ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

Abstract

Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition.

FIG 1

Phylogenetic tree and antibiotic resistance profiles of the ARE ABC-F proteins found in representative Gram-positive pathogens. The tree was generated using the maximum likelihood method with the MEGA 6.0.6 software package (47). An overview of the antibiotic resistance phenotypes conferred by the different subgroups of determinant is given at the right of the figure, denoted by colored boxes (although variations in individual resistance phenotypes within each subgroup are not shown).

FIG 2

Vga(A) protects an S. aureus-derived T/T assay from inhibition by virginiamycin M. (A) Column 1 shows an uninhibited T/T assay with no addition of exogenous protein, whilst column 2 shows an uninhibited assay with the addition of 4 µM Vga(A). In columns 3 and 5, 4 µM Vga(A) added to a T/T assay mixture containing ≥IC₉₀ of virginiamycin M (VGM [column 3]) rescued protein synthesis. In columns 3, 4, and 8, addition of 4 µM heat-denatured (Denat.) Vga(A) (column 4) or 4 µM fusidic acid resistance protein FusB (column 8) failed to rescue protein synthesis from inhibition by virginiamycin M (column 3). In columns 6 and 7, addition of 4 µM Vga(A) to a T/T assay mixture containing ≥IC₉₀ of fusidic acid (FA) did not rescue protein synthesis. (B) Dose-dependent rescue of protein synthesis by Vga(A) from inhibition with ≥IC₉₀ of virginiamycin M. Results are means from at least three independent determinations, and error bars show standard deviations.

FIG 3

Lsa(A) mediates dose-dependent protection of a S. aureus-derived transcription/translation assay from inhibition by virginiamycin M (A) and lincomycin (B). Results are means from at least three independent determinations, and error bars show standard deviations.

FIG 4

Recapitulation of resistance phenotypes associated with ARE ABC-F proteins in vitro. (A) When expressed in E. coli, Vga(A) does not confer resistance to virginiamycin M (21); addition of Vga(A) to an E. coli T/T assay containing ≥IC₉₀ of virginiamycin M (VGM) also failed to restore translational activity. (B) ATPase activity is essential for Vga(A) function (21), and abrogation of ATPase activity of the N-terminal ABC domain rendered Vga(A) inactive when expressed in S. aureus RN4220; the purified ATPase-deficient Vga(A)_E105Q protein also failed to protect staphylococcal translation from inhibition by virginiamycin M in vitro. (C) A single-amino-acid substitution in the interdomain linker expands the resistance spectrum of Vga(A) to encompass lincomycin (20). Addition of the purified Vga(A)_K219T to a staphylococcal T/T assay inhibited with a >IC₉₀ of lincomycin (LNC) restored translational activity, while addition of the wild-type protein did not. Results are means from at least three independent determinations, and error bars show standard deviations.

FIG 5

Lsa(A) prevents binding of lincomycin to staphylococcal ribosomes, and displaces ribosome-bound lincomycin. (A) Preincubation of increasing concentrations of Lsa(A) with 0.5 µM staphylococcal ribosomes caused a reduction in binding of [³H]lincomycin. (B) Preincubation of 0.5 µM ribosomes with a 50× excess of unlabeled lincomycin (LNC) decreased subsequent binding by [³H]lincomycin (column 2 versus 1). Preincubation with 4 µM BSA did not protect ribosomes from binding by [³H]lincomycin (columns 3 and 1). Addition of 4 µM Lsa(A) resulted in decreased association of [³H]lincomycin with ribosomes (column 4 versus 1). (C) Addition of a 50× excess of unlabeled lincomycin caused dissociation of [³H]lincomycin prebound to staphylococcal ribosomes (column 2 versus 1), as did addition of 4 µM Lsa(A) (column 4 versus 1); however, addition of BSA did not (column 3 versus 1). Results are means from at least three independent determinations, and error bars show standard deviations.