Targeting Rna Polymerase Primary σ⁷⁰ As A Therapeutic Strategy Against Methicillin-resistant Staphylococcus Aureus By Antisense Peptide Nucleic Acid

AIMStaphylococcus aureus （SA）, an important pathogenic bacteria belonged tothe Staphylococcus genus, is widely distributed in nature, causing a variety ofinfections, such as skin infections, postoperative wound infections, pneumoniaand sepsis. After the advent of penicillin in the1940s, infections caused by SAhave been well controlled, but some SA strains developed resistance topenicillin not long after the application of penicillin. Penicillin resistance in SAhas become increasingly serious with the extensive use of penicillin. In1959,researchers developed methicillin, a penicillinase-resistant drug which once inclinical application showed effective infection control of SA strains producingpenicillinase. However, in1961methicillin-resistant Staphylococcus aureus（MRSA） was first isolated in United Kingdom, and MRSA infections has beenobserved throughout the world ever since. Currently, the most effectivetherapeutic for MRSA is vancomycin, whereas vancomycin-resistant MRSAstrains have been isolated around the world since1997. Therefore, it is urgentto find a new strategy for the effective control of MRSA infections.In order to effectively combat the infections caused by MRSA, we mustfind a new breakthrough. Unlike classical antibiotics targeting proteins in bacteria, antisense antibacterial agents, generally described as RNA silencers,are synthetic nucleic acid oligomer mimetics to specifically inhibit essentialgene expression in bacteria and achieve gene-specific antibacterial effects,representing one promising strategy with fundamenatal breakthrough. Previousstudies have confirmed that antisense antibacterial agents demonstrated reliablegene-specific antisense growth inhibitory effects.The molecular weight of antisense molecule is too large to be effectivelyuptaken by bacterial cells. Thus, we aimed to discover a gene target thatshows potential for developing potent antisense antibacterial agents; and tostudy the delivery strategy that improves the cellular uptake of antisenseantibacterial agents. Based on the above aims, we design and synthesize a set ofcell penetrating peptide （CPP） conjugated PNAs （PPNAs） targeting the rpoDgene in SA (encoding RNA polymerase σ⁷⁰) and evaluate their antibacterialactivities in vivo to confirm their efficacy.METHODS1. Design and synthesis of anti-rpoD PPNAsSequence alignment of rpoD gene among different SA strains （includingthe resistant strains） obtained from Genebank was performed by BLAST.Secondary structure of SA rpoD mRNA predicted by RNA structure4.6software, and Oligo walk5.0and PNA Tm software were used to analyze thetargeting sites in mRNA and calculate the binding parameters forcomplementary antisense PNAs. Based on the parameter analysis, severalanti-rpoD antisense PNA sequences with conjugated peptide （anti-rpoD PPNAs）were selected and then automatically synthesized by Fmoc chemistry on solidphase supporter. Together, mismatched PPNA, naked PNA and peptide ascontrols were also synthesized and purified by RT-HPLC. The molecular weightof crude product was confirmed by MALDI-TOF.2. in vitro screening and optimization of effective targeting sites for anti-rpoD PPNAs.SA standard strain ATCC29213, MRSA strains of WHO-2and Mu50, andthe multidrug-resistant MRSA strain isolated by the department of LaboratoryMedicine, Xijing Hospital were used as subjects. And the most potent anti-rpoDPPNA was screened out by modified minimal inhibitory concentration （MIC）test, and further modified by a set of optimized designs including orientationexchange of CPP and PNA conjugation, adding spacer between PNA and CPP,and cutting PNA length. In vitro potency screening was carried out thereafter foroptimized PPNAs.3. in vitro antibacterial activity evaluation of screened-out anti-rpoDPPNA.MRSA strain Mu50was used as subject, and the antibacterial activity ofscreened-out anti-rpoD PPNA was further evaluated by a set of tests, includingdose-effect dependence curve and live bacteria colony forming unit （CFU）.Target specificity and antibacterial mechanism of the screened-out anti-rpoDPPNA was demonstrated by comparing expression of gene rpoD at mRNA andprotein levels in inhibited and uninhibited bacterial cells determined by RT-PCRand western blotting.4. Protective effect of anti-rpoD PPNA on human gastric epithelial cellinfected by MRSA Mu50.Human gastric epithelial cell was co-cultured with MRSA Mu50toestablish the cell infection model. Different concentrations of anti-rpoDPPNA2332were added together with mismatched PPNA2332, peptide2332andgrowth control. Bacterial accounts were determined for each group at a2hinterval and cell morphology was observed under optical microscope at24h toexamine the protective effect of anti-rpoD PPNA2332on infected eukaryoticcells.Results 1. Design and synthesis of anti-rpoD PPNAs.The homology analysis results demonstrated that rpoD gene encodingbacterial RNAP primary σ70is highly similar in sequence among SA strains,indicating its highly conserved characteristic as an ideal antisense target.Unstable regions like the expanding rings were selected as possible targetingsites in rpoD mRNA predicted by RNA structure4.6software. And fiveplausible PNA sequences were identified based on the optimal bindingparameters predictively calculated by Oligo walk5.0and PNA Tm software.Together, mismatched PPNA, naked PNA and peptide as controls wereautomatically synthesized by Fmoc chemistry.2. in vitro screening and optimization of effective targeting sites foranti-rpoD PPNAs.The effect of5anti-rpoD PPNAs was determined by measuring MIC inliquid culture. The results demonstrated that PNAs complementary to the SAmRNA nucleotides （nt） encoding233to244（anti-rpoD PPNA233） showed themost potent growth inhibitory effect, as illustrated by the lowest MIC values on4SA strains （including ATCC29213, Mu50, WHO-2, and MRSA Xijing）. Toincrease the antibacterial potency of PPNA233, we further modified its designby optimized methods including orientation exchange of CPP and PNAconjugation, adding spacer between PNA and CPP, and cutting PNA length.MIC test was carried out thereafter to screen out anti-rpoD PPNA2332as themost potent conjugate, with an equal MIC value of12.5μM against four SAstrains.3. in vitro antibacterial activity evaluation of screened-out anti-rpoDPPNA.The results from growth curve test and colony forming unit （CFU） testdemonstrated that anti-rpoD PPNA2332showed excellent antibacterial activity,which realized full-course growth inhibition at≥MIC value and bactericidaleffect at40μM, as compared with growth control. RT-PCR and western blotting results showed that level of σ⁷⁰-specific mRNA in SA Mu50was greatlydiminished in PPNA2332treated cultures compared with untreated cultures, aswas also the case with the level of the σ⁷⁰protein, in a concentration-dependentmanner.4. Protective effect of anti-rpoD PPNA on human gastric epithelial cellinfected by MRSA Mu50.Optical microscope observation showed that anti-rpoD PPNA2332did notvisibly affect epithelial cell growth at the highest concentration tested （10μM）,indicating no cell toxicity. It appeared to fully cure the culture of MRSA Mu50infection at1μM or higher by exerting bactericidal antisense effect on bacterialcells, protecting epithelial cell from morphological lesion and death caused byMRSA Mu50infectionConclusion1. Bacterial RNAP primary σ⁷⁰is verified as a highly promising target forantisense antimicrobial drug development against pathogenic MRSA strains.And a conserved target sequence （233-242nt） within the native SA rpoD mRNAis identified for potent antisense PNA targeting.2. CPP （KFF）₃K was successfully used to conjugate with anti-rpoD PNA,showing high efficiency in mediating the delivery of PNA into SA. Theanti-MRSA activity of PNA2332and its protective effect on eukaryotic cellswas demonstrated and verified by in vitro and ex vivo tests.