Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

et al. (2012) Targeting RNA Polymerase Primary s70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid. PLoS ONE 7(1): e29886. doi:10.1371/journal.pone.0029886 70 Targeting RNA Polymerase Primary s as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid Hui Bai 0 Guojun Sang 0 Yu You 0 Xiaoyan Xue 0 Ying Zhou 0 Zheng Hou 0 Jingru Meng 0 Xiaoxing Luo 0 Stefan Bereswill, Charite-University Medicine Berlin, Germany 0 1 Department of Pharmacology, School of Pharmacy, Fourth Military Medical University , Xi'an, Shaanxi, China, 2 No. 451 Hospital, Xi'an, Shaanxi , China , 3 Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences , Beijing , China , 4 Department of Neurosurgery, No. 309 Hospital , Beijing , China Background: 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 s70 (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. Methodology/Principal Findings: 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 s70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of s70 were observed for 40 mM or 12.5 mM PPNA2332 treatment, respectively, but not for 40 mM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 ($1 mM) 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 mM. Conclusions: The present result suggested that RNAP primary s70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections. - Funding: This work was supported by National Natural Science Foundation of China (National Fund for Natural Science 30973666 to X.X.L.) and Graduate School of Fourth Military Medical University (special fund for doctoral dissertation 2009D11 to H.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. As a frightening superbug, methicillin-resistant Staphylococcus aureus (MRSA) has long been an overwhelming human pathogenic threat in healthcare-associated infections [1]. Its prevalence and adaptability in both community and hospital environment makes healthy patients and immune-deficient patients [2] at high risk of infection [3,4]. Its continued pathogenicity and virulence [5,6] causes invasive infection in bloodstream [7], essential organs, and tissues [8,9], therefore leads to severe clinical presentations and high mortality rate [4,10]. This is primarily due to the high incidence of methicillin-resistance that has failed almost all available antibiotics [11]. Furthermore, there has been an increase in reports of isolated MRSA strains developing multi-drug or vancomycin-(intermediate) resistance [12,13], which exacerbated antibiotic paucity. Interventions like vigilant monitoring of antibiotic susceptibilities and judicious use of culture-directed antibiotic agents have been a long-sought endeavor, yielding limited success [14]. Meanwhile, researchers and pharmaceutical industry have been driven to discover new MRSA-active agents (i.e. new chemical derivatives or compounds with new targets [15], virulence inhibitors [16], natural products, and vaccines [17]) and combination therapies, resulting in few ideal drugs or solutions [18]. Thus, antibacterial strategies that provide timely and effective therapeutic countermeasures are urgently required for possible outbreaks of MRSA infections. Particularly, specific RNA silencing in bacteria by antisense antibacterial strategies can contribute to both aspects of the problem [19]. Antisense antibacterials are short (about 10- to 20- bases), synthetic DNA analogs that inhibit essential genes expression at mRNA level in a sequence-specific manner [20]. Thereafter, antisense inhibition leads to bacteriocidal/bacteriostatic effect or restoration of bacterial susceptibility, which depends on the function of targeted gene. Synthetic antisense oligomers, especially peptide nucleic acid (PNA) [21] and phosphorodiamidate morpholino (PMO) [22], possess favorable properties in light of antisense antibacterial application, including improved targeting specificity, binding affinity, biological stability and access to a variety of chemical modification. Meanwhile, instead of simple mixture, cell penetrating peptides (CPP) can be covalently attached/conjugated at the end of PNA or PMO chain to enhance cellular uptake of antisense oligodeoxynucelotides (ASODNs) without affecting Waston-Crick base paring between antisense oligomers and targeted RNAs [23]. Synthetic peptidePNA or peptide-PMO conjugates targeting growth-essential genes have shown to inhibit bacterial growth in pure culture and in infected tissue culture, Thus, a range of functional genes have been identified as potential targets [24]. However, only a few early reports provided preliminary proof-of-principle evidence on antisense targeting of S. aureus genes for growth inhibitory effect (i.e. peptide-PNA targeting fabI [25], and phoB, fmhB, gyrA, plus hmrB [26]) or restoration of antibiotic susceptibility (i.e. liposomecapsulated phosphorothioate oligodeoxynucleotides targeting mecA [27]) in pure culture. Targeting resistance mechanism in MRSA relies on elucidation of subtle intracellular self-regulation among related genes, the consequences of its antisense inhibition bei (...truncated)