Biology subjects

Murina, V.

Publications and source records attributed to Murina, V..

7 recordsLinked to original sources

bioRxiv2022

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

microbiology↗

bioRxiv2021

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9-3.1 [A], as well as the complete E. faecalis 70S ribosome at 2.2-2.5 [A]. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 [A] from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by [~]4 [A] out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

microbiology↗

bioRxiv2020

Structural basis of resistance to lincosamide, streptogramin A, and pleuromutilin antibiotics by ABCF ATPases in Gram-positive pathogens

Target protection proteins bind to antibiotic targets and confer resistance to the host organism. One class of such proteins, termed antibiotic resistance (ARE) ATP binding cassette (ABC) proteins of the F-subtype (ARE ABCFs), are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition by antibiotics that target the large ribosomal subunit. Using single-particle cryo-EM, we have solved the structure of ARE ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a comparative approach to understanding how these proteins mediate antibiotic resistance on the ribosome. We present evidence of mechanistically diverse allosteric relays converging on a few peptidyltransferase center (PTC) nucleotides, and propose a general model of antibiotic resistance mediated by these ARE ABCFs.

microbiology↗

bioRxiv2020

Structural basis for bacterial ribosome quality control

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality-control (RQC) pathways. RQC begins with splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal tails. How such tailing can occur without the regular suite of translational components is, however, unclear. Using ex vivo single-particle cryo-EM, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with YabO, a protein homologous to, yet distinct from, Hsp15. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

biochemistry↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.

biochemistry↗

bioRxiv2018

Elimination of ribosome inactivating factors improves the efficiency of Bacillus subtilis and Saccharomyces cerevisiae cell-free translational systems

Cell-free translational systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translation regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translational system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translational systems. A possible next step in developing strains for production of even more efficient cell-free translation systems could be achieved by combining a genomic disruption of the ribosome hibernation machinery with inactivation of the genes responsible for proteolysis and RNA degradation.

biochemistry↗

bioRxiv2017

ABCF ATPases involved in protein synthesis, ribosome assembly and antibiotic resistance: structural and functional diversification across the tree of life

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterised VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase centre and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance factors from other ABCFs have not been explored. To address this, we analysed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting they were present in the last common ancestor of both. Surprisingly, currently known antibiotic resistance (ARE) ABCFs are not confined to a distinct lineage of the ABCF family tree. This suggests that either antibiotic resistance is a pervasive feature of bacterial ABCFs, or it is relatively easy to evolve antibiotic resistance from other ABCF functions. Our data suggest there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that Bacillus subtilis VmlR is a ribosome-binding resistance factor localised to the cytoplasm.\n\nAuthor summaryIsolated members of the ABCF protein family of ATP-hydrolysing enzymes have been found to have important roles in protein synthesis and antibiotic resistance. However, their full diversity across the tree of life, and their evolutionary histories have never been examined. Therefore, we analysed the presence of ABCFs and their constituent domains in genomes across the tree of life, discovering 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla. This includes several subfamilies that we predict comprise novel antibiotic resistance (ARE) ABCFs, present in antibiotic producers and important human pathogens. There are significant gaps in our knowledge about the functional capabilities of different ABCF families. To address this, we have made ATPase domain mutants of all four Escherichia coli ABCFs, showing that they inhibit protein synthesis and indicating a role on the ribosome. Furthermore, we demonstrate a genetic interaction of two E. coli ABCFs with the GTPase BipA, involved in ribosome assembly. Finally, we show that Bacillus subtilis VmlR in the ARE2 subfamily is a ribosome-binding resistance factor localised to the cytoplasm. As more is discovered about the function of individual ABCFs, the more it will be possible to predict functions of uncharacterised members, using the ABCF family tree as a framework.

molecular biology↗