Identification of novel loci associated with mycobacterial isoniazid resistance

Tuberculosis 96 (2016) 21e26

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Identification of novel loci associated with mycobacterial isoniazid resistance Gopinath Viswanathan, Sangya Yadav, Tirumalai R. Raghunand* CSIR e Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India

a r t i c l e i n f o

s u m m a r y

Article history: Received 21 May 2015 Received in revised form 24 August 2015 Accepted 28 September 2015

Despite the known association of several genes to clinical Isoniazid (INH) resistance, its molecular basis remains unknown in ~16% of clinical isolates of Mycobacterium tuberculosis (M. tb). While screening a set of Mycobacterium smegmatis (M. smegmatis) transposon mutants with altered colony morphology for differential susceptibility to INH, we found six resistant mutants and mapped their transposon insertion sites. The disrupted genes in six INH resistant mutants were homologs of M. tb ctaE, rplY, tatA, csd and tatB with one insertion mapping to the promoter region of M. smegmatis ctaE. MIC measurements indicated a wide spectrum of INH resistance in these mutants, with complementation analyses of four selected mutants with the cognate M. smegmatis genes and their M. tb homologs confirming the association of the disrupted genes with INH resistance. Our discovery of novel genes associated with INH resistance could lead to the identification of novel INH resistance mechanisms and possibly new diagnostic modalities as well. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Mycobacterium tuberculosis Mycobacterium smegmatis Isoniazid Drug resistance Transposon mutagenesis Resazurin

1. Introduction Isonaizid (INH), a pro-drug, is an integral part of first line TB treatment in conjunction with rifampicin, pyrazinamide and ethambutol [1]. Upon conversion into the NAD adduct by the catalase-peroxidase KatG, INH-NAD inhibits the enoyl-ACP reductase InhA and blocks mycolic acid biosynthesis, leading to bacillary killing [2]. It is also believed that the NO
released during INH activation is partially responsible for the killing by targeting respiratory enzymes in M. tb [3]. Resistance to INH is frequently associated with mutations in katG [4], inhA promoter, and ndhII which encodes NADH dehydrogenase [5]. Mutations in other genes such as ahpC, Rv0340-0343, fadE24, efpA and kasA are also associated with clinical INH resistance, but since they either occur in conjunction with mutations in katG and/or inhA promoter or are present in INH sensitive isolates of M. tb, their roles in resistance remain ambiguous [5]. Current drug susceptibility tests for INH involve phenotypic methods including monitoring bacillary growth, enzyme assays and microscopic observation of drug susceptibility. Since the turnaround time for these assays is long, more rapid genotypic methods like MTBDR and MTBDR-Plus which test for mutations in katG and inhA apart from rpoB, have recently come into practice [6]. However, these tests exhibit a lower sensitivity for

* Corresponding author. Tel.: þ91 40 27192924; fax: þ91 40 27160591. E-mail address: [email protected] (T.R. Raghunand). http://dx.doi.org/10.1016/j.tube.2015.09.008 1472-9792/© 2015 Elsevier Ltd. All rights reserved.

detecting INH resistance since these mutations are absent in approximately 16% of INH resistant clinical isolates [7]. Consequently, there is a need to identify new loci linked to INH resistance to improve the sensitivity of the above genotypic approaches. M. smegmatis transposon mutant screens have been previously used to establish the role of several mycobacterial genes including katG, nudC (involved in hydrolysis and deactivation of INH-NAD adduct), arr (involved in inactivation of rifampin) and roxY (involved in host antimicrobial peptide resistance) in conferring differential susceptibility to TB drugs and stress causing agents [8e11]. In this study, from a set of M. smegmatis transposon mutants with altered colony morphologies [12], we isolated six INH resistant mutants carrying insertions in genes not documented to be associated with INH resistance thus far. MIC determination and complementation studies with the corresponding M. smegmatis and M. tb genes validated their association with INH resistance. We believe that our findings will lead to new insights into the mechanism of INH resistance and may help in developing more sensitive diagnostic tests for resistance detection.

