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…
Antibacterial peptide nucleic acids (PNAs) are typically utilized as antibiotics by binding to specific mRNA sequences, thereby inhibiting translation initiation. However, the precise cellular mechanisms responsible for bacterial resistance to these PNAs have remained largely elusive. In a recent study, researchers have uncovered a novel ribosome-centered mechanism of resistance to antibacterial PNAs in Escherichia coli.
A mutation in the rpsLI82N gene was found to significantly increase the minimum inhibitory concentration (MIC) of an arginine-rich cell penetrating peptide-conjugated PNA targeting the acpP gene, augmenting resistance eightfold. This resistance was not influenced by alterations in the carrier peptide or observed in an enzyme-permeable strain of bacteria.
Similarly, rpsLI82N-associated resistance was unrelated to the targeted mRNA sequence, as it persisted when PNAs were designed to target either the Shine-Dalgarno or AUG regions of acpP or ftsZ mRNAs.
Further investigation revealed that several substitutions within residues 74-82 of ribosomal protein uS12 also conferred PNA resistance. This resistance was observed in cells harboring both error-restrictive and ribosomal-ambiguity alleles, suggesting that it does not correlate with classical decoding-fidelity phenotypes measured by stop-codon readthrough. Proteomic analysis revealed widespread alterations in proteins regulated post-transcriptionally in rpsLI82N cells, with corresponding effects on sRNA-regulated genes.
Interestingly, the presence of rpsLI82N led to increased levels of 30S and 50S ribosomal fractions and reduced levels of 70S fractions. This alteration appeared to weaken the ribosome-mRNA interface, limiting the access of PNAs and sRNAs to overlapping sequences within the translation initiation regions of mRNAs. As a result, PNAs and sRNAs encountered greater difficulty in binding to these regions, leading to increased resistance to PNAs and reduced repression by sRNAs.
Conversely, the presence of a longer-lived 30S initiation state may enhance sRNA binding to flanking regions, thereby strengthening repression. These findings suggest that the ribosome-mRNA interface plays a crucial role as an intracellular determinant of antibacterial PNA susceptibility.
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