Ectopic Expression of Rv0023 Mediates Isoniazid/Ethionamide Tolerance via Altering NADH/NAD+ Levels in Mycobacterium smegmatis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) accounts for nearly 1.2 million deaths per annum worldwide. Due to the emergence of multidrug-resistant (MDR) Mtb strains, TB, a curable and avertable disease, remains one of the leading causes of morbidity and mortality. Isoniazid (INH) is a first-line anti-TB drug while ethionamide (ETH) is used as a second-line anti-TB drug. INH and ETH resistance develop through a network of genes involved in various biosynthetic pathways. In this study, we identified Rv0023, an Mtb protein belonging to the xenobiotic response element (XRE) family of transcription regulators, which has a role in generating higher tolerance toward INH and ETH in Mycobacterium smegmatis (Msmeg). Overexpression of Rv0023 in Msmeg leads to the development of INH- and ETH-tolerant strains. The strains expressing Rv0023 have a higher ratio of NADH/NAD+, and this physiological event is known to play a crucial role in the development of INH/ETH co-resistance in Msmeg. Gene expression analysis of some target genes revealed reduction in the expression of the ndh gene, but no direct interaction was observed between Rv0023 and the ndh promoter region. Rv0023 is divergently expressed to Rv0022c (whiB5) and we observed a direct interaction between the recombinant Rv0023 protein with the upstream region of Rv0022c, confirmed using reporter constructs of Msmeg. However, we found no indication that this interaction might play a role in the development of INH/ETH drug tolerance.


INTRODUCTION
Tuberculosis (TB) remains a major cause of death worldwide and the leading cause by a single infectious agent (World Health Organisation, 2018). Even though the disease can be cured and managed by several multidrug regimens, the emergence of multidrug-resistant (MDR) TB is proving to be a major challenge for complete eradication of the disease. Worldwide, MDR TB constitutes 3.5% of new TB cases and 18% of previously treated cases (World Health Organisation, 2018). To overcome these challenges and to better counter resistance in Mycobacterium tuberculosis (Mtb), understanding the mechanisms and deciphering the pathways majorly responsible for generating resistance are greatly required. Isoniazid (INH), in combination with rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA), forms the firstline therapy for TB. Cycloserine, ethionamide (ETH), and amikacin/capreomycin are used as second-line drugs 1 . INH was first used as an anti-TB drug in 1952 and shortly the first INHresistant Mtb (INHr) clinical isolates were reported (Bernstein et al., 1952;Fox, 1952;Middlebrook and Cohn, 1953). INH and ETH are prodrugs that are converted into their active forms by proteins encoded by katG (catalase peroxidase KatG) and ethA (monooxygenase EthA), respectively (Johnsson and Schultz, 1994;Johnsson et al., 1997;Baulard et al., 2000;DeBarber et al., 2000;Vannelli et al., 2002;Fraaije et al., 2004). Although katG and inhA (encoding an NADPH-dependent enoyl-ACP reductase) are the main genes involved in INH resistance, clinical isolates with mutations in the ndh gene have been identified in INH-resistant Mtb strains (Lee et al., 2001). While in the study by Lee et al. (2001), INH resistance mutations were observed only in the ndh gene, in other studies, ndh mutations occur simultaneously with mutations in other genes (Hazbón et al., 2006;Cardoso et al., 2007). The role of the ndh gene mutations in INH and ETH co-resistance in Mycobacterium smegmatis (Msmeg) and Mycobacterium bovis (Mbovis) has been shown (Vilcheze et al., 2005), while the role of ndh in conferring resistance in Mtb is yet to be determined. The gene ndh encodes the type II NADH dehydrogenase and its ortholog in Escherichia coli (E. coli) has been characterized. In E. coli, it exists in a monomeric state bound to the membrane, where it oxidizes NADH, reduces quinone, and catalyzes the transfer of electrons from reduced flavin to quinone (Jaworowski et al., 1981;Matsushita et al., 1987;Yagi, 1993;Gennis and Stewart, 1996;Kerscher, 2000). In Msmeg, mutations in ndh lead to an increase in NADH cellular concentration and inhibition of INH-NAD and ETH-NAD adducts formation (Vilcheze et al., 2005).
Xenobiotic response element (XRE) family of transcription factors are one of the most frequently occurring families of regulators in bacteria. Among the well-studied members of the XRE family are the lambda and Cro repressors, from lambda bacteriophage, and the prophage repressor Xre from Bacillus subtilis. The XRE family of regulators share a conserved N-terminal helix-turn-helix (HTH) DNA binding domain, while the C-terminal regulatory region is highly variable. The XRE family of regulators control diverse metabolic functions; e.g., SinR regulates developmental process in B. subtilis (Gaur et al., 1991), ClgR regulates Streptomyces growth and controls Clp proteolytic complex (Bellier and Mazodier, 2004), PuuR regulates putrescine utilization pathway in E. coli K-12 (Nemoto et al., 2012), and BzdR is involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB (Barragán et al., 2005). There are seven members of the XRE family of transcription regulators in Mtb: Rv0023, Rv0465c, Rv0474, Rv1129, Rv2017, Rv2021, and EspR (Rv3849). Except for EspR, which positively regulates the ESX-1 protein secretion system, the principal virulence determinant of Mtb (Raghavan et al., 2008), the remaining XRE transcription regulators in Mtb are uncharacterized. 1 https://www.cdc.gov/tb/education/corecurr/ Rv0023 is a regulator from the XRE family of transcriptional regulators, known to induce 488 genes and repress 404 genes (Rustad et al., 2014). Rv0023 regulon is enriched for the regulation of NAD reductases (Rustad et al., 2014). Rv0023 is transcribed in an operon together with Rv0024, a gene that codes for an NLPC/p60 family protein and is transcribed divergently from whiB5, belonging to the WhiB family of transcriptional regulators. The whiB5 gene product is a positive regulator of transcription and contributes to Mtb virulence and reactivation (Casonato et al., 2012). At present, very little is known about Rv0023 functions and its effect on Mtb physiology.
Here, we studied the effects of Rv0023 overexpression in Msmeg. The results show that ectopic expression of Rv0023 confers enhanced INH and ETH tolerance in Msmeg. Rv0023 ectopic expression downregulates the expression of the ndh gene and increases NADH/NAD + levels, which are known to be mediators of INH and ETH resistance in Msmeg (Miesel et al., 1998;Vilcheze et al., 2005). We further studied the regulation of the whiB5-Rv0023 locus and identified Rv0023 as a negative regulator of whiB5. We have characterized its binding site and identified the promoters of Rv0023 and whiB5.

