Ribosome Protection as a Mechanism of Lincosamide Resistance in Mycobacterium abscessus

ABSTRACT

Mycobacterium abscessus has emerged as a successful pathogen owing to its intrinsic drug resistance. Macrolide and lincosamide antibiotics share overlapping binding sites within the ribosome and common resistance pathways. Nevertheless, while M. abscessus is initially susceptible to macrolides, they are completely resistant to the lincosamide antibiotics. Here, we have used RNA sequencing to determine the changes in gene expression in M. abscessus upon exposure to the lincosamide, clindamycin (CLY). We show that Mab_1846, encoding a putative ARE-ABCF protein, was upregulated upon exposure to macrolides and lincosamides but conferred resistance to CLY alone. A Mycobacterium smegmatis homologue of Mab_1846, Ms_5102, was similarly found to be required for CLY resistance in M. smegmatis. We demonstrate that Ms5102 mediates CLY resistance by directly interacting with the ribosomes and protecting it from CLY inhibition. Additional biochemical characterization showed that ribosome binding is not nucleotide dependent, but ATP hydrolysis is required for dissociation of Ms5102 from the ribosome as well as for its ability to confer CLY resistance. Finally, we show that in comparison to the macrolides, CLY is a potent inducer of Mab_1846 and the whiB7 regulon, such that exposure of M. abscessus to very low antibiotic concentrations induces a heightened expression of erm41, hflX, and Mab_1846, which likely function together to result in a particularly antibiotic-resistant state.

INTRODUCTION

Mycobacterium abscessus is the most frequently encountered rapidly growing nontuberculous mycobacterium (NTM) causing pulmonary disease in patients with chronic lung damage (1, 2). Treatment of M. abscessus infections is challenging due to its intrinsic resistance to most FDA-approved antibiotics (3, 4). The current regimen involves a combination of an oral macrolide in conjunction with amikacin and one or more of the injectables cefoxitin, imipenem, and tigecycline for a period of 9 to 12 months (2, 5). The antibiotics used for therapy are noxious, and despite their use, the average rate of eradication is only ∼45% (6–8). A particularly vexing aspect of M. abscessus therapy is that there is poor correlation between in vitro antibiotic susceptibility and in vivo efficacy, a large part of which is attributed to induction of resistance genes upon exposure to sublethal doses of an antibiotic (2, 9–12). Inducible resistance is known to contribute to limited efficacy of the macrolides (12–14) and unavailability of several other antibiotics, most prominently the tetracyclines (15) and the rifamycins (3, 16–19). The expression of antibiotic-inducible pathways can be either dependent or independent of the master regulator, whiB7 (20).

The bacterial ribosome is a target of the vast majority of antibiotics currently in use which bind at surprisingly few locations. The peptidyl transferase center (PTC) and the adjacent nascent polypeptide binding site (NPET) in the 50S subunit is one of the main regions in the ribosomes that is bound by the macrolide, lincosamide, streptogramin, ketolide, and oxazolidinone classes (MLSKO) of antibiotics (21–23). Bacterial resistance to the MLSKO antibiotics is known to be mediated in 3 ways as follows: (i) mono- or dimethylation of the A2058 residue located within the conserved domain V of the 23S rRNA impairs drug binding and is the most widespread mechanism that provides cross-resistance to MLSK antibiotics, (ii) drug inactivation by esterases, lyases, transferases, and phosphorylases confer resistance to structurally related antibiotics, and (iii) antibiotic efflux by the resistance/nodulation/division (RND) family and the major facilitator superfamily (MFS) (21). Antibiotic efflux by members of the ARE-ABCF subfamily of proteins (antibiotic resistance ATP binding cassette, subfamily F) was long considered a major mechanism of resistance to the MLSKO antibiotics in Gram-positive pathogens, despite their lacking a transmembrane domain. However, studies over the last 6 years have unequivocally refuted the efflux mechanism and have demonstrated that ARE-ABCF proteins bind to the ribosome and function as ribosome protection proteins (24–27). In depth sequence analyses show that ARE-ABCFs belong to the ABC superfamily of proteins and consist of twin ABC cassettes or nucleotide binding domains (NBD) separated by a linker region (28, 29). Each ABC cassette consists of the characteristic motifs as follows: Walker A, Walker B, an LSGGQ signature, D loop, and H loop motifs (22). The linker or antibiotic resistance domain (ARD) varies considerably in length and sequence. Cryoelectron microscopy (CryoEM) structures of VmlR from Bacillus subtilis and MsrE from Pseudomonas aeruginosa show that these proteins bind to the vacant E-site of the ribosome, and the ARD is inserted deep into the PTC and NPET and overlaps with the lincosamide and macrolide binding sites, respectively (25, 26). No single ABCF protein confers concomitant resistance to all MLSKO antibiotics but are instead grouped into 3 categories that mediate resistance to the following: (i) lincosamide and group A streptogramins (represented by vmlR, vga, lsa, and sal genes), (ii) macrolides, ketolides, and group B streptogramins (msr-type genes), and (iii) oxazolidinones (optrA genes) (30).

