Construction Of Antisense Peptide Nucleic Acid Of Pseudomonas Aeruginosa Las Quorum Sensing System And Study On Its Biological Effects

Objective1.Targeting on the lasI/lasR gene of the Pseudomonas aeruginosa quorum sensing system,an antisense oligonucleotides integrated closely with its mRNA was designed and screened for synthesizing the peptide nucleic acid.2.To evaluate the effect of peptide nucleic acid on the growth,the expression of lasR and lasI gene,biofilm formation and virulence factor expression of Pseudomonas aeruginosa PAO1.Methods1.Design and screening for antisense peptide nucleic acid of Pseudomonas aeruginosa PAO1 lasR and lasI gene1.1.Synthesis and Screening of antisense oligonucleotides of Pseudomonas aeruginosa PAO1 lasR and lasI gene1.1.1 Pseudomonas aeruginosa PAO1 DNA was extracted from the Bacterial Genomic DNA Extraction Kit.According to the GeneBank database,with Software Primer Primer 6.0 design primers for lasR and las I gene PCR amplification.The products of amplified lasR and lasI gene were ligated with the plasmid pGEM-T easy vector.1.1.2 Validated of the recombinant plasmid pGEM-T easy vector-lasR/lasIFirst,the preparation and transformation of competent DH5? were carried out,followed by screening of lasR and lasI positive clones.The recombinant plasmid pGEM-T easy vector-lasR/lasI was extracted and the recombinant plasmid pGEM-T easy vector-lasR/lasI Not I digested.1.1.3 Design of lasR and lasI mRNA antisense oligonucleotide sequences using software RNA structure 5.21.1.4 Screening of Pseudomonas aeruginosa PAO1 lasR and lasI gene antisense oligonucleotidesThe recombinant plasmid pGEM-T easy vector-lasR/lasI was opened from the closed loop to a linear state,followed by lasR/las I m RNA transcription in vitro and dot blot hybridization assay for digoxigenin-labeled lasR and lasI gene mRNA transcription product sensitivity,and finally the dot blot hybridization experiments were performed for sreening lasR and lasI gene mRNA antisense oligonucleotide sequences.1.2.Design and synthesis of antisense peptide nucleic acids(asPNA)based on the selected antisense oligonucleotide sequences.2.Study on biological effects of Pseudomonas aeruginosa PAO1 lasR and las I gene antisense peptide nucleic acid2.1.Effect of antisense peptide nucleic acid on the expression of PAO1 lasR and las I gene in Pseudomonas aeruginosa by qPCR.2.2.OD600 determination and colony count detection of antisense peptide nucleic acid on the growth of Pseudomonas aeruginosa PAO12.3.The effect of antisense peptide nucleic acid on the formation of Pseudomonas aeruginosa PAO1 biofilm was observed by optical microscopy,scanning electron microscopy and laser scan confocal microscopy.2.4.ELISA to detect the effect of antisense peptide nucleic acid on virulence factors of exotoxin A and pyocyanin.The expression of elastase B was detected by qPCR.Result1.Design and screening for asPNA of Pseudomonas aeruginosa PAO1 lasR and las I gene1.1.The recombinant plasmid pGEM-T easy vector-lasR/lasI was constructed successfully,and the antisense oligonucleotide sequences were selected to bind to Pseudomonas aeruginosa PAO1 lasR and lasI m RNA closely.1.2.Synthesis of asPNA)and antisense peptide-peptide nucleic acids(asPPNA)bound to cell-penetrating peptides(CPPs)based on screened antisense oligonucleotide sequences.2.Study on biological effects of Pseudomonas aeruginosa PAO1 lasR and las I gene antisense peptide nucleic acid2.1.The results of OD600 assay and colony count showed that lasR/las I-as PPNA have a significant inhibitory effect on the growth of Pseudomonas aeruginosa at a concentration of be equal or greater than 40?M,The higher the concentration,the more obvious the inhibitory effect on growth.While the CPPs and the asPNA itself have no significant inhibitory effect on the growth of the bacteria.2.2.The expression of lasR/lasI mRNA was inhibited by lasR/las I-asPPNA,and the expression of lasR/lasI mRNA was enhanced with the increase of asPPNA concentration compared with the control group.2.3.Optical microscopy,scanning electron microscopy and laser scan confocal microscopy showed that the lasR/lasI-asPPNA inhibited the formation of Pseudomonas aeruginosa PAO1 biofilm.2.4.Virulence factors of elastase B,pyocyaninin and exotoxin A were down-regulated by the lasR/lasI-asPPNA,and the difference was significant compared with the control group statistically.Conclusion1.RNA structure 5.2 design and in vitro hybridization experiments can screen out the target gene lasR/las I mRNA effectively integrated antisense oligodeoxynucleotide sequence closely,and the synthesis of asPNA and conjuncted to CPPs successful.2.The expression of lasR and lasI mRNA was inhibited by asPPNA significantly,and achieved the quenching of Pseudomonas aeruginosa las quorum system.3.Biofilm formation,which plays an important role in the pathogenesis of Pseudomonas aeruginosa PAO1,is also inhibited by lasR/las I-asPPNA and its possible mechanism is that through the quenching las quorum sensing system.4.Growth of bacteria was inhibited,the expression of the virulence factors pyocyanin?exotoxin A and elastase B down regulated with as PPNA.5.The asPPNA designed in this study can inhibit the growth significantly and the expression of virulence-related factors by quenching las quorum sensing system,which offers new ideas for treatment infection of Pseudomonas aeruginosa.