Plasmids and DNA Manipulation
Cloning, genomic, and plasmid DNA isolations were done as per standard molecular biology procedures (Sambrook et al., 1989). The plasmids and primers used in this study are listed in Tables 2, 3, respectively. Overlapping extension PCR was performed to generate site-directed mutations for promoter and critical residues studies. Sequences of all clones generated were confirmed by Sanger sequencing.

Protein Expression and Purification
pET23a-0023 plasmid containing Rv0023 ORF was used to transform E. coli BL21 (DE3) strain. Cells were grown in LB broth at 37 • C till mid-log phase, and then 1 mM IPTG (isopropyl 1-thio-β-D-galactopyranoside) was added to induce the expression of protein. Cells were grown for another 4 h at 37 • C, after which cells were harvested and purified using Ni-NTA affinity chromatography as described earlier . The purity of recombinant protein was analyzed

β-Galactosidase Assay
Mycobacterium smegmatis mc 2 155 strain was transformed with various constructs as required and grown in 7H9 media. The cultures were grown to mid-log phase and β-galactosidase activity was measured in Miller Units (MU) and all experiments were done in triplicate (Miller, 1972).

Electrophoretic Mobility Shift Assay
To verify the interaction between Rv0023 and the upstream region of whiB5, electrophoretic mobility shift assay (EMSA) was performed. The 500-bp upstream region and the 50-bp downstream region of whiB5 were PCR amplified and labeled with γ-32 P ATP (3000 Ci mmol −1 ) using T4 polynucleotide kinase as per manufacturer's instructions (New England Biolabs). Labeled DNA was purified using nucleotide purification kit (Qiagen). Purified labeled DNA was then incubated with increasing concentrations of recombinant Rv0023 protein in an EMSA reaction buffer (25 mM HEPES, pH 7.9, 0.1 mM EDTA, 10 mM MgCl 2 , 20 mM KCl, and 5% glycerol) with 50 ng/µl poly(dI-dC) as a non-specific DNA competitor. The protein-DNA complex was incubated for 45 min and was resolved on a 6% non-denaturing polyacrylamide gel in 0.5 × tris-borate (TBE) buffer. The gel was run for 3-4 h to allow sufficient resolution of protein-DNA complex. Electrophoretic mobility shift assay with commercially synthesized overlapping oligonucleotide and motifs was performed to identify the exact binding region of Rv0023.

