Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin-resistant Staphylococcus aureus by antisense peptide nucleic acid.
Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin-resistant Staphylococcus aureus by antisense peptide nucleic acid.
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DOI：
10.1371/journal.pone.0029886
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Luo X
中科院分区：
文献类型：
--
作者：
Bai H;Sang G;You Y;Xue X;Zhou Y;Hou Z;Meng J;Luo X
耐甲氧西林金黄色葡萄球菌（MRSA）在全球范围内引起威胁性的感染相关死亡率。目前，多药耐药性（MDR）MRSA的传播限制了治疗选择，需要新的方法来发现“可药物化”的靶点，以及开发新的MRSA活性抗生素。RNA聚合酶初级σ70（由基因rpoD编码）是一个高度保守的原核因子，在不同的S.金黄色葡萄球菌，暗示反义抑制的潜力。通过软件预测参数合成一系列细胞穿透肽结合肽核酸（PPNAs），并进一步优化设计，我们在rpoD mRNA保守区3.0内发现了一个对反义抑制更敏感的靶序列（234 ~ 243 nt）。开发了一种（KFF）3 K肽缀合的10聚体互补PNA（PPNA 2332），用于有效的微摩尔范围内对四种致病性S.具有不同耐药表型的金黄色葡萄球菌，包括临床上对万古霉素中等耐药的金黄色葡萄球菌。金黄色葡萄球菌和MDR-MRSA分离株。PPNA 2332在对MRSA/VISA Mu 50的MIC值的3.2倍下显示出杀菌反义作用，并且其序列特异性被证明为具有乱序PNA序列的PPNA（Scr PPNA 2332）在较高浓度下不表现出生长抑制作用。此外，PPNA 2332特异性干扰rpoD mRNA，以浓度依赖性方式抑制其蛋白产物σ70的翻译。在MRSA/VISA Mu 50菌株的纯培养物中，40 µM或12.5 µM PPNA 2332处理分别观察到mRNA完全衰减和σ70表达抑制，但40 µM Scr PPNA 2332处理未观察到。PPNA 2332（≥1 µM）基本上清除了上皮细胞培养物中的致死性MRSA/VISA Mu 50感染，并以时间和浓度依赖性方式消除了活细菌细胞，在10 µM时未显示任何明显毒性。目前的结果表明RNAP主要σ70是一个非常有前途的候选目标，为开发新的反义抗生素，以治疗严重的MRSA感染。
Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to “druggable” target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ70 (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ70 were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. The present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.
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