One of the earliest genes expressed in M. abscessus in response to ribosome targeting antibiotics is the transcriptional activator whiB7, which in turn upregulates the expression of ∼80 genes, including erm41, hflX, and Mab_2355c (20, 31). Erm41 methylates the A2058 residue of 23S rRNA preventing macrolide/lincosamide binding, and HflX dissociates ribosomes stalled in the presence of these drugs (11, 31); these comprise known intrinsic resistance determinants to both classes of antibiotics. Mab2355c was recently demonstrated to contribute to macrolide resistance in M. abscessus by a ribosome protection mechanism (32). Despite sharing overlapping binding sites and common mechanisms of intrinsic resistance, the susceptibility of M. abscessus to macrolides and lincosamides is vastly different. M. abscessus isolates appear initially sensitive to macrolides but rapidly become resistant upon macrolide exposure, limiting treatment efficacy (33). In contrast, M. abscessus shows high resistance to lincosamides, making them completely unavailable for therapy. To understand the basis of high-level lincosamide resistance, we performed a transcriptomic analysis of M. abscessus exposed to the lincosamide, clindamycin (CLY). This showed an ∼1,000× induction of Mab_1846, consistent with our previous findings showing CLY hypersensitivity of ΔMab_1846 (31). Herein, we have performed a biochemical characterization of Ms5102, a functional homologue of Mab1846 in Mycobacterium smegmatis. We demonstrate that Ms5102 binds directly to ribosomes and rescues the activity of CLY-inhibited ribosomes, thereby functioning as a bona fide ribosome protection protein.

RESULTS

Mab_1846 is highly induced upon CLY exposure and specifically confers CLY resistance.

To decipher the high-level lincosamide resistance in M. abscessus, we determined the transcriptional changes accompanying exposure to various sublethal doses of the lincosamide, clindamycin (CLY), using RNA sequencing (RNA-seq). A representative RNA-seq analysis of cells exposed to 0.05× MIC (10 μg/ml) of CLY for 30 min (Fig. 1a; see also Data Set S1 in the supplemental material) shows a >1,000-fold induction of whiB7 and Mab_1846 expression. Consistent with our previous studies, we also observed an induction of erm41, hflX, and Mab_2355c; however, while Δerm41 and ΔhflX strains are known to display CLY sensitivity, we find that the ΔMab_2355c strain does not (Fig. 1c; Table 1) (20, 31). Moreover, in contrast to the WhiB7-dependent expression of erm41 and hflX, significant levels of Mab_1846 induction by CLY continued to occur even in a ΔwhiB7 deletion strain, suggesting that Mab_1846 expression may be augmented by WhiB7 but is not dependent on it (Fig. 1b) (31).

FIG 1.

Mab_1846 is highly induced upon CLY exposure and confers CLY resistance. (a) Volcano plot of differentially expressed genes in M. abscessus ATCC 19977 upon exposure to CLY (10 μg/ml for 30 min). whiB7 and Mab_1846 are the two most highly induced genes and are circled. (b) Expression of whiB7, Mab_1846, erm41, and Mab_2355c upon exposure to CLY (10 μg/ml for 30 min) in a WT and ΔwhiB7 strain background was determined using quantitative PCR (qPCR) and expressed as fold-change over an unexposed control. Data represent mean ± standard deviation (SD); n = 3. sigA was used as an endogenous control. Statistical significance was analyzed by one-tailed t test between indicated samples. *, P < 0.05; **, P < 0.01; ns, not significant. (c) The sensitivity of the WT Mab, ΔMab1846, ΔMab1846+pMab1846, and ΔMab2355c strains to CLY was estimated by spotting 10-fold serial dilutions of an OD₆₀₀ of 0.8 of the strains onto a Middlebrook 7H10 OADC plate containing indicated concentration of antibiotics. Data are representative of >5 replicates. (d) M. smegmatis mc²155, ΔMs5102, ΔMs5102+pMs5102, and ΔMs3140 (a homologue of Mab_2355c) strains were similarly spotted on a Middlebrook 7H10 ADS plate with indicated concentration of antibiotics. Data are representative of >5 replicates. ΔMab1846 and ΔMs5102 strains are specifically sensitive to CLY, and the mutant phenotype can be successfully complemented by the constitutive expression of the respective genes.

