Biology subjects

Lobner-Olesen, A.

Publications and source records attributed to Lobner-Olesen, A..

5 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↗

bioRxiv2022

Robust control of replication initiation in the absence of DnaA-ATP {leftrightharpoons} DnaA-ADP regulatory elements in Escherichia coli

Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the bacterial master replication initiator protein, exists in both ATP and ADP forms, despite only the ATP form being essential for initiation. We engineered the {Delta}4 Escherichia coli strain, devoid of all known external elements facilitating the DnaA-ATP/ADP conversion, and found that these cells display nearly wild-type behaviors under non-overlapping replication cycles. However, during rapid growth with overlapping cycles, {Delta}4 cells exhibit initiation instability. This aligns with our model predictions, suggesting that the intrinsic ATPase activity of DnaA alone is sufficient for robust initiation control in E. coli and the DnaA-ATP/ADP conversion regulatory elements extend the robustness to multifork replication, indicating an evolutionary adaptation. Moreover, our experiments revealed constant DnaA concentrations during steady-state cell elongation in both wild-type and {Delta}4 cells. These insights not only advance our understanding of bacterial cell-cycle regulation and DnaA, but also highlight a fundamental divergence from eukaryotic cell-cycle controls, emphasizing protein copy-number sensing in bacteria versus programmed protein concentration oscillations in eukaryotes.

microbiology↗

bioRxiv2021

An amphipathic and cationic antimicrobial peptide kills colistin resistant Gram-negative pathogens in vivo

New antibiotics are needed against multidrug resistant Gram-negative pathogens that have compromised global health systems. Antimicrobial peptides are generally considered promising lead candidates for the next generation of antibiotics but have not fulfilled this expectation. Here we demonstrate activity of a cationic amphipathic undecapeptide (ChIP; Charge change Independent Peptide) against a wide panel of multidrug resistant Gram-negative pathogens. Importantly, the antimicrobial activity of ChIP is independent of the surface charge changes that confer colistin resistance through modification of Lipid A, while decreased activity of ChIP correlates with GlcN1 tri-acylation of Lipid A. In an in vivo peritonitis mouse model ChIP displays excellent activity against both colistin sensitive and resistant Escherichia coli and Acinetobacter baumannii strains. Author SummaryAntimicrobial peptides hold promise as novel treatment options for diseases caused by multidrug resistant bacteria. Here we present evidence that the ChIP peptide, comprised of 11 D-amino acids, is active against a variety of Gram-negative bacteria that ranks high on the WHO list of critically important pathogens. ChIP initially interacts with the Gram-negative outer membrane, independent of its surface charge, followed by entry into the periplasm and permeabilization of the inner membrane, leading to bacterial cell death. Detailed analyses of the outer membrane indicate that the acylation pattern of lipopolysaccharides plays an important role for ChIP activity. In a mouse infection model, ChIP display excellent activity in reducing bacterial numbers for both Escherichia coli and Acinetobacter baumannii. Importantly, ChIP is highly efficient against bacteria resistant to colistin, an antibiotic normally considered as a last resort treatment of infections caused by multidrug resistant Gram-negative bacteria.

microbiology↗

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↗

bioRxiv2018

Comparative activity of Ceftriaxone, Ciprofloxacin and Gentamicin as a function of bacterial growth rate probed by Escherichia coli chromosome replication in the mouse peritonitis model

Commonly used antibiotics exert their effect predominantly on rapidly growing bacterial cells, yet growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as readout for in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (qPCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average and on a single-cell level; from one single biological specimen. Here, we asked whether the in situ growth rate could predict antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as proof-of-concept. Our data demonstrate that the activity of commonly used antibiotics Ceftriaxone and Gentamicin correlated with pre-treatment bacterial growth rate; both drugs performing better during rapid growth than during slow growth. Conversely, Ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotics dependency upon active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.\n\nImportanceMost antibiotics in clinical use exert their effect predominantly on rapidly growing bacterial cells, yet there is a lack of insight into bacterial growth dynamics taking place during infection in vivo. We have applied inexpensive and easily accessible methods for extraction of in situ bacterial growth rate from bacterial chromosome replication during experimental murine infection. This approach not only allows for a better understanding of bacterial growth dynamics taking place during the course of infection, but also serves as a platform to test the activity of different antibiotics as a function of pre-treatment in situ growth rate. The method has the advantage that bacterial growth rate can be probed from a single biological sample, with the potential for extension into clinical use in pre-treatment infected biological specimens. A better understanding of commonly used antibiotics level of dependency upon bacterial growth, combined with measurements of in situ bacterial growth rate in infected clinical specimens, could prove helpful in evaluating future antibacterial treatment regimens.

microbiology↗