2. Materials and methods 2.1. Bacterial strains, media and growth conditions M. smegmatis mc26 and Escherichia coli (E. coli) DH5a were cultured as described [13]. The following antibiotics were added

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when necessary - Kanamycin (15 mg/ml for M. smegmatis), Hygromycin (200 mg/ml for E. coli and 50 mg/ml for M. smegmatis). 2.2. Whole genome transposon mutagenesis and isolation of INH resistant strains The Himar1 based transposon mutant library of M. smegmatis was generated as described earlier [14]. To phenotype INH resistance, 1 ml of exponential phase cultures of WT and mutant strains were spotted on Middlebrook 7H10 agar plates containing INH. As a control for growth, all cultures were spotted onto plates lacking INH. 2.3. Transposon insertion site mapping A modified genome walking protocol was followed to identify transposon insertion sites. The sequences obtained were aligned using the BLAST program (NCBI) and the point of insertion (POI) of the transposon for each mutant was determined to be 1 bp before the 50 coordinate of insertion, as described earlier [14]. 2.4. Mutant complementation To generate complementation constructs, ORFs of ctaEMs, rplYMs, and rplYM. tb were PCR amplified with ~300bp of their putative promoter using gene specific primers (Table S1) and cloned into pMV306h, an integrating shuttle vector encoding hygromycin resistance. ORFs corresponding to ctaEM. tb, csdMs, csdM. tb, tatBMs and tatBM. tb were amplified and cloned downstream of an hsp60 promoter in pMV261h, an episomal plasmid encoding hygromycin resistance. Wild type and mutant strains were transformed with the cognate recombinant plasmids or empty vectors and selected on Middlebrook 7H10 agar containing Kanamycin and/or Hygromycin. 2.5. Resazurin based microplate assay For MIC determination a resazurin based microplate assay was performed in 96-well plates as described earlier [15] with the following modifications e after adding cells to the antibiotic containing wells, the plates were incubated with the corresponding antibiotic for 60 h. After addition of 30 ml resazurin and 12.5 ml of 20% Tween-80 per well, the plates were incubated for 24 h and the colours of all wells were recorded. Blue was interpreted as no growth, and pink or pinkish purple was scored as growth. The MIC was defined as the lowest drug concentration which prevented a colour change from blue to pink or pinkish purple. 2.6. E-test assay The INH E-test (Biomerieux) assay was performed according to the manufacturer's instructions with the following modifications 200 ml of cultures with A600 of 1.0 were spread on Middlebrook 7H10 agar plates and dried. After applying the E-test strips the ellipse of intersection was measured after 2 days of incubation. 2.7. Microscopy To document colony phenotypes, M. smegmatis strains were grown to stationary phase and streaked on Middlebrook 7H10 agar containing the appropriate antibiotics. Colony phenotypes of the strains were visualized using a Zeiss Axioplan-2 imaging microscope at 50 magnification.

2.8. NAD cycling assay The intracellular concentrations of NADH and NADþ and their ratios were calculated using a NAD cycling assay which was performed as described earlier [16]with following modifications. The reaction mixtures were incubated at 30  C for 15 min and the concentration of NADþ and NADH was obtained by measuring spectrophotometrically the extent of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide reduction by the yeast type II alcohol dehydrogenase at 570 nm in 96-well plates. The assay was calibrated using NADþ and NADH standards. 2.9. Genotypic analysis of INH resistant clinical strains of M. tb The genomes of 741 INH resistant clinical isolates from the GMTV database [17] (http://mtb.dobzhanskycenter.org) were screened for the presence of mutations in the genes identified in our study. A quality threshold of 20 (which indicates a probability of 1 in 100 that the observed mutation is incorrect) was set to filter out unreliable mutations that may arise due to errors in sequencing. The clinical isolates showing mutations in the genes identified in this study were further checked for the presence of mutations already reported to be associated with INH resistance [4,5]. 2.10. In vitro growth kinetics To profile their growth characteristics, M. smegmatis strains grown to late exponential phase were diluted to an A600 of 0.2 and cultured in Middlebrook 7H9 broth. Growth curves were generated by plotting A600 measurement against time. 2.11. Real-time PCR analysis To determine the relative fold difference in ctaEMs transcript levels in M. smegmatis TR67 [PMSMEG_4260 (ctaEMs)::Tn] vs the WT strain, cells were harvested at the A600 value of 1 and total RNA isolated from each culture using TRIzol reagent (Invitrogen) as per the manufacturer's protocol. Following treatment with RNAse free DNAse I, cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad) and subsequently used as a template for SYBR green based PCR amplification using ctaEMs gene specific primers (Table S1) to generate 200 bp amplicons. Gene specific transcript levels were normalised to the M. smegmatis sigA transcript in each sample before calculating the fold change. At least 2 biological and 6 technical replicates were performed. 3. Results and discussion 3.1. Isolation of novel INH resistant mutants of M. smegmatis In order to identify novel mycobacterial loci associated with INH resistance we observed the growth profiles of 69 colony morphotype mutants of M. smegmatis on plates containing 10 mg/L INH, a concentration higher than the reported MIC for M. smegmatis [18]. These were originally isolated in a colony morphology screen from a library of 5000 transposon mutants [12]. Since changes in colony morphologies were likely to result from variations in cell envelope composition, we initially hypothesised that these mutants may show differential susceptibilities to INH by virtue of their altered permeability. We obtained six mutants resistant to INH (Figure 1A) and mapped the Tn insertion sites to identify the disrupted genes in these mutants (Figure 1B). All the mutants contained ORF disrupting insertions except TR67, where the Tn was inserted in the promoter of MSMEG_4260. None of the M. tb homologues of the

