Antisense inhibition of the Escherichia coli NrdAB aerobic ribonucleotide reductase is bactericidal due to induction of DNA strand breaks

Background: Antisense peptide nucleic acids (PNAs) constitute an alternative to traditional antibiotics, by their ability to silence essential genes.

Objectives: To evaluate the antibacterial effects of antisense PNA-peptide conjugates that target the gene encoding the alpha subunit (NrdA) of the Escherichia coli ribonucleotide reductase (RNR).

Methods: Bacterial susceptibility of a series of NrdA-targeting PNAs was studied by MIC determination and time-kill analysis. Western-blot analysis, gene complementation and synergy with hydroxyurea were employed to determine the efficiency of NrdA-PNA antisense treatment. The effect on chromosome replication was addressed by determining the DNA synthesis rate, by flow cytometry analysis, by quantitative PCR and by fluorescence microscopy. The use of DNA repair mutants provided insight into the bactericidal action of NrdA-PNA.

Results: Treatment with NrdA-PNA specifically inhibited growth of E. coli, as well as NrdA protein translation at 4 μM. Also, the DNA synthesis rate was reduced, preventing completion of chromosome replication and resulting in formation of double-stranded DNA breaks and cell death.

Conclusions: These data present subunits of the NrdAB RNR as a target for future antisense microbial agents and provide insight into the bacterial physiological response to RNR-targeting antimicrobials.