| 1. |
- Aleksandrova, Elena V, et al.
(författare)
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Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion
- 2023
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
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| 2. |
- Aleksandrova, Elena V., et al.
(författare)
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Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it
- 2024
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Ingår i: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469.
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.
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| 3. |
- Juhas, Mario, et al.
(författare)
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In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii
- 2019
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Ingår i: Journal of Antimicrobial Chemotherapy. - : OXFORD UNIV PRESS. - 0305-7453 .- 1460-2091. ; 74:4, s. 944-952
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.Methods: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.Results: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.Conclusions: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.
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| 4. |
- Pantel, Lucile, et al.
(författare)
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Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site
- 2018
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Ingår i: Molecular Cell. - : CELL PRESS. - 1097-2765 .- 1097-4164. ; 70:1, s. 83-94
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Tidskriftsartikel (refereegranskat)abstract
- Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.
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| 5. |
- Svetlov, Maxim S., et al.
(författare)
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Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance
- 2021
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Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 17:4, s. 412-420
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Tidskriftsartikel (refereegranskat)abstract
- Structural analysis of the A2058-dimethylated and unmethylated 70S ribosome complex alone and in combination with macrolides reveals the role of the desosamine moiety of macrolides in drug binding and resistance. Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 angstrom resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.
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| 6. |
- Tamman, Hedvig, et al.
(författare)
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A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes
- 2020
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Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 16:8, s. 834-840
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Tidskriftsartikel (refereegranskat)abstract
- Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 ' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3 ' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (Rel(Tt)). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel(Tt) (Rel(Tt)(NTD)) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.
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| 7. |
- Travin, Dmitrii Y., et al.
(författare)
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Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition
- 2019
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.
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