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Open accessJournal ArticleDOI: 10.1073/PNAS.2013315118

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

02 Mar 2021-Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences)-Vol. 118, Iss: 9
Abstract: The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

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Topics: Aminoglycoside (55%), Translation (biology) (54%), Protein biosynthesis (53%) ... show more

5 results found

Journal ArticleDOI: 10.1016/J.BIOORG.2021.105227
Himani Chaurasia1, Vishal K. Singh1, Richa Mishra1, Aditya K. Yadav1  +3 moreInstitutions (1)
Abstract: A series of new N-1-(β- d -ribofuranosyl) benzimidazole derivatives has been designed using in silico methods and synthesized as probable antimicrobial agents. Further, the compounds were assessed for their antibacterial and antifungal activity. Antibacterial screening was done by employing broth micro-dilution method and compounds exhibited excellent inhibitory activity (MIC, 50–1.56 µg/mL) against different human pathogenic bacteria, viz. B. cerus, B. subtilis, S. aureus, E. coli and P. aeruginosa and drug resistant strain (DRS) of E. coli. A great synergistic effect was observed during evaluation of ∑FIC, where a combination study was performed using standard references, viz. chloramphenicol and kanamycin. The MIC data obtained from different methods of combination approach revealed 4–128 fold more potency compared to compounds tested alone. The results clearly indicated the possibility of these compounds as active ingredients of drug regimen used against MDR strains. Antifungal screening were also performed employing two different methods, viz. serial dilution method and zone inhibition method, clearly indicated that compounds were also potentially active against several species of pathogenic fungal strains, viz. A. flavus, A. niger, F. oxysporum and C. albicans. The assessment of structure activity relationship (SAR) clearly revealed that presence of less polar and more hydrophobic substituents positively favours the antibacterial activity, conversely, more polar and hydrophilic substituents favours the antifungal activities. Thus, the results positively endorsed the compounds as potent antibacterial and antifungal agents which could be developed as possible drug regimens.

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Topics: Antibacterial activity (54%), Antimicrobial (52%), Benzimidazole (50%)

1 Citations

Open accessJournal ArticleDOI: 10.1016/J.JMST.2021.08.089
Lijun Fan1, Wenxin Sun1, Yu-Hong Zou1, Qian-qian Xu1  +3 moreInstitutions (2)
Abstract: Implant-associate infection (IAI) is a major cause of failure of bone implant materials, and one of the significant challenges in clinical managements. A synergistic coating strategy combining montmorillonite (MMT) sustained release, adsorption of bacteria and gentamicin (GS) bactericidal is proposed herein to tackle infection issues. Surface morphology, microstructure and chemical composition of the samples were investigated using SEM, XRD, FT-IR and XPS. Electrochemical experiments and immersion experiments reveal that corrosion resistance of Mg samples with GS/MMT coatings was higher than that of bare Mg alloy substrate in DMEM solution. In vitro studies demonstrated that the GS/MMT coating had a significant inhibitory effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The viability of MC3T3-E1 cells was 92.7% after a co-culturing for 72 h. After a subcutaneous transplantation of 90 days, the survival rate was 100% for GS/MMT-coated Mg alloy specimens with no infection at the implantation sites and no toxic damage to liver, kidney and local muscles pathological sections. This study provides a novel method for the preparation of sustained-release antimicrobial coatings on biodegradable Mg alloys as promising candidates for orthopedic implant materials.

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Topics: Transplantation (52%)

