PNA Length Restriction of Antibacterial Activity of Peptide-PNA Conjugates in Escherichia coli Through Effects of the Inner Membrane

Abstract

Peptide Nucleic Acid (PNA)-peptide conjugates targeting essential bacterial genes are showing promise as antisense antimicrobials in drug discovery. Optimization has focused on selection of target genes and exact localization around the ribosome binding site, but surprisingly a length optimum around 10-12 nucleobases has been found. Addressing this observation, we have investigated the relationship between PNA-length, PNA-RNA duplex stability and antimicrobial activity in E. coli in more detail. For PNAs of identical length of ten nucleobases the expected reverse correlation between the thermal stability (Tm) of the PNA-RNA duplex and the MIC for single mismatched PNAs was found. Also the expected direct correlation between the length of the PNA and the PNA-RNA duplex stability was found. Nonetheless, 10-mer PNAs [in a 6-18 mer extension series of (KFF)₃K- and (RXR)₄ conjugates] were the most active as antisense antimicrobials in both wild type E. coli MG1655 and AS19, suggesting that the size constraint is related to the bacterial uptake of PNA-peptide conjugates. This conclusion was supported by flow cytometry data showing higher bacterial uptake of shorter PNA fluorophore labeled conjugates. Interestingly, the size-limited uptake seems independent on outer membrane integrity (AS19), and thus the results suggest that the inner membrane limits the molecular size for peptide-PNA passage.

Keywords: Escherichia coli; antisense antimicrobials; bacterial envelope; peptide conjugates; peptide nucleic acid.

FIGURE 1

Thermal stability of PNA–RNA duplex correlates with MIC-value. (A) MIC and melting temperature for (KFF)₃K-PNAs of increasing PNA length. (B) Correlation between melting temperature and MIC for (KFF)₃K-PNA with single base mismatches.

FIGURE 2

Uptake of PNA is limited by the inner membrane in E. coli spheroplasts. Survival of E. coli spheroplasts after incubation with peptide-PNA conjugates (A,B) of different PNA length.

FIGURE 3

Peptide-PNA uptake monitored by flow cytometry. E. coli culture incubated with BODIPY fluorophore labeled peptide-PNA with different PNA length was profiled by flow cytometry. (A) Full profiles of E. coli cultures incubated with each of the three peptide-PNA conjugates or without PNA. Background fluorescence extends to approx. 130 fluorescence units. (B) Excerpt from (A, box) showing only the part of the bacterial population containing the labeled peptide-PNA-conjugate. (C) Percentage of bacterial cells with peptide-PNA uptake.