rRNA Methyltransferases and their Role in Resistance to Antibiotics

Morić, Ivana; Savic, Milan; Ilić-Tomič, Tatjana; Vojnović, Sandra; Bajkić, Sanja; Vasiljević, Branka

doi:10.2478/v10011-010-0030-y

Cited by 17 publications

(16 citation statements)

References 73 publications

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“…For example, it is known that 16S rRNA methylation is a common mechanism by which pathogenic bacteria develop resistance to common aminoglycosides [56–58]. Analytical methods such as LC/MS that can detect changes in methylation status, including the specific type of methylation, would be attractive options for examining the progression of antibacterial resistance within rRNA.…”

Section: Discussionmentioning

confidence: 99%

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

Russell

Limbach

2013

Journal of Chromatography B

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Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less.

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Section: Discussionmentioning

confidence: 99%

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

Russell

Limbach

2013

Journal of Chromatography B

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“…Methyltransferases are able to catalyze the methylation of ribosomal RNAs (rRNAs). The conformational modification of the rRNAs, which are the molecular targets of tigecycline, may result in an alteration of the antibiotic target interaction, suggesting that trm disruption could be a valid tigecycline resistance mechanism (41). We also sequenced the trm gene of our XDR strain panel and found in 9 out of 10 clinical isolates a disrupted trm gene (Table 2).…”

Section: Discussionmentioning

confidence: 99%

A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism

Trebosc

Gartenmann²,

Royet³

et al. 2016

Antimicrob Agents Chemother

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bInfections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC 90 of tigecycline from 25 g/ml in the parental strains to 3.1 g/ml in the ⌬adeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains. O ne of the greatest global health problems results from the limited treatment options to fight bacterial infections caused by multidrug-resistant (MDR) organisms. The group of ESKAPE organisms that is comprised of Enterobacter spp., Staphylococcus aureus/epidermidis, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis/faecium is considered to cause the vast majority of, often untreatable, nosocomial infections (1). Among these ESKAPE pathogens A. baumannii is most difficult to treat due to its multiple intrinsic and acquired resistance mechanisms that resulted in the development of MDR, extensively drug resistant (XDR), or even pan-drug-resistant (PDR) phenotypes (2-5).Bacteria have evolved multiple ways to evade antibiotic-mediated cell death, such as (i) enzymatic modification/cleavage of the antibiotic (e.g., beta-lactams), (ii) modification/protection of the antibiotic target (e.g., fluoroquinolones), or (iii) reduction of the intracellular concentration by antibiotic efflux or reduced influx (e.g., tetracyclines) (6). The expression of such defense mechanisms may require an extensive metabolic investment, often leading to a reduced fitness of these resistant bacteria in the absence of the external selection pressure (7). To overcome these ecol...

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“…8,9 Furthermore, methylation of A2058 of 23S rRNA in the large subunit of the Escherichia coli ribosome by MTases have been shown to promote intrinsic resistance to three distinct classes of antibiotics including macrolides, lincosamides, and quinupristin. 10–12 For these reasons, there is great interest in an enzymatic characterization of MTase function as well as development of novel inhibitors for MTases. Such compounds could be used as antimicrobials or anticancer therapeutics.…”

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confidence: 99%

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

et al. 2018

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S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5′-methylthioadenosine, AMP, and ADP.

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rRNA Methyltransferases and their Role in Resistance to Antibiotics

Cited by 17 publications

References 73 publications

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

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