TABLE 1.

Survival of wild-type ATCC 19977 and mutant M. abscessus strains in a 2-fold dilution series of antibiotics in Middlebrook 7H9 medium^a

Antibiotic	MIC (μg/ml) for:
	WT Mab strain	ΔMab_whiB7 strain	ΔMab_erm41 strain	ΔMab_1846 strain	ΔMab_2355 strain
Erythromycin	2	0.625–0.125	0.25	2	1
Clarithromycin	1	0.0625	0.125	1	0.5
Azithromycin	8	0.5	2	8	2
Clindamycin	200	12.5	25	25	200

^aThe minimum concentration of antibiotic required to inhibit 99% of growth is shown. Data is representative of 3 independent experiments.

Previously, we showed that a ΔMab_1846 strain was sensitive to CLY on Middlebrook 7H10 agar (31). Constitutive expression of Mab_1846 from a chromosomally integrated location in the ΔMab_1846 strain restored CLY sensitivity to wild-type levels (Fig. 1c). The MIC of CLY determined by broth dilution was 8-fold lower for the ΔMab_1846 mutant compared to that of wild-type M. abscessus (Table 1). Since Mab_1846 was also induced in the presence of macrolides (Data Set S1), we determined the susceptibility of the ΔMab_1846 strain to three macrolide antibiotics—erythromycin (ERT), clarithromycin (CLR), and azithromycin (AZI). As seen in Fig. S1a in the supplemental material and Table 1, the macrolide susceptibility of the ΔMab_1846 strain was indistinguishable from wild-type (WT) bacteria. Susceptibility of the ΔMab_1846 strain to other ribosome-targeting antibiotics was also unchanged (Fig. S1a). Additionally, we constructed an isogenic deletion of Ms_5102, the homologue of M. abscessus Mab_1846, in M. smegmatis. The ΔMs_5102 mutant displayed an ∼16-fold hypersensitivity to CLY, which could be restored by constitutive expression of Ms_5102 from a chromosomally integrated copy (Fig. 1d; Table 2).

TABLE 2.

Survival of wild-type and mutant M. smegmatis in a 2-fold dilution series of antibiotics in Middlebrook 7H9 medium^a

Antibiotic	MIC (μg/ml) for:
	WT M. smegmatis	ΔMs_5102 strain
Erythromycin	2.5	2.5
Clarithromycin	0.25	0.25
Azithromycin	2	2
Clindamycin	16	1

^aThe minimum concentration of antibiotic required to inhibit 99% of growth is shown. Data are representative of 3 independent experiments.

Ms5102 directly interacts with ribosomes and offers ribosome protection from CLY inhibition in in vitro translation assays.

A BLASTP analysis of Mab_1846 showed sequence similarity to the ABCF subfamily of proteins. Multiple sequence alignment of Mab_1846 showed the presence of all conserved motifs within twin ABC domains, as well as a distinct lack of a transmembrane domain (Fig. 2a; see also Fig. S2 in the supplemental material). ABCF proteins were long thought to be involved in antibiotic efflux owing to the presence of the ABC cassette but were subsequently shown to be ribosome protection proteins. To determine if Mab_1846 interacts with the ribosome, we attempted to overexpress and purify this protein. However, after several attempts, we were not able to obtain Mab1846 in the soluble fraction. Since Ms_5102 and Mab_1846 are functionally homologous, we overexpressed and successfully purified soluble Ms5102 protein. Consistent with the presence of ABC domains, purified Ms5102 displayed both ATPase and GTPase activities (Fig. 2b). The interaction of Ms5102 with M. smegmatis ribosomes was then analyzed using cosedimentation through a sucrose cushion. As seen in Fig. 2c, Ms5102 was capable of interacting with 70S ribosomes in the absence of added nucleotide. However, addition of ATP, but not the nonhydrolysable adenylyl-imidodiphosphate (AMP-PNP), resulted in a reduced binding of Ms5102 to 70S ribosomes, suggesting that ATP hydrolysis results in dissociation of Ms5102 from the ribosomes (Fig. 2c).