Primer Extension
To map the (+1) transcription start site (TSS), primer extension was performed as described earlier and in Cold Spring Harbor protocols (Carey et al., 2013;Angara et al., 2018). As the ORFs and intergenic region of whiB5-Rv0023 locus are 100% conserved between Mtb and Mbovis, we used Mbovis RNA for primer extension studies. RNA was isolated with the Qiagen RNeasy kit as per manufacturer's instructions. SuperScript III reverse transcriptase (Invitrogen) was used to transcribe cDNA from total RNA (10-15 µg) using 5 -end labeled γ-32P ATP primers (Table 4). Primer extension products were resolved on 6% polyacrylamide/8 M urea gel in TBE buffer. The size of primer extension product was determined by generating a dideoxy sequencing ladder using pUC19 plasmid as template and M13 universal primer (USB; 70140 KT).

Stress Assay
To check the sensitivity of Msmeg toward various stress conditions, MsmegWT, MsmegpVV16, and MsmegpVV0023 cells were grown in complete 7H9 media (supplemented with 10% OADC, 0.2% glycerol, and 0.05% Tween80) at 37 • C overnight. Then, the cells were centrifuged and washed with PBS. The cells were then suspended in 7H9 media and the cell concentration was adjusted to 0.02 OD at 600 nm. The sensitivity of bacterial cultures toward 0.1% SDS (Sigma Aldrich) and 5 mM hydrogen peroxide (Merck) was measured at 37 • C for 6 h. Sensitivity to 250 µg/ml lysozyme (Sigma Aldrich) was measured at 37 • C for 24 h. Susceptibility of Msmeg toward the antibiotics (Sigma Aldrich) INH (10 µg/ml, MIC of 5 µg/ml), RIF (10 µg/ml, MIC of 1 µg/ml), and ETH (2.5 µg/ml, MIC of 10 µg/ml) was determined at 37 • C for 24 h. For determining CFU numbers for each stress condition and antibiotics, the cells were serially diluted (10-fold) and plated onto 7H10 agar plates. Further, the strains MsmegWT, MsmegpVV16, and MsmegpVV0023 were serially diluted (10-fold) and spotted on the 7H10 agar plates containing increasing concentrations of INH (0, 5, 10, and 15 µg/ml) and ETH (0, 2.5, and 5 µg/ml). The plates were incubated at 37 • C for 48 h.

qRT-PCR
Total RNA from MsmegpVV16 (vector control) and MsmegpVV0023 (Rv0023 protein overexpressed) cultures were isolated using Qiagen RNeasy kit. SuperScript III reverse transcriptase (Invitrogen) was used to generate cDNA using random hexamers. Real-time PCR was carried on the BioRad CFX96 system using gene-specific primers ( Table 5) and EvaGreen qPCR mastermix (Applied Biological Materials Inc.) as per standard protocol. The fold change in expression relative to MsmegpVV16 (vector control) was calculated after normalizing to sigA. The 2 − CT method was used to calculate relative changes in gene expression (Livak and Schmittgen, 2001).

NADH/NAD + Cellular Concentration
To measure the cellular concentration of NADH and NAD + , MsmegpVV16 and MsmegpVV0023 cells were grown to an OD 600 of 0.8-1.2. Cells were collected (1 ml) by centrifugation (11,000 rpm for 2 min). The supernatant was removed and 300 µl of 0.2 M HCl (for NAD + extraction) or 0.2 M NaOH (for NADH extraction) was added to the cells. The cells were resuspended and incubated at 50 • C for 10 min after which extracts were cooled to 0 • C. The bacterial suspensions were neutralized by adding 0.1 M HCl (for NADH extraction) or  (Leonardo et al., 1996;San et al., 2002;Vilcheze et al., 2005). The concentration of nucleotide (NADH/NAD + ) is proportional to the rate of MTT reduction.

