Resistance to antibacterial peptide nucleic acids through altered ribosome function

When used as antibacterial agents, Peptide Nucleic Acid (PNAs) are generally designed to base-pair with complementary sequences of an essential mRNA and block translation initiation. Although bacterial susceptibility to peptide-conjugated PNAs is strongly influenced by cellular uptake, intracellular determinants of PNA activity remain poorly understood. Here, we identify a ribosome-centered mechanism of resistance to antibacterial PNAs in Escherichia coli. The rpsLI82N mutation increased the minimum inhibitory concentration (MIC) of an arginine-rich cell penetrating peptide-conjugated PNA targeting acpP eightfold. The effect of rpsLI82N was additive with mutations that reduce PNA entry into bacterial cells PNA resistance conferred by rpsLI82N was independent of carrier peptide and also applied when naked PNA was tested in an envelope-permeable strain. Similarly, rpsLI82N-associated resistance was independent of the targeted mRNA, as it applied to PNAs targeting either the Shine–Dalgarno or AUG region of acpP or ftsZ mRNA. Several additional substitutions within residues 74–82 of ribosomal protein uS12 conferred PNA resistance. Because resistance occurred among both error-restrictive and ribosomal-ambiguity alleles, it did not correlate with the classical decoding-fidelity phenotypes measured by stop-codon readthrough. Proteomic analysis revealed widespread changes in proteins under post-transcriptional control in rpsLI82N cells. The effect on selected sRNA-regulated genes correlated with the location of the sRNA-binding site: repression was less efficient when binding overlapped the translation-initiation region whereas it was more efficient when regulatory sites were located outside this region. In parallel, rpsLI82N increased the 30S and 50S ribosomal fractions and reduced the 70S fraction. Both loss of KsgA, which disrupts 30S maturation, and treatment with kasugamycin, which perturbs translation initiation, increased PNA resistance. We propose that rpsLI82N alters uS12-dependent 30S assembly and initiation-complex dynamics, thereby changing the effective occupancy of mRNA translation-initiation regions. This limits access of PNAs and sRNAs to overlapping sequences, resulting in PNA resistance and reduced sRNA-mediated repression. Conversely, a longer-lived 30S initiation state may facilitate sRNA binding to flanking regions and strengthen repression. These findings identify the ribosome–mRNA interface as an intracellular determinant of antibacterial PNA susceptibility.