FIG 2.

Ms5102 directly interacts with ribosomes and rescues ribosomes from CLY inhibition. (a) Domain organization of Ms5102 containing two nucleotide binding domains flanking an antibiotic resistance domain (ARD) is shown. The location of conserved motifs is indicated. (b) Buffer subtracted ATPase and GTPase activity of WT Ms5102 is shown. Data represent mean ± SD; n = 3. (c) Association of purified WT Ms5102 with 70S M. smegmatis ribosome was examined using cosedimentation through a 33% sucrose cushion followed by Western blotting using an anti-His monoclonal antibody. Data are representative of 3 replicates. (d) Effect of addition of a 10-fold molar excess of Ms5102 to 70S ribosomes inhibited with a 50% inhibitory concentration of CLY is shown. Control reactions lacking CLY and Ms5102 are included. Data represent mean ± SD; n = 3. Statistical significance was analyzed by one-tailed t test between indicated samples.

The ABCF proteins such as Staphylococcus aureus VgaA and LsaA, Pseudomonas aeruginosa MsrE, and Mycobacterium abscessus Mab2355c have previously been shown to protect their respective translation apparatus from antibiotic inhibition (24, 26, 32). To determine if Ms5102 behaves similarly, we assayed Ms5102 for its ability to protect purified M. smegmatis ribosomes from CLY inhibition using the Δ ribosome transcription-translation system (NEB) and purified M. smegmatis 70S ribosomes. Addition of a 10-fold molar excess of Ms5102 to 70S ribosomes inhibited with a 50% inhibitory concentration of CLY restored the activity to that of the untreated control (Fig. 2d).

ATP hydrolysis and the linker domain are required for CLY resistance.

To determine the role of ATP hydrolysis on the ability of Ms5102 to interact with ribosomes and influence CLY resistance, we constructed point mutations in the conserved catalytic glutamic acid residues within both the Walker B motifs (E165Q/E483Q), designated Ms5102-EQ₂. Walker B EQ₂ mutations in VmlR have previously been shown to result in minimal ATPase activity without interfering with ATP binding (25). The binding of Ms5102-EQ₂ to the ribosome remained unaffected in the presence of ATP due to the inability of Ms5102-EQ₂ to hydrolyze ATP, which suggested that ATP hydrolysis is required for dissociation of Ms5102 from 70S ribosomes (Fig. 3a). Interestingly, the Ms5102-EQ₂ mutant was also incapable of restoring the CLY sensitivity of the ΔMs_5102 strain (Fig. 3c).

FIG 3.

ATP hydrolysis and the linker domain are required for CLY resistance. (a) Association of purified Ms5102-EQ2 and Ms5102-mLinker with 70S M. smegmatis ribosome was examined using cosedimentation through a 33% sucrose cushion followed by Western blotting using an anti-His monoclonal antibody. Data are representative of 3 replicates. (b) Model of Ms5102 (magenta) using B. subtilis VmlR (green) as a template are superimposed on the CryoEM structure of the 70S ribosome of M. smegmatis. Location of CLY in the PTC is indicated in the box and is zoomed out for clarity. The residues of Ms5102 that are replaced by the residues of EttA in mLinker are shown and are indicated in blue. (c) Growth of a 10-fold serial dilution of a 0.7 OD₆₀₀ culture of M. smegmatis mc²155, ΔMs5102 strain, and the complementing strains containing the pMs5102, pMs5102EQ₂, and pMs5102-mLinker plasmids integrated at the Bxb1 attB site of the ΔMs5102 strain on Middlebrook 7H10 containing 8 μg/ml CLY is shown. Data are representative of >5 replicates.

