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

Abstract

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.

Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes

“…Our attempts to reconstitute HflXr-50S complexes in vitro were unsuccessful due to difficulties obtaining active soluble L. monocytogenes HflXr protein. Therefore, to generate homogenous L. monocytogenes HflXr-50S complexes for structural analysis, we employed an in vivo pull-down approach using the L. monocytogenes Δ hflXr strain overexpressing a C-terminally FLAG 3 -tagged HflXr protein, as used recently to generate ARE-ABCF–ribosome complexes ( 11 , 12 ). As a specificity control, we also performed the in vivo pull-down experiment using the L. monocytogenes Δ hflXr strain transformed with the empty pIMK3 plasmid.…”

Persistence of transferable oxazolidinone resistance genes in enterococcal isolates from a swine farm in China

“…Since the initial description of the optrA gene, it and its many variants have been reported in several countries, revealing that it is more difficult to treat oxazolidinone-resistant bacteria ( Schwarz et al, 2021 ). Unfortunately, another ribosomal protection protein of the ABC-F family, poxtA , was reported in MRSA of clinical origin in 2018 ( Antonelli et al, 2018 ; Crowe-McAuliffe et al, 2022 ). The poxtA2 variant was also reported in 2021 ( Baccani et al, 2021 ).…”

“…Although oxazolidinone antibiotics have never been approved for use in livestock breeding, we often detect the transferable determinants of oxazolidinone resistance in enterococci isolated from animals used in food production ( Wang et al, 2020 ). Florfenicol is a phenicol compound that has broad spectrum antibacterial activity and few side effects and is used to treat respiratory and intestinal bacterial infections in animals used in food production ( Crowe-McAuliffe et al, 2022 ). It was reported that the abundance of oxazolidinone resistance genes in livestock feces was related to florfenicol residue ( Kim, 2021 ).…”

Detection of an Enterococcus faecium Carrying a Double Copy of the PoxtA Gene from Freshwater River, Italy

“…Cfr and Cfr-like methylases confer resistance to five classes of antimicrobial agents, including phenicols, lincosamides, oxazolidinones, pleuromutilines, and streptogramin A (PhLOPS A phenotype) via the post-transcriptional methylation of the 23S rRNA [ 10 ]. The ABC-F proteins OptrA and PoxtA lead to a decreased susceptibility to phenicols and oxazolidinones (including tedizolid) by a ribosomal protection mechanism [ 11 ].…”