 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 12, 2022 |
| Title |
Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics |
| Organism |
Enterococcus faecalis |
| Experiment type |
Other
|
| Summary |
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 by investigating 5'P mRNA decay intermediates, that provide ribosome protection data, we show that PoxtA protects against Linezolid specific stalls. Furthermore, we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9–3.1 Å, as well as the complete E. faecalis 70S ribosome at 2.2–2.5 Å. 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 Å 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 Å 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.
|
| |
|
| Overall design |
Here we investigate 5'P mRNA decay intermediates in Enterococcus faecalis to investigate context-specific ribosome stalls in response to Linezolid in presence or absence of PoxtA. We analyzed 12 samples using HT-5PSeq (Zhang and Pelechano, https://doi.org/10.1016/j.crmeth.2021.100001) optimized for bacteria (Huch, Nersisyan et al, https://doi.org/10.1101/2021.04.08.439066). We performed three biological replicates for two strains (with and without PoxtA expression) in the presence and absence of Linezolid treatment at sub-MIC concentrations.
|
| |
|
| Contributor(s) |
Crowe-McAuliffe C, Murina V, Kasari M, Turnbull KJ, Takada H, Polikanov YS, Sunsfjord A, Hegstad K, Atkinson GC, Hauryliuk V, Wilson DN, Huch S, Nersisyan L, Pelechano V |
| Citation(s) |
35387982 |
| |
| Submission date |
Jul 02, 2021 |
| Last update date |
Apr 27, 2022 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
|
| Organization name |
ScilifeLab - Karolinska Institutet
|
| Department |
MTC
|
| Street address |
Nobels väg 16
|
| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
| |
|
| Platforms (1) |
| GPL30343 |
NextSeq 2000 (Enterococcus faecalis) |
|
| Samples (12)
|
|
| Relations |
| BioProject |
PRJNA743321 |
| SRA |
SRP326687 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE179348_RAW.tar |
7.3 Mb |
(http)(custom) |
TAR (of BEDGRAPH) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...