The linker/ARD domains of ARE-ABCF proteins fold into two α-helices that traverse the E site and penetrate into the NPET/PTC. The sequence composition and length of the ARD is known to determine the specificity of antibiotic resistance. A model of Ms5102 was generated using Swiss-Model using VmlR as the template and superimposed on the structure of M. smegmatis 70S ribosome (PDB accession number 5O61). The crystal structure of the Escherichia coli ribosome bound to CLY (PDB accession number 4V7V) was used to locate CLY on the M. smegmatis 70S ribosome (Fig. 3b). As seen in Fig. 3b, the loop region between the two Ms5102 linker α-helices is located within the PTC in close proximity to the CLY binding site and can presumably interfere with CLY binding. We constructed a mutant of Ms5102 (Ms5102-mLinker), where 17 residues (amino acids [aa] 265 to 281) of the loop (in blue) were deleted and replaced with 8 amino acids that constitute the EttA loop, resulting in a shortened loop region (Ms5102-mLinker) with altered amino acid composition (Fig. 3b). Alteration of the loop resulted in a protein incapable of restoring the antibiotic sensitivity of ΔMs_5102 (Fig. 3c), underscoring the importance of the Ms5102 ARD in CLY resistance. Binding characteristics of Ms5102-mLinker to the 70S ribosome was however unaltered (Fig. 3a).

CLY is an exceptionally potent inducer of antibiotic resistance genes.

To study the basis of differential sensitivity of M. abscessus to macrolide and lincosamide antibiotics, we compared the transcriptional changes accompanying exposure to various sublethal doses of CLY and to the macrolides, erythromycin (ERT) and clarithromycin (CLR), using RNA sequencing (RNA-seq). A representative sample of transcriptional changes occurring upon exposure of M. abscessus to 10 μg/ml CLY (0.05× MIC), 10 μg/ml ERT (5× MIC), and 2 μg/ml CLR (2× MIC) is presented in Data Set S1. Figure 4a to c show that low CLY concentrations (0.025 to 0.05× MIC) for short time periods are sufficient to strongly induce Mab_1846, whiB7, and the whiB7-regulated erm41. In comparison, macrolides are weaker inducers of whiB7, requiring higher antibiotic concentrations (2 to 5× MIC) and greater exposure times (3 h) in order to obtain a similar induction of whiB7 and its regulon (Fig. 4a and b; see also Data Set S1). This strong upregulation of Mab_1846 and the whiB7-dependent erm41 may contribute, at least in part, to the high-level lincosamide resistance of M. abscessus.

FIG 4.

CLY is an exceptionally strong inducer of Mab_1846 and the WhiB7 regulon. (a) Heat map showing fold changes in expression of whiB7, Mab_1846, erm41, and hflX when exposed to CLY, ERT, and CLR for 30 min and 3 h determined using RNA-seq. The P_adj values are available in Data Set S1 in the supplemental material. (b) Expression of MAB_whiB7, Mab_1846, erm41, and Mab_2355c was determined as a function of CLY and ERT concentration over a fixed exposure time (30 min) using qPCR and is expressed as a fold change over the unexposed control. (c) Time course of induction of Mab_whiB7 and Mab_1846 upon CLY (10 μg/ml = 0.05× MIC) and ERT (10 μg/ml = 5× MIC) exposure were determined using qPCR. Data represent mean ± SD; n = 3. sigA was used as an endogenous control.

DISCUSSION

In the present study, an RNA-seq analysis demonstrated that Mab_1846 expression is rapidly turned on upon exposure to very small concentrations of CLY (0.05× MIC) and is consistent with the CLY hypersensitivity of the ΔMab_1846 strain. An earlier study by Guo et al. demonstrated that Mab_1846 and Mab_2355c were CLR inducible and concluded that they were involved in CLR efflux due to the presence of conserved ABC protein motifs (34). Our results show that although CLR/ERT can induce the expression of an Mab_1846, a ΔMab_1846 strain is hypersensitive only to CLY but not to CLR, ERT, or AZI. Ms_5102, a homologue of Mab_1846 similarly confers CLY resistance but not ERT/CLR resistance in M. smegmatis. A previous RNA-seq study in our laboratory comparing the expression profile of the ΔMab_whiB7 strain and the complemented strain in the absence of antibiotic had suggested that Mab_1846 is part of the WhiB7 regulon (20). Interestingly, we note that while the low-level macrolide-induced expression of Mab_1846 appears to be dependent on WhiB7, the high-level CLY-induced expression of Mab_1846 appears to be largely, but not entirely, independent of WhiB7, suggesting the presence of additional mechanisms of CLY-mediated Mab_1846 induction (Fig. 1b; see also Fig. S3 in the supplemental material).