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Figure 1. INH resistant mutants of M. smegmatis. A. Growth profiles of wild type (WT) and INH resistant transposon mutants of M. smegmatis mc26 spotted on Middlebrook 7H10 agar (Control) and Middlebrook 7H10 agar containing 10 mg/L INH. Results shown are representative of two biological replicates. B. Transposon insertion sites of INH resistant M. smegmatis mutants, their gene lengths, M. tb homologues and their predicted functions. POI e Point of transposon insertion.

disrupted M. smegmatis genes have been described in the literature as being related to INH resistance, signifying the novelty of this finding. Our finding that two sets of mutants contained insertions in genes involved in related functions (TR34 [MSMEG_3887 (tatAMs)::Tn] and TR59 [MSMEG_5069 (tatBMs)::Tn]) or in different regions of the same gene (TR18 [MSMEG_4260 (ctaEMs)::Tn] and TR67 [PMSMEG_4260 (ctaEMs)::Tn]) provided validation for the association of these genes with INH resistance. To test if the resistance in TR67 was due to compromised ctaE expression resulting from a promoter insertion, we determined the levels of the ctaEMs transcript in the mutant strain with respect to wild type M. smegmatis. TR67 showed a near complete loss (731 ± 25 (Mean ± SD) fold reduction) in ctaE mRNA levels compared to the wild type strain illustrating that the transposon insertion in this mutant phenocopies TR18 [ctaEMs::Tn] by reducing transcript levels of ctaEMs. Although, several genes/mutations identified to be associated with drug resistance in M. smegmatis have been demonstrated to be functionally homologous in M. tb [8,19,20], not all drug resistance mutations are observed to behave the same in M. tb as in M. smegmatis [21e23]. Hence, the genes we have identified to be involved in INH resistance in M. smegmatis, will require functional validation in M. tb, prior to their designation as bonafide resistance associated genes. 3.2. Mutants exhibit variable resistance to INH INH MIC measurements in the above mutants revealed twofold (TR20 [MSMEG_5431 (rplYMs):Tn]), fourfold (TR34 [tatAMs::Tn]), eight fold (TR18 [ctaEMs::Tn] and TR67 [PctaEMs::Tn]) and 16 fold (TR59 [tatBMs::Tn]) increases in INH MIC compared to WT M. smegmatis. TR51 [MSMEG_3125 (csdMs):Tn] displayed a > 16 fold higher MIC in comparison to the control (Table 1). These results indicated that the mutants were variably but significantly resistant to INH. In order to test the effect of growth rate of these mutants on the observed variability in INH resistance, we performed in vitro growth assays of the mutants vs the WT strain. Although the

mutants were observed to grow at a slower rate (consistent with their small colony sizes, Figure S2A) as compared to the WT strain, we found no correlation between the growth rates and INH resistance patterns in these mutants. Importantly, the INH sensitive strain TR62 (parAMs::Tn) [12] also showed a slow growth phenotype, indicating that reduced growth rate itself does not lead to an increase in INH MIC (Figure S2B). These results imply that alternate mechanisms contribute to the observed resistance in these mutants. As increase in the NADH/NADþ ratio has been previously associated with INH resistance in mycobacteria [16] we

Table 1 INH MIC values for WT, INH resistant mutants of M. smegmatis and their corresponding vector controls, Ms and M. tb complements. Results shown are representative of two biological replicates and four technical replicates. Strain

INH MIC (mg/L)

WT WT:pMV261h WT:pMV306h TR18 TR18:pMV261h TR18:pMV306h TR18:ctaEMs TR18:ctaEMtb TR20 TR20:pMV306h TR20:rplYMs TR20:rplYMtb TR51 TR51:pMV261h TR51:csdMs TR51: csdMtb TR59 TR59:pMV261h TR59:tatBMs TR59:tatBMtb TR34 TR67

