Mutations in pepQ Confer Low-Level Resistance to Bedaquiline andClofazimine in Mycobacterium tuberculosis

Deepak Almeida,a Thomas Ioerger,b Sandeep Tyagi,a Si-Yang Li,a Khisimuzi Mdluli,c Koen Andries,d Jacques Grosset,a

Jim Sacchettini,e Eric Nuermbergera,f

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAa; Department of Computer Science,Texas A&M University, College Station, Texas, USAb; Global Alliance for TB Drug Development, New York, New York, USAc; Janssen Pharmaceutica, Beerse, Belgiumd;Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texase; Department of International Health, Johns Hopkins Bloomberg School ofPublic Health, Baltimore, Maryland, USAf

The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuber-culosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multi-drug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, >4-fold upward shifts in be-daquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increasebedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presum-ably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofaziminecross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putativeXaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with orwithout clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parentalH37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant andparent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR(qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains.Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is suf-ficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer be-daquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant furtherevaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

Multidrug-resistant tuberculosis (MDR-TB) is a major threatto global control of tuberculosis (TB). When multidrug re-

sistance is not diagnosed, patients respond poorly to standardizedfirst-line regimens and additional resistance may develop. WhenMDR-TB is diagnosed, current second-line regimens require pro-longed treatment durations and are less effective, more toxic, andfar more expensive than first-line therapy (1).

The diarylquinoline drug bedaquiline (B) received acceler-ated approval from the U.S. Food and Drug Administrationas part of combination therapy for MDR-TB when otheralternatives are not available (2). It is now being studied as acomponent of novel short-course regimens for MDR aswell as drug-susceptible TB (ClinicalTrials.gov identifiersNCT02333799, NCT02193776, NCT02589782, NCT02409290,and NCT02454205 [https://clinicaltrials.gov/]). For new drugssuch as bedaquiline, it is essential to define and catalog the mech-anisms conferring bacterial resistance in order to design appropri-ate diagnostic tests (including rapid molecular tests), to bettermanage the treatment of patients who fail therapy or relapse afterreceiving the drug, and to conduct population level surveillancefor changes in drug susceptibility.

The principal mechanism of action of bedaquiline is inhibitionof the mycobacterial ATP synthase (3, 4). Strains selected in vitrofor resistance to bedaquiline often have mutations in atpE, whichencodes the ATP synthase subunit to which bedaquiline binds (5).These target-based mutations cause relatively large (i.e., 10� to128�) shifts in MIC. However, it was noted that many bedaqui-line-resistant isolates selected in vitro, typically with smaller shifts

in MIC, do not have mutations in the ATP synthase complex (5).A new non-target-based mechanism conferring low-level be-daquiline resistance and cross-resistance to clofazimine (C) wasrecently identified among isolates from patients with delayed spu-tum culture conversion while receiving bedaquiline for MDR-TB,among mice treated with bedaquiline-containing combinations,and in vitro (6, 7). Although both bedaquiline and clofazimine (7)can select for non-target-based mutants in vitro, such mutantshave not yet been isolated from mice or patients treated with clofazi-mine. The responsible mutations were found in Rv0678, which en-codes a negative regulator of mmpL5 and mmpS5. Increased tran-scription of these genes, which comprise a membrane transporter inthe resistance-nodulation-division (RND) family, is putatively asso-ciated with increased efflux of bedaquiline and clofazimine.

Identification of mutations conferring cross-resistance to clo-

Received 4 April 2016 Returned for modification 24 April 2016Accepted 7 May 2016

Accepted manuscript posted online 16 May 2016

Citation Almeida D, Ioerger T, Tyagi S, Li S-Y, Mdluli K, Andries K, Grosset J,Sacchettini J, Nuermberger E. 2016. Mutations in pepQ confer low-level resistanceto bedaquiline and clofazimine in Mycobacterium tuberculosis. Antimicrob AgentsChemother 60:4590 –4599. doi:10.1128/AAC.00753-16.

Address correspondence to Eric Nuermberger, enuermb@jhmi.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00753-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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fazimine and bedaquiline is important because clofazimine is acomponent of some short-course regimens currently under studyfor treatment of MDR-TB (8–10) and the two drugs are being usedtogether in the ongoing STREAM trial (ClinicalTrials.gov identi-fier NCT02409290 [https://clinicaltrials.gov/]). Herein, we reporta new genetic determinant of low-level bedaquiline and clofazi-mine cross-resistance in Mycobacterium tuberculosis, conferred bymutations in pepQ.

MATERIALS AND METHODSBacterial strains. M. tuberculosis H37Rv was passaged in mice, subcul-tured in Middlebrook 7H9 (Fisher Scientific) supplemented with 10%oleic acid-albumin-dextrose-catalase (OADC) complex (Becton-Dickin-son) and 0.05% Tween 80 (Sigma-Aldrich), and used for aerosol infectionwhen the optical density at 600 nm (OD600) was approximately 1.0.

Antimicrobials. Pretomanid (Pa), moxifloxacin (M), bedaquiline,and linezolid (L) were provided by the Global Alliance for TuberculosisDrug Development (New York, NY), Bayer (Leverkusen, Germany), Jans-sen (Beerse, Belgium), and Pfizer (Groton, CT), respectively. Rifampin(R), isoniazid (H), pyrazinamide (Z), ethambutol (E), and clofaziminewere purchased from Fisher or Sigma. Dosing formulations were pre-pared and maintained as previously described (11). All drugs were admin-istered once daily by gavage, 5 days per week.

