Abstract
BackgroundMethicillin-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.Methodology/Principal FindingsBy 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.ConclusionsThe present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.
Highlights
As a frightening ‘‘superbug’’, methicillin-resistant Staphylococcus aureus (MRSA) has long been an overwhelming human pathogenic threat in healthcare-associated infections [1]
The present result suggested that RNA polymerase (RNAP) primary s70 is a very promising candidate target for developing novel antisense antibiotic to treat severe Methicillin-resistant Staphylococcus aureus (MRSA) infections
By using four clinical pathogenic S. aureus genus with varying resistance patterns, we report the identification of rpoD as a potent target for markedly bacteriocidal effect in vitro and ex vivo by antisense peptide-peptide nucleic acid (PNA) conjugate
Summary
As a frightening ‘‘superbug’’, methicillin-resistant Staphylococcus aureus (MRSA) has long been an overwhelming human pathogenic threat in healthcare-associated infections [1]. Its continued pathogenicity and virulence [5,6] causes invasive infection in bloodstream [7], essential organs, and tissues [8,9], 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]. 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
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