8 8 8 64 64 64 8 8 16 16 8 8 >128 >128 64 64 128 128 16 16 32 64

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hypothesised that the INH resistance observed in these mutants were due to high NADH/NADþ ratios. We observed no increase in the ratio (Figure S3) in any of the mutants tested, implying that differential permeability or other yet to be identified mechanisms may be the reason for the observed INH resistance in these mutants. Surprisingly, we observed a significant decrease in the NADH/ NADþ ratio in TR20 [rplYMs::Tn] (Figure S3), which carries an insertion in the gene encoding protein L25, a subunit of the 50S ribosome. We speculate that this mutant has reduced translational efficiency, and that the lower NADH/NADþ ratio may result from an overall reduction in metabolic activity caused by global reduction in translation. Further investigations will be required to test this hypothesis as well as to identify a link between the reduced NADH/ NADþ ratio and INH resistance in this mutant. 3.3. Complementation analyses confirms the association of identified mycobacterial loci with INH resistance Complementation of the selected mutants TR18 [ctaEMs::Tn], TR20 [rplYMs:Tn] and TR59 [tatBMs::Tn] with their corresponding M. smegmatis genes as well as the M. tb homologues led to complete or significant restoration of INH MICs to the wild type levels (Table 1). These results ruled out the possibility of polar effects on the genes downstream to the transposon insertion being responsible for the observed phenotype, and also confirmed the association of the M. tb homologues of the disrupted genes with INH resistance. A partial restoration in MIC was observed in complements of TR51 [csdMs:Tn] which carries a disruption in csdMs, one of seven genes present in the suf operon, predicted to be involved in iron sulphur cluster assembly and repair [24]. It is therefore conceivable that in addition to csd, a polar effect on genes in the suf operon partly contributes to INH resistance in this mutant. A qualitative confirmation of these observations was obtained via the INH E-test assay (Figure S1). Additionally, we observed identical INH MIC values in the strains and their corresponding vector controls ruling out the effect of antibiotic treatment used for plasmid selection, on the phenotypic properties of the complemented strains (Table 1). 3.4. TR51 [csdMs::Tn] and TR59 [tatBMs::Tn] show a specific high level resistance to INH In order to test the specificity of their resistance to INH we determined the MICs of these mutants for rifampicin (RIF). We observed that in addition to INH resistance three mutants, TR18 [ctaEMs::Tn], TR20 [rplYMs::Tn] and TR34 [tatAMs::Tn] showed a similar increase in MIC values for rifampicin ranging from 2 to 4 fold (Table 2) suggesting that the resistance phenotype observed in these mutants is not specific to INH. These results led us to speculate that a general phenomenon like altered permeability may cause resistance to both INH and RIF in these mutants. This hypothesis is further supported by the observation that these mutants

Table 2 RIF MIC values for WT and INH resistant mutants of M. smegmatis. Results shown are representative of two biological replicates and four technical replicates. Strain

RIF MIC (mg/L)

