Biology subjects

Nielsen, P. E.

Publications and source records attributed to Nielsen, P. E..

2 recordsLinked to original sources

bioRxiv2026

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 argininerich 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.

molecular biology↗

bioRxiv2020

Activation of the Cpx-envelope stress response system promotes tolerance to antibacterials delivered by arginine-rich peptides and aminoglycosides in Escherichia coli

Cell penetrating peptides (CPP) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. However; their mechanism of action, particularly for prokaryotes is still unknown. Arginine-rich CPPs (R-CPP) efficiently delivers the antimicrobial peptide nucleic acid (PNA) into bacteria. Here, we show that resistance to an R-CPP PNA conjugate in Escherichia coli requires multiple genetic modifications and is specific to R-CPP and not to the PNA-part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx*), which decreased the cytoplasmic membrane potential and thereby indicates an indirectly energy dependent uptake mechanism. Interestingly, cpx* mutants also showed increased tolerance to aminoglycosides and R-CPP conjugated to a peptide targeting the DNA sliding clamp; i.e., similar uptake in E. coli for these antimicrobial compounds. We speculate that the cpx* phenotype could create an evolutionary opportunity to adapt and evolve in the presence of either compounds. Author summaryThe emergence of multidrug resistant bacteria is raising the need for new classes of antibiotics. Peptide nucleic acids (PNAs) may fill this requirement by their ability to block translation of essential mRNAs and hence inhibit growth. PNA needs conjugation to a delivery peptide (cell penetrating peptide; CPP) to enter the bacteria. Arginine-rich CPPs (CPPR) are receiving a lot of attention for use as delivery vessels. Here, we show, for the first time, CPPR-PNA resistance in Escherichia coli directed towards the delivery peptide. Consequently, resistance also applies to other antimicrobial compounds delivered by the same carrier. An integral part of CPPR resistance is due to a constitutive active Cpx-response system, which leads to a decreased electric potential ({Delta}{Psi}) across the inner membrane. The decreased {Delta}{Psi} is a result of down-regulation of two aerobic respiratory operons, namely NADH:ubiquinone oxidoreductase complex I and cytochrome bo3 ubiquinol oxidase. The decreased {Delta}{Psi} also led to increased tolerance to aminoglycosides. This shows that a (large) negative {Delta}{Psi} is important for providing sufficient free energy for membrane translocation of both CPPR and that the inner membrane is the main barrier for entry of both arginine-rich delivery peptides and aminoglycosides.

microbiology↗