Antimicrobial activity of antisense peptide–peptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms

Background: Antisense peptide nucleic acids (PNAs) are synthetic polymers that mimic DNA/RNA and inhibit bacterial gene expression in a sequence-specific manner. Methods: To assess activity against non-typeable Haemophilus influenzae (NTHi), we designed six PNA-peptides that target acpP, encoding an acyl carrier protein. MICs and minimum biofilm eradication concentrations (MBECs) were determined. Resistant strains were selected by serial passages onmedia with a sub-MIC concentration of acpP-PNA. Results: The MICs of six acpP-PNA-peptides were 2.9-11 mg/L (0.63-2.5 mmol/L) for 20 clinical isolates, indicating susceptibility of planktonic NTHi. By contrast, MBECs were up to 179 mg/L (40 mmol/L). Compared with one original PNA-peptide (acpP-PNA1-3' N), an optimized PNA-peptide (acpP-PNA14-5' L) differs in PNA sequence and has a 5' membrane-penetrating peptide with a linker between the PNA and peptide. The optimized PNA-peptide had an MBEC ranging from 11 to 23 mg/L (2.5-5 mmol/L), indicating susceptibility. A resistant strain that was selected by the original acpP-PNA1-3' N had an SNP that introduced a stop codon in NTHI0044, which is predicted to encode an ATP-binding protein of a conserved ABC transporter. Deletion of NTHI0044 caused resistance to the original acpP-PNA1-3' N, but showed no effect on susceptibility to the optimized acpP-PNA14-5' L. The WT strain remained susceptible to the optimized PNA-peptide after 30 serial passages on media containing the optimized PNA-peptide. Conclusions: A PNA-peptide that targets acpP, has a 5' membrane-penetrating peptide and has a linker shows excellent activity against planktonic and biofilm NTHi and is associated with a low risk for induction of resistance.