Open accessJournal ArticleDOI: 10.3389/FMICB.2021.711037
Kexin Zhou1, Jialei Liang1, Xu Dong1, Peiyao Zhang1  +11 moreInstitutions (1)
Abstract: Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we characterized a novel resistance gene named aac(2′)-If from an isolate of a novel Providencia species, Providencia wenzhouensis R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2′-N-acetyltransferase. Whole-genome sequencing and comparative genomic analysis were performed to elucidate the molecular characteristics of the genome and the genetic context of the resistance gene-related sequences. Among the functionally characterized resistance genes, AAC(2′)-If shares the highest amino acid sequence identity of 70.79% with AAC(2′)-Ia. AAC(2′)-If confers resistance to several aminoglycoside antibiotics, showing the highest resistance activity against ribostamycin and neomycin. The recombinant strain harboring aac(2′)-If (pUCP20-aac(2′)-If/DH5α) showed 256- and 128-fold increases in the minimum inhibitory concentration (MIC) levels to ribostamycin and neomycin, respectively, compared with those of the control strains (DH5α and pUCP20/DH5α). The results of the kinetic analysis of AAC(2′)-If were consistent with the MIC results of the cloned aac(2′)-If with the highest catalytic efficiency for ribostamycin (kcat/Km ratio = (3.72 ± 0.52) × 104 M−1·s−1). Whole-genome sequencing demonstrated that the aac(2′)-If gene was located on the chromosome with a relatively unique genetic environment. Identification of a novel aminoglycoside resistance gene in a strain of a novel Providencia species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.

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Topics: Ribostamycin (58%), Providencia (55%), Population (51%) ... show more

Open accessPosted ContentDOI: 10.1101/2021.10.06.463398
06 Oct 2021-bioRxiv
Abstract: Aminoglycoside antibiotics interfere with selection of cognate tRNAs during translation, resulting in the production of aberrant proteins that are the ultimate cause of the antibiotic bactericidal effect. To determine if these aberrant proteins are recognized as substrates by the cells protein quality control machinery, we studied whether the heat shock (HS) response was activated following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan). Levels of the HS transcription factor {sigma}32 increased about 10-fold after exposure to Kan, indicating that at least some aberrant proteins were recognized as substrates by the molecular chaperone DnaK. To investigate whether toxic aberrant proteins therefore might escape detection by the QC machinery, we studied model aberrant proteins that had a bactericidal effect when expressed in E. coli from cloned genes. As occurred following exposure to Kan, levels of {sigma}32 were permanently elevated following expression of an acutely toxic 48-residue protein (ARF48), indicating that toxic activity and recognition by the QC machinery are not mutually exclusive properties of aberrant proteins, and that the HS response was blocked in these cells at some step downstream of {sigma}32 stabilization. This block could result from halting of protein synthesis or from radial condensation of nucleoids, both of which occurred rapidly following ARF48 induction. Nucleoids were similarly condensed following expression of toxic aberrant secretory proteins, suggesting that transertion of inner membrane proteins, a process that expands nucleoids into an open conformation that promotes growth and gene expression, was disrupted in these cells. The 48-residue ARF48 protein would be well-suited for structural studies to further investigate the toxic mechanism of aberrant proteins.

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Topics: Protein biosynthesis (52%), Secretory protein (52%), Gene expression (52%) ... show more

Open accessJournal ArticleDOI: 10.1093/NAR/GKAB495
Abstract: How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.

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Topics: Aminoglycoside (51%)

52 results found

Open accessJournal ArticleDOI: 10.1016/J.CELL.2007.06.049
07 Sep 2007-Cell
Abstract: Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.

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Topics: Hydroxyl radical (53%), DNA damage (50%)

2,101 Citations

Open accessJournal ArticleDOI: 10.1038/NMETH.1237
Khuloud Jaqaman1, Dinah Loerke1, Marcel Mettlen1, Hirotaka Kuwata2  +3 moreInstitutions (2)
20 Jul 2008-Nature Methods
Abstract: Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

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1,459 Citations

Journal ArticleDOI: 10.1038/35030019
21 Sep 2000-Nature
Abstract: The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

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Topics: Transfer RNA (65%), Eukaryotic Large Ribosomal Subunit (64%), Ribosome (61%) ... show more

1,436 Citations

Journal ArticleDOI: 10.1126/SCIENCE.1060612
04 May 2001-Science
Abstract: Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or “wobble,” position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs. During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide

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Topics: Transfer RNA (70%), Ribosome (64%), Eukaryotic Ribosome (64%) ... show more

1,128 Citations

Journal ArticleDOI: 10.1038/327389A0
Danesh Moazed1, Harry F. Noller1Institutions (1)
04 Jun 1987-Nature
Abstract: Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA

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Topics: 50S (60%), Ribosome (58%), Ribosomal RNA (58%) ... show more

1,085 Citations

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