Furthermore, our results unequivocally demonstrate that Ms5102 is a cytoplasmic protein that binds 70S ribosomes and rescues it from CLY inhibition either by protecting it from CLY binding or displacing ribosome-bound CLY, thereby confirming its role as an ARE-ABCF protein. Mab2355c similarly has also been recently shown to function as a ribosome protection factor in macrolide resistance (32). Although Mab_2355c expression is induced upon CLY exposure (see Data Set S1 in the supplemental material; Fig. 4b), it functions solely in macrolide resistance and does not influence CLY resistance (Table 1; see also Fig. S1 in the supplemental material). The ability of Mab1846 and Mab2355c to confer resistance to distinct antibiotics is not unexpected, as most ARE-ABCF proteins are known to confer resistance to a distinct subset of antibiotics; this specificity has been attributed to the length and composition of the linker region that enable them to penetrate different portions of the ribosome (30). A model of Ms5102, using the B. subtilis VmlR protein that confers lincosamide resistance, shows that Ms5102 interacts in a similar manner to position the loop interspersed between the two α-helices of the linker within the PTC in proximity to CLY. Modification of the loop abrogates the ability of Ms5102 to restore CLY resistance of a ΔMs_5102 strain, underscoring the significance of the loop in CLY resistance.

The role of ATP hydrolysis in binding of ABCF proteins to the ribosome and its influence on antibiotic resistance also shows considerable variability. Mutation of the catalytic glutamines of the Walker B motifs in NBD1 and NBD2 of VmlR and EttA results in ribosome binding, whereas the MsrE protein displays ribosome binding in the presence of ATP as well as the nonhydrolysable analogue AMP-PNP (25, 26, 35). Additionally, while mutations in the catalytic glutamates of VgaA abrogate its ability to confer virginiamycin M resistance, macrolide resistance mediated by LmrC appears to be independent of ATP hydrolysis (36, 37). Mutation of the catalytic glutamate residues within Walker B of Ms5102 completely abolished its ability to mediate CLY resistance. Moreover, while Ms5102 binding to ribosomes is not dependent on ATP, addition of ATP results in a decrease in ribosome association of WT Ms5102; binding of Ms5102-EQ2 is unaffected in the presence of ATP. Together these observations suggest that ATP hydrolysis is required for dissociation of Ms5102 as well as CLY from the ribosomes.

Macrolide and lincosamide resistance in M. abscessus therefore appears to be mediated by the common determinants, erm41 and hflX, and distinct ribosome protection proteins but are insufficient to fully explain the vast difference in susceptibility to these antibiotics. Our results demonstrate that <0.05× MIC of CLY brings upon rapid induction of Mab_1846, and the whiB7 regulated erm41 and hflX. In comparison, macrolides are weaker inducers of the whiB7 regulon, requiring higher drug concentrations (2 to 5× MIC) over longer time periods to achieve a similar level of whiB7 and, consequently, erm41 and hflX expression. While it is possible that additional mechanisms of CLY resistance exist, we speculate that the difference in strength of induction of resistance determinants may provide an explanation as to why M. abscessus displays initial susceptibility to macrolides but is resistant to lincosamides. Previously, we showed that amikacin, one of the most effective antibiotics against M. abscessus, is also the poorest inducer of whiB7 and the coregulated eis2 required for amikacin resistance (20). The strength of whiB7 induction can therefore serve as a useful marker for predicting the susceptibility of M. abscessus to a given ribosome-targeting antibiotic. Moreover, since the whiB7 regulon contains resistance determinants for most ribosome targeting antibiotics, treatment with a strong inducer of whiB7 early in therapy can negatively impact the efficacy of other ribosome targeting antibiotics within the regimen. The strength of whiB7 induction by an antibiotic therefore also provides an indication of the preferred timing of its administration within a multidrug regimen.

