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

Abstract

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 uORF4u, 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.

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA

“…To date, twelve cryo electron microscopy structures of ABC-F proteins in complex with the 70S ribosome are known. These structures reveal an overall geometry with similar contacts between the protein and the ribosome, but the corresponding regions of each ABC-F that contact the ribosome differ in sequences 7,10,12,16,19,20,23,27 . The PtIM extension points towards the peptidyl transfer center (PTC) of the ribosome, but shows considerable variation in length 10,16,17,[19][20][21]28 .…”

“…Each ABC-domain carries consensus Walker A and B motifs and in the presence of ATP the two domains come together, binding two ATP molecules to shape an ATP-bound closed conformation 21,23,26 . In this conformation, ABC-F proteins bind to the E site of the 70S ribosome 6,7,10,16,17,19,20 and require the hydrolysis of the ATP molecules to dissociate from the ribosome 6 . Consequently, an ATPase-deficient mutant, in which the two catalytic glutamates of each Walker B motif of the ABC domain are replaced by glutamines (EQ 2 mutant) 26 , forms a stable complex with the ribosome 6,7,10,13,19,20 .…”

“…With the EF-P/eIF-5A protein that has been identified to rescue ribosomes stalled during the synthesis of polyproline-containing proteins [29][30][31] , ABC-F are the only factors known to bind to the ribosomal E site. Some ABC-F have a clear physiological function such as the antibiotic resistance ABC-F (ARE ABC-F) factors 9,10,12,[14][15][16][17][18][19][20]32,33 which provide resistance toward antibiotics that target the PTC and the nascent peptide exit tunnel (NPET) of the ribosome 9,10,12,16,17,19,20 . However, for most of the family, their physiological function remains unclear.…”

“…Some ABC-F have a clear physiological function such as the antibiotic resistance ABC-F (ARE ABC-F) factors 9,10,12,14–20,32,33 which provide resistance toward antibiotics that target the PTC and the nascent peptide exit tunnel (NPET) of the ribosome 9,10,12,16,17,19,20 . However, for most of the family, their physiological function remains unclear.…”

An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance

“…For example, CRM arrested the growth of an E. faecalis isolate known to encode both Erm and LsaA-resistance proteins ( 37 ) (Fig. 2, AR-0671, MIC ≤ 0.06 versus 0.5 μg·ml –1 ), as well as a C. difficile isolate encoding both Erm and CplR-resistance proteins ( 38 ) (Fig. 2, AR-1072, MIC = 2 versus 16 μg·mL –1 ).…”