Aerosol infection. Female BALB/c mice (Charles River, Wilmington,MA) aged 4 to 6 weeks were infected by the aerosol route using an inha-lation exposure system (Glas-col Inc., Terre Haute, IN). Mice were ran-domized to treatment groups (five mice per group per time point) afteraerosol infection and were routinely sacrificed (i) on the day after infec-tion to determine the number of CFU implanted in the lungs, (ii) on theday of treatment initiation to determine the pretreatment CFU count, and(iii) at selected time points during and after treatment. Quantitative cul-tures of lung homogenates were performed in parallel on selective 7H11agar with and without 0.4% activated charcoal to reduce drug carryovereffects, as previously described (11). All procedures involving animalswere approved by the Animal Care and Use Committee of Johns HopkinsUniversity.

Efficacy of combinations containing bedaquiline and clofazimine inmurine models of TB. Beginning 14 days after high-dose aerosol infec-tion, as previously described (12), BALB/c mice received no treatment(negative controls) or treatment with the first-line regimen of R-H-Z(positive controls) or one of the following test regimens: bedaquiline (25mg/kg [of body weight]) alone, the two-drug combination of bedaquilineplus clofazimine (20 mg/kg), or three-drug combinations of bedaquilineplus clofazimine plus one of the following: rifampin (10 mg/kg), isoniazid(10 mg/kg), pyrazinamide (150 mg/kg), ethambutol (100 mg/kg), moxi-floxacin (100 mg/kg), pretomanid (50 mg/kg), and linezolid (100 mg/kg).Lung CFU counts were determined for all treatment groups after 4 weeksof treatment and, for mice receiving bedaquiline alone and bedaquilineplus clofazimine, also after 6 and 8 weeks of treatment.

MIC determination. Determination of bedaquiline and clofazimineMICs and the proportion of drug-resistant mutants was performed usingthe agar proportion method. Serial dilutions of lung homogenates, colonysuspensions, or a broth culture were inoculated in 500-�l aliquots onto7H11 plates with and without the desired drug. MICs were determinedusing doubling bedaquiline and clofazimine concentrations ranging from0.003 to 0.25 �g/ml and 0.06 to 2 �g/ml, respectively. The MIC was de-fined as the lowest drug concentration inhibiting at least 99% of thegrowth observed on drug-free control plates. To investigate the effect ofefflux pump inhibitors on the susceptibility of the strains, the MIC ofbedaquiline was also determined using the broth macrodilution method.Doubling concentrations of bedaquiline from 0.007 to 1 �g/ml weretested in the presence or absence of 40 �g/ml of verapamil or 3 �g/ml ofreserpine. Briefly, tubes containing in 2.5 ml of 7H9 broth plus OADCwith the above-mentioned concentrations of bedaquiline were inoculatedwith 105 CFU of log-phase culture of H37Rv or the B5 mutant. The MIC

was defined as the lowest concentration that prevented visible growthafter 14 days of incubation at 37°C. Controls with and without effluxinhibitors and bedaquiline were included for each test. The experimentwas performed twice.

DNA sequencing. Genomic DNA from the parental wild-type strainand resistant mutants was extracted by using the cetyltrimethylammo-nium bromide (CTAB) protocol (13) and sonicated (Covaris, Inc.). TheDNA library was constructed by using a genomic DNA sample prepara-tion kit (Illumina, Inc.). The samples were sequenced on an IlluminaGenome Analyzer II, which was operated in paired-end mode, collectingpairs of 51-bp reads from opposite ends of �250- to 350-bp fragments.Image analysis and base-calling were done by using the Illumina GA Pipe-line software (v0.3). Genome assembly was performed by a comparativeassembly method using software developed in-house. Briefly, reads werealigned to the genome of H37Rv as a reference sequence, and then localcontig building was used to identify insertions and deletions (14). Muta-tions in pepQ (Rv2535c) were confirmed by PCR amplification using spe-cific primers (see Table S1 in the supplemental material).

In vivo confirmation of low-level cross-resistance to bedaquilineand clofazimine. BALB/c mice were infected with either the H37Rv par-ent strain or an isogenic strain (B5) with a nonsynonymous pepQ muta-tion and randomized to receive no treatment, isoniazid (10 mg/kg) alone,bedaquiline (12.5, 25, or 50 mg/kg) alone, clofazimine (20 mg/kg) alone,or bedaquiline at 25 mg/kg plus clofazimine. Treatment began 4 days afterinfection and was administered for 4 weeks for all groups, except thegroups receiving bedaquiline at 25 mg/kg, which received 8 weeks of treat-ment.

Complementation studies to confirm that a pepQ mutation is suffi-cient for bedaquiline and clofazimine cross-resistance. The B5 strainwas complemented with pDT-Rv2535c (see Fig. S1 in the supplementalmaterial) containing the wild-type pepQ gene from H37Rv. Complemen-tation was confirmed by showing a loss of susceptibility to hygromycinand PCR for presence of the hygromycin resistance gene. Bedaquiline andclofazimine MICs were determined in vitro. Following aerosol infectionwith the H37Rv parent strain, the B5 mutant, or the B5::pepQ comple-mented strain, BALB/c mice were randomized to receive no treatment,isoniazid at 10 mg/kg, bedaquiline at 25 mg/kg, or clofazimine at 20 mg/kg. Treatment began 3 days after infection and continued for 4 weeksbefore mice were sacrificed to determine lung CFU counts.

