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Updated: May 25, 2026
MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin-resistant Staphylococcus aureus by
1Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi, 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 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 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 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.
Conclusions:
The present result suggested that RNAP primary σ⁷⁰ is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.
Insights
Novel peptide nucleic acids (PNAs) targeting RNA polymerase sigma 70 (σ⁷⁰) show potent antibacterial activity against multi-drug resistant Staphylococcus aureus (MRSA). This antisense approach offers a promising new strategy for treating severe MRSA infections.
Area of Science:
- Microbiology
- Antimicrobial Resistance
- Molecular Biology
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a global health threat due to its high mortality and increasing multi-drug resistance (MDR).
- Limited therapeutic options necessitate the discovery of new drug targets and the development of novel antibiotics against MRSA.
- The essential transcription factor RNA polymerase primary σ⁷⁰ (encoded by rpoD) is a conserved target in S. aureus, suitable for antisense inhibition.
Purpose of the Study:
- To develop and evaluate novel antisense peptide nucleic acids (PNAs) targeting the rpoD gene in MRSA.
- To assess the efficacy and specificity of these PNAs against various MRSA strains, including MDR and vancomycin-intermediate strains.
- To investigate the mechanism of action of the lead PNA candidate.
Main Methods:
- Synthesis of cell-penetrating peptide-conjugated PNAs (PPNAs) designed to target a conserved region of rpoD mRNA.
- In vitro testing of PPNA efficacy against multiple S. aureus strains, including determination of minimum inhibitory concentration (MIC) and assessment of bactericidal activity.
- Evaluation of PPNA specificity using a scrambled sequence control and analysis of rpoD mRNA and σ⁷⁰ protein levels post-treatment.
- Assessment of PPNA efficacy in a MRSA-infected epithelial cell culture model.
Main Results:
- A specific PPNA (PPNA2332) demonstrated potent, micromolar-range growth inhibition against diverse S. aureus strains, including MDR-MRSA and vancomycin-intermediate strains.
- PPNA2332 exhibited a bactericidal antisense effect and specifically targeted rpoD mRNA, leading to decreased σ⁷⁰ protein expression in a concentration-dependent manner.
- The PPNA effectively cleared MRSA infection in epithelial cell cultures without apparent toxicity at effective concentrations.
Conclusions:
- Antisense inhibition of RNA polymerase primary σ⁷⁰ is a highly promising strategy for developing new antibiotics against severe MRSA infections.
- The developed PPNA2332 demonstrates significant potential as a therapeutic agent for combating challenging MRSA infections.
- Targeting essential prokaryotic factors like σ⁷⁰ offers a viable avenue for overcoming antimicrobial resistance.
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