Suppression Of Virulence Factors Of Pseudomonas Aeruginosa By Artificial Trans-encoded Srna

As a common clinical opportunistic pathogen, Pseudomonas aeruginosa requires the discovery of new therapeutic approaches for its resistance to available antibiotics. Targeting bacterial virulence is a novel approach of antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacteria. Certain virulent factors and pathogenic mechanisms have been shown to be potential targets for drug design and therapeutic intervention. Targeting virulence represents new opportunities and challenges for treating bacterial infectious diseases more effectively.Small noncoding RNAs (sRNAs) are widespread in prokaryotes, primarily as regulators of translation and message stability with the participation of RNA chaperone Hfq. Their lengths are between 50-200 nucleotides and they play an important regulatory role in bacteria metabolism, environmental adaptation, quorum sensing and bacterial virulence and other biological functions. In eukaryotes, RNA interference (RNAi) has become an extraordinarily powerful RNA silencing tool for treating diseases. In our previous study (Dr. Cheng Rubin, etc.), we firstly developed the principle and process for artificial trans-encoded sRNAs (atsRNAs) design based on the common structural characteristics of the natural bacterial sRNAs and used successfully atsRNAs for specific gene silencing in E.coli. This work aims to use artificial tyans-encoded sRNAs in Pseudomonas aeruginosa to specifically interfere the expression of virulent factors, which provides a promising approach to treat infectious disease.Firstly, based on the common structural characteristics of the endogenous small RNAs in Pseudomonas aeruginosa, we developed the principle for atsRNA design in Pseudomonas aeruginosa. The virulent factors elastase and exotoxin A were used as targets to design SRLB atsRNAs and SRTX atsRNAs. The pRPA vector was used to overexpress atsRNAs in Pseudomonas aeruginosa and the results indicated that SRLB2 yielded a 75% reduction on elastase production, and a 34.9% reduction on lasB mRNA; SRTX1 yielded a 30.6% reduction on toxA mRNA.Secondly, another virulence pyocyanin, synthesized by phzABCDEFG operon of Pseudomonas aeruginosa, was used as a target to design SRphF and SRpzA atsRNAs, PhzF is the sixth gene in phz operon and encodes a key enzyme for synthesizing pyocyanine; phzA is the first gene and closely linked with phz promoter. The results showed that SRphF atsRNAs failed to reduce pyocyanin production, and as the best one of all SRphF atsRNAs, SRphF2 only yielded a 15.8% reduction on pyocyanin production; On the contrary, SRpzA2 yielded a 56% reduction and SRpzA3 yielded a 37% reduction. This illustrated thatthe first gene's RBS in operon might be the best target region, when atsRNAs were designed to interfere the factor controlled by an operon. Based on these results, a target-fluorescence fusion vector pACYC-T7-phzF-EGFP was constructed and co-expressed with SRphF atsRNAs in E.coli BL21. Fluorescence results showed SRphF atsRNAs, which failed to interfere phzF in Pseudomonas aeruginosa, inhibited the expression of phzF effectively in E.coli BL21. SRphF2 caused a 83.5% reduction on phzF gene expression, which was much higher than 15.4% in Pseudomonas aeruginosa. This indicated that there were some differences between the two bacteria in atsRNA's effect.Finally, we designed SRlaR and SRrhR atsRNAs targeting Pseudomonas aeruginosa quorum sensing system regulator RhlR and LasR. RT-PCR indicated SRrhlR4 yielded a 79% reduction on rhlR mRNA, and a 37% reduction on elastase production, which has been reported to be controlled by RhlR; SRlaR3 yielded a 63% reduction on lasR mRNA, and a 32.7% reduction on elastase production. Recent studies have revealed that bacterial quorum sensing system can be a potential new anti-infection target to control bacterial pathogenicity. We made use of SRlaR and SRrhR atsRNAs to down-regulate rhlR and lasR expression successfully, and reduce elastase production to some extent. In conclusion, a primary method was developed for inhibiting expression of Pseudomonas aeruginosa virulence factors by using atsRNAs, Virulent fators controlled by a single gene, an operon or quorum sensing system were effectively suppressed, and Pseudomonas aeruginos pathogenesis were weakened to some extend. This method brings forward a new approach for preventing the spread of drug-resistant strains and researching a new generation of anti-infective drugs.