Expression analysis of mmpS5, mmpL5, and Rv0678. The H37Rvparent strain and the B5 mutant were grown to mid-log phase (OD600 �0.6) in 7H9 broth, and cells were pelleted by centrifugation. RNA wasextracted by bead beating in TRIzol and column purified according to themanufacturer’s instructions (Qiagen, Valencia, CA). RNA was reversetranscribed using the iScript Reverse Transcription Supermix (Bio-Rad,Hercules, CA). Real-time PCR was performed with iQ SYBR green super-mix on an iCycler (Bio-Rad, Hercules, CA) using primers specific forRv0676c, Rv0677c, and Rv0678 and 16S rRNA (see Table S1 in the supple-mental material). All experiments were performed in biological and tech-nical triplicates. Cycle threshold (CT) values were normalized against 16SrRNA expression, and fold change was calculated by the �2��CT method(15).

Expression, purification, and activity assays of PepQ. The entirecoding region of Rv2535c was cloned into the pET28b expression vector(Novagen) containing an in-frame N-terminal 6� His tag with the to-bacco etch virus (TEV) cleavage site using the NdeI and HindIII restric-tion sites. The plasmid was transformed into Escherichia coli BL21(DE3)cells for expression of Rv2535c. Cells containing the plasmid were grownat 37°C for 7 h in LB medium with 50 �g/ml of kanamycin followed byinduction with 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) andgrown overnight at 18°C. The cells were lysed via an M-100P microfluid-izer (Microfluidics, Worcestershire, United Kingdom) in lysis buffer (50mM Tris [pH 7.5], 500 mM NaCl, 5% glycerol, DNase, and 2 mM MgCl2)and centrifuged at 15,000 rpm for 1 h. The supernatant was chromato-graphed over a His tag affinity column (GE Healthcare) charged with Ni.

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The protein was eluted with 0 to 300 mM imidazole gradient, and the 6�His tag was cleaved using the tobacco etch virus protease. The final puri-fication step was gel filtration chromatography on the standardized s200Superdex (GE Healthcare) column. The PepQ protein was eluted in twopeaks: a large-molecular-mass aggregate that came out near the void vol-ume, followed by a distinct peak with calculated molecular mass corre-sponding to a tetramer (�160 kDa). Formation of this tetramer is consis-tent with other related proteins, e.g., the protein with PDB code 3Q6D.The protein was 95% pure, as observed by SDS-PAGE and was concen-trated to 7.5 mg/ml, flash frozen, and stored in dialysis buffer (50 mM Tris[pH 7], 50 mM NaCl, 5% glycerol, 1 mM dithiothreitol [DTT]) at �80°C.The purified recombinant PepQ tetramer in 50 mM Tris (pH 7) was testedfor both creatinase activity using the creatinase assay kit (Sigma) andendopeptidase activity using commercially available fluorescent peptides.As the peptides were all labeled with a p-nitroanilide group, the assaymonitored for a shift in absorbance at 410 nm, resulting from the cleavedproduct.

Statistical analysis. CFU counts were log10 transformed before anal-ysis. Group means for experimental treatment groups were comparedwith that of the standard treatment control by one-way analysis of vari-ance (ANOVA) with Dunnett’s posttest to adjust for multiple compari-sons. All analyses were performed with GraphPad Prism v.4.01 (Graph-Pad, San Diego, CA).

RESULTSBedaquiline and clofazimine have additive activity against M.tuberculosis in mice but select for cross-resistant mutants. Usinga well-established high-dose aerosol model of TB in mice, we eval-uated the activity of novel drug combinations containing be-daquiline and clofazimine (Fig. 1). Consistent with prior observa-tions (3, 11, 16), treatment with the standard first-line R-H-Zregimen reduced the mean lung CFU count by 2.46 log10 over thefirst 4 weeks and treatment with bedaquiline alone reduced thelung CFU counts by an additional 1.08 log10. The addition ofpyrazinamide and clofazimine each significantly increased the ac-tivity of bedaquiline (P 0.001), whereas the addition of preto-manid had antagonistic effects (P 0.05). The addition of rifam-

pin, isoniazid, ethambutol, or moxifloxacin did not significantlyaffect the activity of the bedaquiline-clofazimine combination,whereas addition of pyrazinamide significantly increased the ac-tivity (P 0.001) and addition of linezolid or pretomanid reducedthe activity (P 0.05). After 8 weeks of treatment, 2 of 5 micereceiving bedaquiline alone had more than 3 log CFU in the lungs,while the mean log CFU count was 0.98 � 0.53 in the remaining 3mice. Likewise, 3 of 5 mice receiving bedaquiline plus clofaziminehad a mean log CFU count of 2.12 � 0.69, while the remaining 2mice were culture negative, as were all 5 mice treated for just 6weeks (Fig. 1, inset). Unlike those from mice with lower CFUcounts at week 8, the isolates from the lungs of mice with higherCFU counts grew equally well on 7H11 agar with and withoutactivated charcoal, indicating that their growth was not impairedby bedaquiline or clofazimine carried over in the lung homoge-nates.

While bedaquiline and clofazimine MICs for the wild-typeH37Rv parent strain were 0.03 and 0.25 �g/ml, respectively, MICsfor the isolates from the 2 mice treated with bedaquiline alone for8 weeks (named B4 and B5) and the 3 mice treated with bedaqui-line plus clofazimine for 8 weeks (named BC2, BC3, and BC4)with high, outlying lung CFU counts were 0.12 to 0.25 and 0.5 to 1�g/ml, respectively, indicating that treatment with bedaquiline,with or without clofazimine, selected for strains with reduced sus-ceptibility to both drugs.