In conclusion, M. abscessus employs multiple mechanisms of resistance to CLY, all of which are rapidly turned on upon exposure to very low antibiotic concentrations. In addition to the well-accepted mechanism of lincosamide resistance by erm41-mediated target modification, ribosome protection constitutes a significant and previously undescribed mechanism of resistance to the lincosamide antibiotics in M. abscessus. The genome of M. abscessus encodes >40 ATP binding ABC transporters; a future in-depth analysis will help to distinguish if these ABC proteins function in efflux or function as ribosome protection factors.

MATERIALS AND METHODS

Media and strains.

The bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. Gene replacement mutants were constructed using recombineering as described previously (20). The recombineering construct was generated by cloning in the multiple cloning sites flanking the apramycin cassette of pYUB854. Mutant clones were checked using F_check and R_check primers flanking the deletion site. Mycobacterium smegmatis mc²155 and the mutant strains were grown in Middlebrook 7H9 (Difco) supplemented with 1% ADC, 0.5% glycerol, and 0.05% Tween 20. Mycobacterium abscessus ATCC strain and the mutant strains was grown in Middlebrook 7H9 (Difco) supplemented with 1% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 20. E. coli strains were grown in Luria-Bertani broth or LB agar. E. coli and mycobacteria were grown at 37°C (unless stated otherwise) with shaking at 225 rpm.

Cloning and expression of mycobacterial genes.

Mab_1846 was PCR amplified from MAB ATCC genomic DNA and cloned in pMH94 under a constitutively expressed hsp60 promoter. Similarly, Ms_5102 was cloned in the integrative vector pSJ25 under the hsp60 promoter. Complementing strains were constructed by transforming the above plasmids in their respective mutant backgrounds. Ms5102-EQ2 and Ms5102-mLinker were synthesized (Gene Universal) and cloned into pSJ25 under the control of the hsp60 promoter.

Protein overexpression and purification.

WT and mutant Ms_5102 genes were cloned into pET21b and transformed into E. coli BL21(DE3) competent cells. The transformants were grown to an optical density at 600 nm (OD₆₀₀) of 0.6 in 250 ml LB medium at 30°C and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 3 h at 30°C. The hexahistidine-tagged protein was purified with Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography by harvesting the cells by centrifugation at 5,000 × g for 15 min at 4°C, followed by lysing the cells with sonication in buffer A (50 mM Tris, 300 mM NaCl, 5% glycerol) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.25% sodium deoxycholate. The cells were centrifuged at 18,000 × g for 20 min. The clear supernatant was then loaded with 5 mM imidazole onto a Ni-column preequilibrated in buffer A plus 5mM imidazole. The column was incubated with protein at 4°C for 1 h. on a rocker with slow shaking, followed by washing with 20 column volumes of buffer A plus 35 mM imidazole. The protein was finally eluted in buffer A containing 100 mM imidazole and analyzed by 10% SDS-PAGE. The mutant proteins 5102 LS and 5102 EQ2 were also purified under similar conditions.

Antibiotic sensitivity assays.

All M. smegmatis strains and M. abscessus strains were grown to an A₆₀₀ of 0.8. Ten-fold serial dilutions of the cells were spotted on Middlebrook 7H10 (Difco) plates containing required concentrations of antibiotics. MICs in liquid medium were determined by inoculating the strain in a 2-fold dilution series of each antibiotic with an initial OD₆₀₀ of 0.0004. The cultures were incubated at 37°C, and the OD₆₀₀ was measured after 72 h for M. abscessus.

RNA sequencing and real-time PCR.

Wild-type M. abscessus ATCC 19977 was grown to exponential phase (0.7 OD) in Middlebrook 7H9-OADC and exposed to varying concentrations of ERT/CLR/CLY for varying periods of time (0 to 3 h) and evaluated for lethality. Total RNA was prepared from wild-type strains exposed to 10 μg/ml of CLY for 30 min, 10 μg/ml of ERT for periods of 30 min and 3 h, and 2 μg/ml of CLR for periods of 30 min and 3 h using the Qiagen RNA preparation kit followed by DNase I treatment. Unexposed samples were used as controls. Total RNA samples were treated with the Ribo-Zero rRNA removal procedure, and 500 ng of RNA was used for library preparation using the NEBNext Ultra II (NEB) RNA-seq kit and high-throughput sequencing on the Illumina NextSeq platform. The sequence data were analyzed using Rockhopper in which the data are normalized by upper quartile normalization, and transcript abundance is reported as reads per kilobase per million (RPKM). Differential gene expression is tested for each transcript, and q values are then reported that control the false discovery rate (38, 39).

