MAB_2355c Confers Macrolide Resistance in Mycobacterium abscessus by Ribosome Protection

Abstract

Macrolide resistance is always a concern when treating Mycobacterium abscessus infections. MAB_2355c was identified previously as a possible new factor that confers the intrinsic resistance of 194 clinical M. abscessus isolates to clarithromycin. Herein, the potential mechanism by which MAB_2355c exerts macrolide resistance was explored by bioinformatics analysis, MAB_2355c cloning and protein purification, ATP hydrolysis assay, gene knockout and complementation, antibiotic sensitivity, and transcription-translation assays. MAB_2355c is a putative ATP-binding cassette F (ABC-F) family protein. Purified MAB_2355c protein exhibits ATP hydrolysis activity, which can be inhibited by ribosome-targeting antibiotics. MAB_2355c mRNA expression is upregulated more significantly after exposure to macrolides than after exposure to other ribosome-targeting antibiotics. MAB_2355c deleted strains showed increased sensitivity to macrolides, which was reduced by MAB_2355c complementation. Finally, MAB_2355c rescued the transcription and translation activities affected by macrolides in vitro. These findings suggest that MAB_2355c confers the resistance of M. abscessus to macrolides by ribosome protection, thus complementing other known resistance mechanisms.

Keywords: ABC-F protein; MAB_2355c; Mycobacterium abscessus; macrolides; resistance; ribosome.

FIG 1

MAB_2355c exhibits similarities to ABC-F subfamily proteins. (A) Schematic diagram of the conserved domains of MAB_2355c predicted by the Pfam database. ABC_tran (PF00005) and ABC_tran_Xtn (PtIM, PF12848) were found in MAB_2355c. (B) The amino acid sequences of the ABC-F subfamily proteins were aligned and shaded using DNAMAN software. Dark blue represents 100% homology, and light blue represents 75% homology. The amino acid sequences used for alignment include MAB_2355c (GenBank accession numbers CAM62436), Rv2477c (CCP45271), MsrA (ADM29228), and EttA (P0A9W3). The annotation above the MAB_2355c sequence indicates the following conserved motifs: Walker A, ABC signature, Walker B.

FIG 2

Characterization of purified MAB_2355c ATPase activity and changes in MAB_2355c expression after exposure to ribosome-targeting antibiotics. (A) Coomassie-stained SDS-PAGE confirmed the accuracy of MAB_2355c induction and purification. MW, molecular weight marker. Lane 1, before IPTG induction; lane 2, after IPTG induction; and lane 3, purified MAB_2355c protein. (B) The MAB_2355c protein exhibits ATP, TTP, CTP, and GTP hydrolase activity. (C) MAB_2355c protein (1 μM) was preincubated with 1 mM antibiotic for 15 min at room temperature prior to the addition of ATP. ATP hydrolysis was quantified after deducting the background (solvent rather than antibiotic added). Data in panels C and D represent the mean ± standard deviation (SD), and statistical significance was analyzed by one-tailed t test versus buffer controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Fold MAB_2355c transcript induction in wild-type M. abscessus ATCC 19977 after 30 min and 3 h exposure to 0.5 MIC ribosome-targeting antibiotics. The results are expressed as fold increased expression compared to unexposed samples. Data represent the mean ± SD, sigA was used as an endogenous reference gene. All experiments were repeated independently three times.

FIG 3

Construction of knockout and complementation strains. (A) Schematic representation of the MAB_2355c deletion created using phage recombineering. (B) Using LYZFP/LYZRP and RYZFP/RYZRP primer pairs, 1,270 bp LYZ and 1,292 bp RYZ were amplified, respectively, confirming MAB_2355c deletion; the knockout strain (MUT) was used as a template. No targeted DNA fragment was amplified using the wild-type strain (WT) as a template. (C) Three complementation clones were randomly selected (numbers 1 to 3), and the JDP DNA fragment was amplified using the JDLP and JDRP primers and confirmed by sequencing.

FIG 4

MAB_2355c deletion renders M. abscessus sensitive to macrolides. Ten-fold dilutions of ATCC 19977 wild-type, ATCC 19977ΔMAB_2355c mutant, and ATCC 19977ΔMAB_2355c:pMV361_MAB_2355c complementation strains were spotted onto Middlebrook 7H10 agar plates containing the indicated antibiotic concentrations. MAB_2355c deletion rendered M. abscessus ATCC 19977 more sensitive than the wild-type parental strain to erythromycin, azithromycin, clarithromycin, and linezolid. An integrated, constitutively expressed copy of MAB_2355c in the complementation strain partially restored antibiotic resistance.

FIG 5

MAB_2355c rescues in vitro transcription-translation from erythromycin inhibition in a dose-dependent manner. (A) Erythromycin inhibitory activity profile in an in vitro coupled transcription-translation luciferase assay. (B) Transcription-translation activity in the absence of erythromycin and MAB_2355c (column 1) or the presence of 3 μM erythromycin (E) and increasing concentrations of MAB_2355c protein (columns 2 to 5). Results are the means ± SD of data from three independent experiments.