Isolates with bedaquiline and clofazimine cross-resistanceharbored mutations in pepQ. Duplicate samples of genomicDNA from four cross-resistant strains isolated in the first mouseexperiment (i.e., B5, BC2, BC3, and BC4) were sequenced on anIllumina GenomeAnalyzer IIx in paired-end mode using a readlength of 51 bp. The mean depth of coverage ranged from 118 to161� (number of reads covering each site, averaged over thewhole genome), and the completion was 99% (fraction of sitesin 4.4-Mb genome covered by at least 1 read; sites lacking coveragewere primarily restricted to PPE and PE_PGRS genes, due to veryhigh GC contents). The parental strain used in these experiments(H37RvJH) was also sequenced, to identify any differences fromthe public reference genome sequence for H37Rv (17). Comparedto the sequence of the parental strain, only one mutation wasobserved in each strain (Table 1). All 4 strains had a mutation inpepQ (Rv2535c), which encodes a putative cytoplasmic peptidase.The mutations observed included 2 frameshift mutations (�C inArg271 and �C in Ala14) in mice treated with the bedaquiline-clofazimine combination, as well as a nonsynonymous single nu-cleotide polymorphism causing a Leu44Pro mutation, obtainedfrom a mouse treated with bedaquiline alone. All mutations wereconfirmed by PCR amplification and sequencing.

TABLE 1 Sequencing results for the resistant strainsa

Mouse no. Treatment Coverage % completion Mutation

B5 B 142.1� 99.25 pepQ: L44PBC2 B � C 118.4� 99.20 pepQ: �C in

Ala14BC3 B � C 154.3� 99.43 pepQ: �C in

Ala14BC4 B � C 161.1� 99.14 pepQ: �C in

Arg271a For all strains, the MIC of bedaquiline (B) was 0.12 �g/ml and that of clofazimine (C)was 0.5 to 1 �g/ml.

Day 0 RH

Z B BC BCLBCPa BC

RBCH

BCEBCM

BCZ

0

1

2

3

4

5

6

7

8

Day 0Week 4Week 8

Log 10CFU/lung

Log 10CFU/lung

B BC0

1

2

3

4

FIG 1 Mean lung log10 CFU counts (�SD) after 4 and 8 weeks of treatmentwith bedaquiline- and clofazimine-containing regimens. The main graph doesnot include results from mice in which bedaquiline and clofazimine cross-resistant mutants were selected after 8 weeks of treatment: 2 and 3 mice fromthe B and BC groups with mean lung CFU counts of 3.22 � 0.16 and 2.12 �0.69 log10, respectively (inset). The CFU counts at the time of treatment initi-ation are indicated at day 0. Abbreviations: R, rifampin; H, isoniazid; Z, pyr-azinamide; B, bedaquiline; C, clofazimine; L, linezolid; Pa, pretomanid; E,ethambutol; M, moxifloxacin.

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A pepQ mutant is virulent and less susceptible to bedaquilineand clofazimine in a murine TB model. Because transposon mu-tagenesis experiments have suggested that inactivation of pepQmay reduce the fitness of M. tuberculosis (18), and because thepepQ mutants displayed only low-level resistance to bedaquilineand clofazimine in vitro, we sought to confirm their virulence andreduced susceptibility in vivo. We compared the parental H37Rvstrain and the B5 mutant on the basis of their abilities to multiplyin untreated BALB/c mice and their susceptibilities to treatmentwith isoniazid (positive control), bedaquiline, and/or clofazimine.The aerosol infectious dose was approximately 1 log10 higher forthe parent strain than for the B5 mutant (Fig. 2). By 4 weekspostinfection, untreated (UT) control mice infected with H37Rvhad died, and therefore, none were available for CFU counts (al-though lung CFU counts are typically 8 log10, or approximately4 log10 higher than baseline, at the time of death). Untreated miceinfected with B5 did not die within 4 weeks of infection becausethe infectious dose was not high enough. However, the approxi-mately 4-log10 increase in lung CFU counts over this period isevidence that the strain multiplies more or less normally in mice.The treatment with isoniazid (positive control) was at least asbactericidal against the B5 strain as against H37Rv, reducing thelung CFU counts by more than 1 log10 in both strains. Dose-dependent activity of bedaquiline was observed against bothstrains. However, while bedaquiline at 12.5 mg/kg was bacterio-static and 50 mg/kg reduced the lung CFU counts by more than 2log10 against H37Rv, even 50 mg/kg of bedaquiline did not fullyinhibit multiplication of the B5 mutant but rather allowed a nearly2-log10 increase in CFU counts. Clofazimine enabled growth ofboth strains in this acute infection model, as previously observed(19), but was more effective against the H37Rv strain than againstthe B5 mutant.