Mycobacterium abscessus WT and the ΔwhiB7 mutant were exposed to different concentrations of antibiotics for varying periods of time. Total RNA was extracted using the Qiagen RNA preparation kit followed by Turbo DNase digestion. cDNA was generated using Maxima reverse transcriptase (Fisher Scientific). Reverse transcriptase PCR (RT-PCR) was performed using the cDNA and gene-specific primers (generated using Primer Quest Software from IDT) in the Applied Biosystems 7300 real-time PCR system with cycling conditions of 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Ribosome purification.

Ribosomes were purified as described previously (31). Briefly, wild-type M. smegmatis mc²155 was grown in 250 ml of 7H10 plus ADS plus Tween plus glycerol medium to an OD₆₀₀ of 0.8. The cells were harvested and lysed using The CryoMill (Retsch) in 20 mM HEPES, pH 7.5, 30 mM NH₄Cl, and 10 mM MgCl₂ (HMA-10 buffer). The samples were clarified three times by centrifugation, and crude ribosomes were collected by ultracentrifugation at 42,800 rpm for 2 h and 15 mins using a Beckman 50.2 Ti rotor followed by resuspension in HMA-8 buffer (20 mM HEPES, pH 7.5, 30 mM NH₄Cl, and 8 mM MgCl₂). Crude ribosomes were treated with RNA-seq free DNase I (Ambion) for 4 h at 4°C, layered on 33% wt/vol sucrose cushion and centrifuged using a Beckman 50.2 Ti rotor at 100,000 × g for 16 h at 4°C. Ribosome pellets were washed one time with HMA-8, resuspended, and stored in HMA-8 at −80°C.

In vitro transcription-translation assay.

A New England Biolabs Inc. PureExpress delta ribosome kit was used for the assay. A T7 promoter driven nano-luc reporter was used as the template (40). The reactions were set up as per the manufacturers protocol. To determine the concentration of CLY required to inhibit 50% ribosome activity, 20 nM purified M. smegmatis 70s ribosomes was incubated with 1, 2, 5, 10, 20, or 50 nM CLY and kept at room temperature for 15 min. Four microliters of solution A, 1.2 μl of Factor mix from the kit, and 50 ng of DNA template were then added to the reaction mixture and incubated at 37°C for 1 h. After 1 h, 2 μl from the reaction mixture was mixed with 18 μl of water, 20 μl of luciferase assay buffer, and 0.4 μl of luciferin substrate and incubated for 5 min at room temperature. The luminescence generated from the luciferase was measured in a Veritas microplate luminometer (Turner Biosystems). To determine the effect of Ms5102 on clindamycin inhibition, 20 nM purified M. smegmatis 70s ribosomes was incubated with 200 nM purified Ms5102 for 15 min at room temperature, followed by the addition of 2 nM clindamycin for an additional 10 min. The amount of luciferase activity in each sample was determined as described above.

Cosedimentation assay.

A total of 2.0 μM of purified Ms5102 protein (WT or mutant) was incubated with 200 nM purified 70s ribosome in HMA-8 buffer in a total volume of 50 μl at 37°C for 15 min. The volume was increased to 125 μl and gently layered on top of a 33% wt/vol sucrose cushion (125 μl) and centrifuged at 100,000 rpm for 2.5 h at 4°C in a TLA-100 rotor (Beckman Coulter; Optima Max-TL ultracentrifuge). The supernatant was carefully discarded, and the pelleted ribosome was reconstituted in HMA-8 buffer. The samples were loaded onto 10% SDS-PAGE gel and analyzed by Western blotting using anti-His monoclonal antibodies.

ATP hydrolysis assay.

ATPase activity of Ms5102 was determined using the colorimetric ATPase assay kit (Sigma). The ATP hydrolysis reaction was performed by incubating 0.34 μM Ms5102 with either 1 mM ATP or GTP (NEB) in an HMA-8 buffer for 30 min at room temperature followed by the addition of the malachite green reagent included in the kit. Following incubation for 30 min at room temperature to generate the colorimetric product, the absorbance of the samples was measured at 620 nm. The amount of free phosphate liberated was determined from a standard curve that was generated using phosphate standards provided in the kit.