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Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics

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  • Caillan Crowe-McAuliffe
  • Victoriia Murina
  • Kathryn Jane Turnbull
  • Susanne Huch
  • Marje Kasari
  • Hiraku Takada
  • Lilit Nersisyan
  • Arnfinn Sundsfjord
  • Kristin Hegstad
  • Gemma C. Atkinson
  • Vicent Pelechano
  • Daniel N. Wilson
  • Vasili Hauryliuk
Vis graf over relationer

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

OriginalsprogEngelsk
Artikelnummer1860
TidsskriftNature Communications
Vol/bind13
Udgave nummer1
ISSN2041-1722
DOI
StatusUdgivet - dec. 2022

Bibliografisk note

Funding Information:
We are grateful to Barbara E. Murray for sharing E. faecalis ΔlsaA (lsa ::Kan) strain TX5332 (ref.32), Jose A. Lemos for sharing pCIE plasmid 76, Alberto Antonelli for sharing genetic material containing poxtA AOUC-0915 and optrA-E35048 and Anette M. Hammerum for sharing the plasmid containing optrA ST16 (ref.21) and to Yury S. Polikanov for providing structural insights into the mechanism of chloramphenicol action. We thank Michael Hall for help with cryo-EM data collection. The electron microscopy data was collected at the Umeå Core Facility for Electron Microscopy, a node of the Cryo-EM Swedish National Facility, funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Umeå University. Genomic analysis was performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (grant WI3285/8-1 to D.N.W.), Swedish Research Council (Vetenskapsrådet) grants (2017-03783 and 2021-01146 to V.H., 2019-01085 to G.C.A.), Ragnar Söderbergs Stiftelse (to V.H.), postdoctoral grant from the Umeå Centre for Microbial Research, UCMR (to H.T.), the European Union from the European Regional Development Fund through the Centre of Excellence in Molecular Cell Engineering (2014-2020.4.01.15-0013 to V.H.); and the Estonian Research Council (PRG335 to V.H.). D.N.W. and V.H. groups are also supported by the Deutsche Zentrum für Luft- und Raumfahrt (DLR01Kl1820 to D.N.W.) and the Swedish Research Council (2018-00956 to V.H.) within the RIBOTARGET consortium under the framework of JPIAMR. GCA and VH were also supported by a project grant from the Knut and Alice Wallenberg Foundation (2020-0037 to G.C.A.). V.P. group is supported by Swedish Foundation’s Starting Grant (Ragnar Söderberg Foundation), the Swedish Research Council [VR 2016-01842, 2020-01480 and 2021-06112], a Wallenberg Academy Fellowship [KAW2016.0123], Vinnova [2020-03620] and Karolinska Institutet (SciLifeLab Fellowship, SFO and KI funds). LN was supported by the EU H2020-MSCA-IF-2018 program under grant agreement [845495 – TERMINATOR].

Funding Information:
We are grateful to Barbara E. Murray for sharing E. faecalis ΔlsaA (lsa::Kan) strain TX5332 (ref. ), Jose A. Lemos for sharing pCIE plasmid, Alberto Antonelli for sharing genetic material containing poxtA AOUC-0915 and optrA-E35048 and Anette M. Hammerum for sharing the plasmid containing optrA ST16 (ref. ) and to Yury S. Polikanov for providing structural insights into the mechanism of chloramphenicol action. We thank Michael Hall for help with cryo-EM data collection. The electron microscopy data was collected at the Umeå Core Facility for Electron Microscopy, a node of the Cryo-EM Swedish National Facility, funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Umeå University. Genomic analysis was performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (grant WI3285/8-1 to D.N.W.), Swedish Research Council (Vetenskapsrådet) grants (2017-03783 and 2021-01146 to V.H., 2019-01085 to G.C.A.), Ragnar Söderbergs Stiftelse (to V.H.), postdoctoral grant from the Umeå Centre for Microbial Research, UCMR (to H.T.), the European Union from the European Regional Development Fund through the Centre of Excellence in Molecular Cell Engineering (2014-2020.4.01.15-0013 to V.H.); and the Estonian Research Council (PRG335 to V.H.). D.N.W. and V.H. groups are also supported by the Deutsche Zentrum für Luft- und Raumfahrt (DLR01Kl1820 to D.N.W.) and the Swedish Research Council (2018-00956 to V.H.) within the RIBOTARGET consortium under the framework of JPIAMR. GCA and VH were also supported by a project grant from the Knut and Alice Wallenberg Foundation (2020-0037 to G.C.A.). V.P. group is supported by Swedish Foundation’s Starting Grant (Ragnar Söderberg Foundation), the Swedish Research Council [VR 2016-01842, 2020-01480 and 2021-06112], a Wallenberg Academy Fellowship [KAW2016.0123], Vinnova [2020-03620] and Karolinska Institutet (SciLifeLab Fellowship, SFO and KI funds). LN was supported by the EU H2020-MSCA-IF-2018 program under grant agreement [845495 – TERMINATOR].

Publisher Copyright:
© 2022, The Author(s).

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