Complementation of pepQ restores susceptibility to be-daquiline and clofazimine. Complementation of the B5 and BC2strains with the wild-type pepQ gene from H37Rv restored thesusceptibility of the strains to bedaquiline, as confirmed by theMIC of 0.03 �g/ml, which was no different from that for the pa-

rental H37Rv control. Following this, 3 groups of mice were in-fected with H37Rv, the B5 strain, or the B5::pepQ strain and ini-tiated on treatment with isoniazid at 10 mg/kg, bedaquiline at 25mg/kg, or clofazimine at 20 mg/kg 4 days later. In untreated mice,lung CFU counts increased by approximately 4 log10 over the sub-sequent 4 weeks (Fig. 3) and were accompanied by visible lunglesions (see Fig. S2 in the supplemental material). Isoniazid wasbactericidal, with the greatest effect against the B5 strain, and pre-vented the formation of lung lesions. Like isoniazid, bedaquilinereduced the mean lung CFU counts by 0.5 to 1 log10 CFU againstH37Rv and the B5::pepQ strain, and it did prevent formation ofmacroscopic lung lesions in mice infected with the B5 mutant (seeFig. S2 in the supplemental material). However, bedaquiline al-lowed a 1-log10 increase in mean CFU counts of the B5 mutant.Clofazimine, which has a relatively poor activity in this acute in-fection model when given alone (19), allowed multiplication of allstrains but significantly reduced the multiplication of the H37Rv(P 0.05) and B5::pepQ (P 0.05) strains but not the B5 strain(P 0.05) compared to findings with no treatment. In addition,clofazimine prevented formation of lung lesions in mice infectedwith H37Rv and the B5::pepQ complemented strain but not the B5strain (see Fig. S2 in the supplemental material). These resultsclearly demonstrate that complementation with the wild-typepepQ gene was sufficient to restore susceptibility to bedaquilineand clofazimine in the B5 mutant and thus confirm that the loss ofPepQ function is sufficient for low-level resistance to bedaquilineand clofazimine.

Structural analysis of M. tuberculosis pepQ product. pepQencodes a 372-amino-acid (aa) protein that has two domains: an�100-aa N-terminal alpha/beta domain and an �250-aa C-ter-minal peptidase domain (Fig. 4). Based on homology to Ypdf in E.coli (e.g., 37% amino acid identity over 363 residues), M. tubercu-losis pepQ is predicted to encode a proline-specific aminopepti-dase (prolidase), active on substrates with Xaa1-Pro2 at the aminoterminus. The C-terminal domain of YpdF houses the catalyticactivity and is homologous to the E. coli methionine aminopepti-dase (MetAP; 29% amino acid identity) (20). However, MetAP

FIG 2 Mean lung log10 CFU counts (�SD) (A and B) and change in CFU counts (week 4 � day 0) (C and D) after 4 weeks of treatment in mice infected withthe parental H37Rv strain (A and C) or the B5 mutant (B and D). CFU counts were not determined for untreated mice infected with the parental strain, whichrequired euthanasia prior to the predetermined endpoint. Abbreviations: UT, untreated; H, isoniazid (10 mg/kg); C, clofazimine (20 mg/kg); B, bedaquiline(12.5, 25, or 50 mg/kg).

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lacks the N-terminal domain. In enzyme assays, YpdF was foundto have weak activity on substrates with an N-terminal methio-nine (Met-Xaa) if the second amino acid is alanine, proline, orserine and higher activity for other substrates with a proline in thesecond position (Xaa-Pro) (21). In E. coli, YpdF is encoded in anoperon with YpdE, which is a methionine aminopeptidase. YpdFand YpdE are proposed to work in concert to degrade proteins,with YpdE removing amino acids from the N terminus until aproline is encountered in the second position and YpdF removingthe block (21). The M. tuberculosis pepQ product also has homol-ogy (�30% amino acid identity) to other E. coli proline amino-peptidases, the pepP and pepQ products, which have both the cat-alytic C-terminal domain and a noncatalytic N-terminal domainthat plays a role in oligomerization and substrate specificity. ThepepP product and similar aminopeptidase P enzymes (EC3.4.11.9) may preferentially hydrolyze the Xaa-Pro bond at theterminus of larger oligopeptides (but also dipeptides) and candiscriminate the 3rd and 4th residues in their catalytic specificity.

Proline aminopeptidases have a 2-domain architecture similarto that of creatinases (which catalyze a similar reaction, hydrolysisof creatine into sarcosine and urea), although the peptidase activ-ity of the former is metal ion dependent and they bind cations inthe active site (20), whereas creatinase activity does not requiremetal ions (22, 23). The mutations selected by treatment with

bedaquiline with or without clofazimine in mice occurred in bothdomains. The frameshift mutation in Ala14 and the nonsynony-mous mutation L44P occur in the N-terminal domain, and theframeshift mutation in Arg271 occurs in the catalytic domain.Figure 4 illustrates where these mutations fall in the domain struc-ture of the pepQ product. To better understand the role of thesemutations, we used Phyre2 (24) to build a homology model of thethree-dimensional (3D) atomic structure of the pepQ product.Phyre2 detected the greatest homology to a proline aminopepti-dase from Bacillus anthracis (PDB code 3Q6D; 39% amino acididentity; deposited by Midwest Center for Structural Genomics).Phyre2 created a hidden Markov model based on a PSI-BLASTalignment of closely related sequences and used it to optimallythread the M. tuberculosis pepQ product amino acid sequence ontothe 3Q6D structure as a template, followed by loop optimizationand energy minimization (24). The active site can be inferred to bein a pocket in the C-terminal domain containing a Ca2� metal ion,in the same position as the dinuclear divalent cation sites in pepP(1a16) and MetAP (3mat), which is involved in catalyzing thecleavage of the scissile peptide bond. The cations are coordinatedby D221, D232, E339, E325, and His296 (M. tuberculosis pepQ), allperfectly conserved in all of these structures. Based on the homol-ogy model, Leu44 of M. tuberculosis pepQ appears to be buried inthe core of the N-terminal domain (on a beta strand that shows a

FIG 3 Mean lung log10 CFU counts (�SD) (A to C) and change in lung CFU counts (week 4 � day 0) (D to F) after 4 weeks of treatment in mice infected withthe parental H37Rv strain (A and D), the B5 mutant (B and E), or the complemented B5::pepQ strain (C and F). Abbreviations: UT, untreated; H, isoniazid (10mg/kg); C, clofazimine (20 mg/kg); B, bedaquiline (25 mg/kg).

