conclusion：The PPNA targeted at gyrA gene could inhibit the growth ofCY-623, and it could be a new therapeutic strategy for multidrug-resistentAcinetobacter baumannii infection.