WT TR18 TR20 TR34 TR51 TR59

8 32 16 16 4 16

showed altered colony morphology (Figure S2A) and hence may have alterations in their cell envelope causing differential permeability to antibiotics. Two mutants, TR51 [csdMs:Tn] and TR59 [tatBMs::Tn], showed a specific high level of resistance to INH as they were either sensitive or marginally resistant to RIF (Tables 1 and 2), pointing to an INH specific mechanism of resistance. 3.5. Genes corresponding to TR20 [rplYMs::Tn], TR51 [csdMs::Tn] and TR59 [tatBMs::Tn] carry mutations in a subset of INH resistant clinical isolates of M. tb To determine the clinical significance of our findings, we screened the genomes of INH resistant clinical isolates for the presence of mutations in genes identified in our study in the GMTV database [17] the only comprehensive online resource that brings together detailed information on Mycobacterium tuberculosis genome variations associated with phylogeographic distribution, drug resistance and clinical outcome of TB. Our analyses led to the identification of a total of 11 strains with mutations in rplY (4 strains), csd (3 strains) and tatB (4 strains) out of which 9 strains also carried a katG-S315T mutation (Table 3). Although the resistance in these 9 strains could primarily be attributed to the katG mutation, we speculate that the presence of additional mutations in rplY, csd and tatB in katG-S315T strains may lead to a severity in their INH resistant phenotypes. Two strains with non-synonymous mutations in TatB that led to a P113A conversion did not contain mutations in genes previously reported to be involved in INH resistance, indicating a possible link between this mutation and clinical INH resistance. 3.6. The resistant mutants possibly have a defect in INH activation In section 3.4, we have speculated that altered permeability may be a reason for the observed INH resistance in TR18 [ctaEMs::Tn], TR20 [rplYMs::Tn] and TR34 [tatAMs::Tn]. In addition to this, on the basis of a thorough analysis of the annotated/reported functions of the disrupted M. smegmatis genes as well as their M. tb homologs, we propose several alternate hypotheses as the cause for INH resistance in the isolated mutants. Mycobacterial ctaE encodes the cytochrome C oxidase subunit III (subunit of cytochrome bc1eaa3 (CcO) complex), which transfers an electron to the oxygen molecule in the electron transport chain. It has been reported earlier that mycobacterial strains defective in CcO function show an upregulation of cytochrome bd oxidase (an alternative terminal oxidase) activity. This alternative terminal oxidase does not generate superoxide radicals and shows high affinity for O2 and superoxide [25]. Since superoxide is necessary for INH-NAD adduct formation [26], we speculate that reduced production or increased scavenging of superoxide in TR18 [ctaEMs::Tn] and TR67 [PMSMEG_4260 (ctaEMs)::Tn] as possible explanations for the observed INH resistance in these mutants. A similar mechanism may be the cause for increased INH MIC in TR34 [tatAMs::Tn] and TR59 [tatBMs::Tn] for the following reasons. M. tb tatA and tatB encode subunits of the twin arginine translocase (TAT) which is responsible for the transport of folded proteins across cytoplasmic membrane which includes the predicted substrate QcrC, a subunit of the CcO complex [27]. Using the TatP program [28] we identified a TAT signal peptide in the M. smegmatis homolog of QcrC, suggesting that CcO complex assembly in mycobacteria depends on secretion of QcrC through TAT. We therefore presume that loss of TAT activity in our mutants leads to INH resistance through a defective CcO complex. Mycobacterial csd encodes a cysteine desulfurase predicted to be involved in iron metabolism [24]. Defects in this gene could possibly lead to increased intracellular iron levels leading to downregulation of katG which is under the control of the iron

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Table 3 List of INH resistant clinical isolates from GMTV database showing mutations in genes identified in our study. Gene*

Isolate ID

Coordinate of mutation

Type of mutationy

Mutation in known INHR associated loci?

rplY (648bp, 215aa)

TB0039 TB0048 TB0052 TB0070 TB0618 TB1037 TB1205 TB1206 TB0053 TB0064 TB0534

1134323 1133949 1134323 1134323 1367799 1367799 1367695 1367695 1652631 1652056 1651770

Ins G (frame shift after aa 82) Ins T (frame shift after aa 206) Ins G (frame shift after aa 82) Ins G (frame shift after aa 82) C to G (P113A) C to G (P113A) G to A (R78Q) G to A (R78Q) Ins G (frame shift after aa 371) Ins G (frame shift after aa 179) T to C (F85L)

Yes, Yes. Yes, Yes, No No Yes, Yes, Yes, Yes, Yes,

tatB (396bp, 131aa)

csd (1254bp, 417aa)

* y

katG-S315T katG-S315T katG-S315T katG-S315T

katG-S315T katG-S315T katG-S315T katG-S315T katG-S315T

Length of the corresponding gene as well their encoded protein was shown within the brackets. The position of frameshift in case of insertion and amino acid change in case of missense mutation were indicated within the brackets, Ins e insertion.

responsive protein Fur [29]. This might result in reduced INH activation in TR51 [csdMs:Tn] causing the resistant phenotype. As described earlier in section 3.2 we propose that diminished translational efficiency in TR20 [rplYMs::Tn] may lead to a decrease in KatG levels and the reduced activation of INH might result in the resistant phenotype. Overall our analysis indicates a possible defect in INH activation in all the reported mutants. 4. Conclusions An analysis of a pool of EMS induced and spontaneous INH resistant mutants of M. smegmatis led to the discovery of ndhII in conferring INH resistance [20]. The clinical relevance of this observation was established by the identification of mutations in this gene in 8 of 84 INH resistant isolates of M. tb in Singapore [19]. Similarly, we believe that our results open the possibility for new screens aimed at finding mutations in the genes identified here, in INH resistant clinical isolates of M. tb. In conclusion, this approach is likely to improve the existing detection methods for resistance to INH, and will provide novel insights into mechanisms of INH resistance in mycobacteria. Acknowledgements The authors gratefully acknowledge the assistance of Ms Gauri Navgire in the NAD cycling assays. Funding: This work was supported by a Starting Research Grant from Institut Merieux (to TRR) and the Council of Scientific and Industrial Research (CSIR), Government of India. GV was supported by a Senior Research Fellowship from the CSIR. SY was supported by a fellowship from Institut Merieux. Competing interests: Ethical approval:

None declared. Not required.

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