FIG 4 Locations of mutations and domains in pepQ.

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high level of conservation with E. coli pepP and both prokaryoticand eukaryotic prolidases [25]), not in close proximity to the ac-tive site. Thus, L44P is likely a structure-destabilizing mutation, asis the frameshift mutation in Ala14. A frameshift in Arg271 wouldtruncate half the C-terminal domain (splitting the pseudosym-metric alpha/beta “pita bread” fold in half and destroying the ac-tive site).

The PepQ model can be superimposed with the crystal struc-ture of the E. coli MetAP protein (3mat), which was complexedwith a peptide inhibitor (bestatin analog). The mechanism andspecificity determinants of E. coli MetAP have been well charac-terized (20). MetAP has a dinuclear cation site (2 Co2�) in thesame location as in 3Q6D. The N-terminal residue of the peptideinhibitor of MetAP binds in a P1 pocket, and the second residuebinds in a P2 pocket, with the polar backbone atoms of the scissilebond between them coordinating the cations. The residues of M.tuberculosis PepQ lining the P1 part of the pocket are considerablydifferent than MetAP. For example, F190 is replaced with Cys inMetAP, and I302 is replaced with F. This could potentially explaindifferences in specificity for the N-terminal residue between pro-lidases and methionine aminopeptidases (26). While the inhibitorin the 3mat structure mimics Met1-Ala2, YpdF would be expectedto bind Xaa1-Pro2 as a prolidase. The putative position of the P2pocket can also be inferred by superimposing the crystal structureof E. coli PepP in complex with the inhibitor Pro-Leu (PDB code

1a16), which putatively mimics the 2nd and 3rd residues of apeptide substrate. In both MetAP and the pepP product, the P2pocket is adjacent to residue His-204. While the homology be-tween M. tuberculosis pepQ and E. coli pepP is relatively low (31%amino acid identity), nearly every residue in the vicinity of theactive site was either identical or highly conserved. The residueslining the P1 pocket are F190 (Y), I302 (V), H303, T341 (D), T232,and V398 (L), with residues of the E. coli pepP product shown inparentheses. The residues lining P2 are T234, I193, R337, H204,P203 (L), and H292 (Fig. 5). Thus, we expect M. tuberculosis PepQto be able to coordinate metal ions similarly to E. coli PepP and thespecificity of M. tuberculosis PepQ to be very similar to that of E.coli PepP, i.e., to correspond to a proline aminopeptidase (Xaa-Pro).

Enzymatic characterization of M. tuberculosis PepQ. Recom-binant purified M. tuberculosis pepQ product exhibited no creati-nase activity (data not shown). Of the 6 proline-containing pep-tide substrates tested, only Arg-Pro-p-nitroanilide showedmodest cleavage activity. Several substrates with an N-terminalmethionine were also tested. However, none of these showedcleavage, suggesting that the pepQ product is not a methionineaminopeptidase.

Mass spectrometry showed that recombinant M. tuberculosispepQ product does not bind to or modify the structure of bedaqui-line or clofazimine. Overexpression of M. tuberculosis pepQ in

FIG 5 Multiple amino acid sequence alignment for the products of M. tuberculosis pepQ, Lactococcus lactis pepP, and E. coli pepP, constructed using ClustalW.Residues coordinating the Mn2� ions are shown in red. Asterisks indicate positions which have a single, fully conserved residue. Colons indicate conservationbetween groups of strongly similar properties, scoring 0.5 in the Gonnet PAM 250 matrix. Periods indicate conservation between groups of weakly similarproperties, scoring �0.5 in the Gonnet PAM 250 matrix.

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Mycobacterium smegmatis did not significantly alter the MIC ofbedaquiline or clofazimine. This is not surprising, as the muta-tions presumably result in loss of function. Taken together, theseresults suggest that PepQ is not a target or activating enzyme foreither drug.

Resistance mediated by pepQ mutation may be associatedwith increased drug efflux, but this is not due to upregulation ofmmpL5 and mmpS5 expression. Addition of verapamil and re-serpine reduced the MIC against the H37Rv parent strain from0.03 to 0.007 �g/ml and 0.015 to 0.03 �g/ml, respectively. Bothefflux inhibitors reduced the MIC of both the B5 mutant and theH37Rv parent to the same level that was below the MIC against theparent strain in the absence of the efflux inhibitor, indicating apossible role of one or more efflux pumps in the mechanism oflow-level bedaquiline resistance mediated by loss-of-functionmutations in pepQ (see Table S2 in the supplemental material).Further, quantitative PCR (qPCR) for the genes involved in theefflux-mediated resistance reported in previous studies did notshow any significant fold change in transcription levels (see TableS3 in the supplemental material).

