Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance

Abstract

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.

Keywords: HflX; Mycobacterium abscessus; erm41; macrolides; ribosome.

Fig. 1.

Deletion of M. abscessus hflX confers macrolide-lincosamide sensitivity. (A–C) Ten-fold serial dilutions of M. abscessus ATCC 19977, ΔMab_hflX, ΔMab_whiB7, ΔMab_erm41, ΔMab_hflX/ΔMab_erm41, ΔMab_1846, ΔMab_2355, and the indicated complementing strains were grown to A₆₀₀ of 0.7 and spotted on Middlebrook 7H10 OADC containing 20 μg/mL erythromycin, 1 μg/mL clarithromycin, 10 μg/mL azithromycin, or 400 μg/mL clindamycin.

Fig. 2.

Nucleotide-dependent dissociation of 70S ribosomes by Ms-HflX in vitro. (A–F) Dissociation of 70S ribosomes (0.2 μM) was carried out in the presence of 3.0 μM Ms-HflX_6his in HMA-7 buffer in the presence of 1 mM GTP, ATP, or GMP-PNP at 37 °C for 45 min and examined using a 5-mL analytical 10% to 40% SDGC. Reactions lacking either nucleotide or HflX were included as controls. Percentage area under the curve was calculated for 70S and 50S peaks, using PeakChart (v. 2.08, Brandel), and expressed as a ratio of 70S:50S. Data represent mean ± SD, n = 3. (G) The samples were collected using the Brandel Teledyne ISCO gradient fractionation system, methanol-chloroform precipitated, followed by immunoblotting with anti-his antibody to determine the presence of Ms-HflX_6his in each fraction.

Fig. 3.

(A) In vivo association of Ms-HflX with ribosomes. An M. smegmatis strain in which Ms-HflX was C-terminally tagged with the 3X-FLAG epitope at its native chromosomal location was grown to an OD of 0.7 and treated with either 20 μg/mL erythromycin or 16 μg/mL clindamycin for 1 h. Untreated cells were used as a control. A total of 50 pmoles crude ribosomes isolated from each sample were loaded on a 10-mL, 10% to 40% sucrose gradient, followed by ultracentrifugation in an SW41 rotor. Samples were collected from top to bottom on a Brandel fractionation system, and the distribution of endogenous Ms-HflX_FLAG in ribosome fractions was analyzed by immunoblotting, using anti-FLAG antibody. (B) Ribosome profile of ΔMs_hflX compared with wild-type bacteria. Wild-type M. smegmatis and ΔMs-HflX strains were grown to an OD of 0.7 and treated with 20 μg/mL erythromycin for 1 h. Untreated cells were used as a control. Crude ribosomes were prepared from each sample, and equal quantities (50 pmoles) were loaded on 10-mL, 10% to 40% sucrose gradients. After ultracentrifugation, the samples were fractionated using the Brandel Teledyne gradient fractionation system, and results were normalized based on area under the curve (AUC). AUC values for WT-Untreated, WT-ERT treated, ΔhflX-untreated, and ΔhflX-ERT treated were 87.42, 89.66, 86.81, and 87.23, respectively. Polysome profile of erythromycin-treated samples are in blue, and untreated samples are shown in black.

Fig. 4.

Ribosome splitting function of Ms-HflX correlates with its ability to confer antibiotic resistance. (A) A structural model of Ms-HflX guided by the structure of E. coli HflX was obtained using I-TASSER and overlaid on the structure of E. coli HflX (orange) using PyMOL (https://pymol.org/2/). The Ms-HflX model is color coded by domains, as shown on the Right. (B) Location of truncations are indicated. (C–G) Dissociation of 70S ribosomes (0.2 μM) was carried out in the presence of 3.0 μM of either full-length Ms-HflX_6his, Ms-HflXΔNTD_6his, Ms-HflXΔCTD_6his or Ms-HflX(K258A/S259A)_6his in HMA-7 buffer containing 1 mM GMP-PNP at 37 °C for 45 min and examined using a 5-mL analytical 10% to 40% SDGC and Brandel gradient fractionation. Percentage AUC was calculated for 70S and 50S peaks, using PeakChart (v. 2.08, Brandel), and expressed as a ratio of 70S:50S. Data represent mean ± SD, n = 3. (H) Wild-type, ΔhflX mutant, and complementing strains containing the respective HflX-ΔNTD_, HflX-ΔCTD, and HflX(K258A/S259A) at either the Bxb1 attB site of ΔMs_hflX or the L5 attB site of ΔMab_hflX were assayed for growth on Middlebrook 7H10 containing indicated concentrations of antibiotics. Expression of HflX-ΔNTD and HflX-ΔCTD in the complementing strain was verified using real-time PCR (SI Appendix, Table S2).

Fig. 5.

Overexpression of the ribosome recycling factor (Ms-RRF) partially restores macrolide sensitivity of ΔMs_hflX. Complementing strains were created by integrating either Ms_RRF or Ms_hflX at the Bxb1 attB site of ΔMs_hflX. Expression of Ms-RRF in the complementing strain was verified using real-time PCR (SI Appendix, Table S2). Tenfold serial dilutions of wild-type M. smegmatis, ΔMs_hflX, and the complementing strains were grown to A₆₀₀ of 0.7 and spotted on Middlebrook 7H10 ADC containing indicated concentrations of macrolides.

Fig. 6.

Model of mycobacterial HflX-mediated antibiotic resistance.