DISCUSSION

Bedaquiline is a promising new drug for the treatment of TB. Itslimited clinical usage to date has provided little opportunity toselect for drug-resistant mutants or to assess the impact of suchmutants on treatment outcomes. In vitro-selected target-basedmutations in atpE confer high-level resistance to bedaquiline (e.g.,16 to 128� increase in MIC) (5, 6). However, to our knowledge,no atpE mutant has been isolated from a patient treated with be-daquiline. On the other hand, nontarget mutations in Rv0678conferring low-level resistance to bedaquiline and clofaziminewere recently described for sputum isolates with at least 4-foldincreases in bedaquiline MIC after treatment including bedaqui-line (6). More recently, Rv0678 mutants were identified in thesputa of both MDR-TB and drug-susceptible TB patients that hadnot received bedaquiline or clofazimine (27). Although theseRv0678 mutations cause relatively small (2� to 8�) increases inbedaquiline and clofazimine MICs compared to mutations inatpE, their selection during combination therapy in TB patientsand in mice is a cause of concern (6). In mice, Rv0678 resistantmutants may be selected with 8 weeks of bedaquiline mono-therapy (D. Almeida, unpublished observation) but emerge onlylate in the course of combination therapy and are eventuallycleared, presumably by the action of companion agents (6). Clin-ically, these mutants emerged almost entirely among patients withpre-XDR- and XDR-TB (6), but their isolation has not clearlybeen associated with poorer clinical outcomes among patients re-ceiving bedaquiline with more effective companion agents (28).These results underscore the importance of appropriate combina-tion therapy and adherence and the risk of premature discontin-uation of therapy.

The present study provides the first evidence of nontarget mu-tations in pepQ conferring low-level resistance to bedaquiline andcross-resistance to clofazimine. Zhang et al. (29) recently reportedin vitro selection of a pepQ mutant in M. tuberculosis with clofazi-mine. However, they did not report the results of direct suscepti-bility testing with clofazimine or bedaquiline or confirm the caus-ative role of pepQ mutation in clofazimine resistance. Likemutations in Rv0678 (6), the observed pepQ mutations producemodest increases (up to 4-fold) in bedaquiline and clofazimine

MICs and reduce the efficacy of bedaquiline and clofazimine invivo. However, these mutations did not result in complete resis-tance to these drugs, as increasing doses of bedaquiline and, to alesser extent, combining bedaquiline and clofazimine were asso-ciated with a greater antituberculosis effect. These data and theadditive bactericidal and sterilizing effects of bedaquiline and clo-fazimine against drug-susceptible bacilli (30) indicate that despitethe presence of two shared resistance mechanisms, the combineduse of clofazimine and bedaquiline may be advantageous. How-ever, these two drugs will not protect each other against the emer-gence of resistance, and it is therefore important to use them in com-bination only if they are protected from emergence of resistance byadditional antibiotics that are not substrates of the same effluxpumps. Both Rv0678 and pepQ mutants are selected and able to growin mice treated with bedaquiline, despite plasma bedaquiline concen-trations exceeding the MIC against these resistant organisms (11, 31).This may be due to the high protein binding of bedaquiline, whichresults in free drug concentrations below the MIC at the site of infec-tion. Rv0678 mutants and pepQ mutants may be selected preferen-tially over atpE mutants in vivo during bedaquiline treatment becausethey strike the right balance between reduced susceptibility to be-daquiline and maintenance of fitness.

Our findings may have implications for breakpoint selectionfor bedaquiline susceptibility testing. Although the bedaquilineMIC against the pepQ mutants is below the provisional suscepti-bility breakpoint of 0.5 �g/ml proposed by EUCAST (32), mono-therapy with clinically relevant bedaquiline doses in mice infectedwith a pepQ mutant had limited efficacy. These findings and thelimited clinical data available to date warrant careful monitoringof bedaquiline MICs and treatment outcomes and further consid-eration whether a lower breakpoint would better predict patientresponse to bedaquiline treatment. Polymorphisms in Rv0678have recently been observed among isolates from patients thathave not received bedaquiline or clofazimine and are not alwaysassociated with bedaquiline MICs that exceed the provisionalbreakpoint (27), and it remains unclear whether such Rv0678 mu-tations are associated with poorer clinical outcomes among pa-tients receiving bedaquiline-containing regimens (6, 28). Thus,the clinical impact of the small shift in MIC conferred by pepQmutations remains to be determined. Further surveillance is re-quired to identify Rv0678 and pepQ mutants and to correlate theirpresence with MICs and clinical outcomes among patients receiv-ing therapy that includes one or both drugs.

It is interesting that single nucleotide polymorphisms in pepQwere occasionally observed in clinical isolates that we have se-quenced. In particular, pepQ::Ser66Pro was observed in twoXDR-TB isolates (X16 and X23; Beijing strain family) and anMDR-TB isolate (R1792; LCC strain family) from the WesternCape of South Africa, all isolated prior to the introduction ofbedaquiline (33). No other strains were observed to have muta-tions in pepQ among over 50 clinical isolates from South Africaand South America. It is possible that use of clofazimine to treatMDR-TB in this region could have contributed to selective ampli-fication of these mutants. However, this seems unlikely given thelimited usage of clofazimine in South Africa during the period inwhich such selection would have occurred. The clinical isolateswith polymorphisms in pepQ were not available for drug suscep-tibility testing. However, the presence of Rv0678 and pepQ muta-tions in isolates from patients naive to bedaquiline and clofazi-

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mine raises the possibility that they may also be selected by otherdrugs used to treat TB or other infections.

What is the function of PepQ in M. tuberculosis, and how mightloss of function result in reduced susceptibility to bedaquiline andclofazimine? While further characterization of the enzymatic ac-tivity of PepQ is clearly required before drawing conclusions, thegenomic and structural characterization described here suggeststhat the pepQ product is a proline-specific aminopeptidase andortholog of the pepP product in E. coli and Lactococcus lactis andPapA (YqhT) in Bacillus subtilis (34). This is supported by homol-ogy in both genetic sequence and predicted protein structure. Al-though the DNA sequence homology is not particularly high,there is a high degree of conservation of amino acids in the pre-dicted active site, including strict conservation of metal-bindingresidues between PepQ and PepP from both E. coli and L. lactis.Moreover, each of these orthologous genes is situated next to andcoexpressed with elongation factor P (EF-P; Rv2534c), one of thefew universally conserved elongation factors (34). Conserved ho-mologs also exist in other pathogenic mycobacteria (M. bovis, M.leprae, and M. paratuberculosis) (35).

Proline-specific aminopeptidases are among only 80 enzymesconserved in the 3 major kingdoms of life (36). Such high conser-vation may stem from the exceptional conformational rigiditythat proline residues introduce into peptide chains, which pres-ents challenges to proteolytic enzyme systems. For example, in E.coli, the essential methionine aminopeptidase (PepM) responsiblefor cotranslational N-terminal methionine excision readily liber-ates the N-terminal methionine when proline or an amino acidwith a similarly small side chain occupies the penultimate posi-tion. However, a proline in the third position inhibits PepM ac-tivity (37, 38), making PepP required for efficient N-terminal me-thionine excision in such cases (39).

The conformational constraints of polyproline motifs alsopresent challenges during ribosomal peptide synthesis by stallingtranslation. As EF-P is required to prevent such stalling and pro-mote efficient translation of polyproline-containing peptides (40,41), the genomic organization of a putative proline-specific ami-nopeptidase like the pepQ product with EF-P is likely no coinci-dence. Work with Gram-negative enteric pathogens has revealedthat proteins with EF-P target motifs (and hence dependent onEF-P for optimal translation) are enriched for metabolic enzymes,membrane-associated proteins, transporters, and two-compo-nent regulatory systems (42). The same appears to be true for M.tuberculosis. This system may have evolved for rapid remodelingof the cellular proteome in response to changing environmentalconditions, such as carbon source (43, 44). The highly conservedorganization of genes for proline-specific aminopeptidases andEF-P in bacteria thus supports a potential role for pepQ in regu-lating maturation and/or turnover of specific proteins in a mannerthat is sensitive to the metabolic status of M. tuberculosis.

Proline-specific aminopeptidases also have important roles inthe utilization of exogenous and endogenous proteins as sourcesof essential amino acids useful for protein synthesis, energy pro-duction, osmoprotection, and recycling of reduced cofactors. Pro-line itself is increasingly recognized as having a critical role inenergy metabolism, redox control, and bacterial virulence (45). Inearly transposon mutagenesis studies (18), disruptions of pepQwere observed to cause a growth defect (thus pepQ is neither ab-solutely essential nor nonessential in vitro). More recent high-resolution transposon mutagenesis studies suggest that pepQ is

conditionally essential when grown on glycerol as a carbon sourcebut not essential when grown on cholesterol (P value 0.01 forcomparison of transposon insertion counts) (46). This is interest-ing given recent evidence linking mycobacterial proline catabo-lism to protection from the toxic effects of methylglyoxal pro-duced during growth on glycerol (47). If mutation of pepQreduces intracellular availability of proline, this might increasesusceptibility to methylglyoxal and reduce growth on glycerol.

The mechanism by which pepQ mutations result in reducedsusceptibility to bedaquiline and clofazimine is unclear.Rv0678 mutations appear to confer cross-resistance to be-daquiline and clofazimine via derepression of mmpL5-mmpS5(Rv0676c-Rv0677c), which encode an RND transporter capableof exporting bedaquiline and clofazimine (6, 7). Reversion of be-daquiline susceptibility of a pepQ mutant to that of its wild-typeparent in the presence of efflux pump inhibitors suggests that drugefflux is involved in the mechanism of resistance. However, unlikethe mutations in Rv0678, the mutation in pepQ is not associatedwith overexpression of mmpL5 or mmpS5. One possibility is thatpepQ mutations increase efflux through this transporter by a dif-ferent means, such as preventing degradation of MmpL5, whichcontains a Val-Pro-Pro stretch near the amino terminus.

In conclusion, we demonstrate that loss-of-function muta-tions in pepQ confer reduced susceptibility to bedaquiline andclofazimine. While the shift in susceptibility is relatively small, it isof a magnitude similar to those of mutations in Rv0678 that havebeen selected in patients and mice receiving combination therapyincluding bedaquiline. Therefore, these results should provide theimpetus to include pepQ in the genetic analysis of bedaquiline-and clofazimine-resistant strains identified during clinical usage.Further study of the biological function of pepQ in M. tuberculosisand the mechanism by which pepQ mutations confer reduced sus-ceptibility is clearly warranted.

ACKNOWLEDGMENTS

This research was funded by the Global Alliance for TB Drug Develop-ment and the National Institutes of Health (R01-AI111992 to E.N.).

K.A. is employed by, and holds stock in, Janssen Pharmaceutica. Noneof the other authors has a financial relationship with a commercial entitythat has an interest in the subject of this article.

FUNDING INFORMATIONThis research was funded by the Global Alliance for TB Drug Develop-ment and the National Institutes of Health (R01-AI111992 to E.N.).

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