Bioinformatics
Rv0023 protein sequence (UniProt P9WMI3) was used as a query in the SynTax web server 2 for synteny analysis within the Mycobacteriaceae family (Oberto, 2013); a conserved domain database search was performed to identify HTH_3 and XRE domains (Marchler-Bauer et al., 2015). Orthologous sequences of the whiB5-Rv0023 intergenic region from different mycobacterial species were retrieved from KEGG genome database and aligned using CLUSTAL OMEGA (Sievers et al., 2011).

Statistical Analysis
Data were presented as mean ± SD. Student's t-test and oneway ANOVA were used to determine the statistical significance between groups and values with p < 0.05 were considered to be significant. GraphPad Prism software version 5.02 was used for the statistical analysis.

Overexpression of Rv0023 Confers Increased INH and ETH Tolerance in Msmeg
Rv0023 is a non-essential gene in Mtb, yet it regulates a high number of genes in the genome (Sassetti et al., 2003;Rustad et al., 2014). Msmeg genome does not harbor the ortholog of Rv0023 and hence serves as a good model to study the function of Rv0023.
To ascertain the role of Rv0023, we constructed an overexpressing strain of Rv0023 in Msmeg, MsmegpVV0023. The wild-type MsmegWT, vector control MsmegpVV16, and MsmegpVV0023 strains were subjected to acid fast staining to rule out the effect of vector insertion on cellular integrity. The cells were found to be intact and acid fast (Supplementary Figure 1). To further study the role of Rv0023 in cellular physiology, MsmegWT, MsmegpVV16, and MsmegpVV0023 were subjected to different stress conditions as depicted in Figure 1A, and tolerance was measured in terms of CFU. The CFU was counted for all conditions and we observed that overexpression of Rv0023 leads to increased tolerance with regard to INH and ETH, and no effect was observed with any other stress conditions. Tolerance to INH and ETH was not seen either on the wild type or in vector control strains ( Figure 1A). The role of Rv0023 in generating tolerant strains was further confirmed via spotting the wild type, vector control, and Rv0023 overexpressed strains in the presence of increasing concentrations of INH and ETH. MsmegWT, MsmegpVV16, and MsmegpVV0023 cells were serially diluted and spotted on plates containing 5, 10, and 15 µg/ml of INH and 2.5 and 5 µg/ml of ETH. Even at higher concentrations, MsmegpVV0023 showed tolerance toward INH and ETH (Figures 1B,C and  Supplementary Figure 2). The results indicate that Rv0023 expression specifically contributes toward the higher tolerance of INH and ETH in Msmeg.

Rv0023 Expression in Msmeg Alters the NADH/NAD + Levels
Isoniazid and ethionamide are both prodrugs and they are activated by the protein catalase-peroxidase KatG and the NADPH-specific flavin adenine dinucleotide-containing monooxygenase, EthA, respectively. After activation, they react with NAD + to form INH-NAD and ETH-NAD adduct. This species then inhibits the enoyl ACP reductase InhA, which leads to the inhibition of mycolic acid biosynthesis and eventual mycobacterial cell death (Figure 2A). The major common genes to both pathways are the ndh (maintains NADH/NAD + ratio) and inhA. Msmeg orthologs of these and other important genes  involved in INH and ETH resistance were subjected to qRT-PCR quantification. Total RNA was isolated from MsmegpVV16 and MsmegpVV0023 and the expression levels compared between the two strains. We found that the expression level of ndh (MSMEG_3621) in MsmegpVV0023 was approximately twofold lower than that of MsmegpVV16. No changes in the expression levels were observed in inhA (MSMEG_3151), katG (MSMEG_6384), and ethA (MSMEG_6440) between both strains ( Figure 2B). As ndh encodes NdhII, which oxidizes NADH to NAD + , we reasoned that the ratio of NADH/NAD + might be altered in MsmegpVV0023 strain. So, we measured the cellular ratio of NADH and NAD + in MsmegpVV16 and MsmegpVV0023 strains and found that the ratio of NADH/NAD + is increased in MsmegpVV0023 (Table 6). These results indicate that Rv0023 expression alters both the transcript levels of ndh gene and NADH/NAD + levels. To find out whether these effects are directly mediated by Rv0023 or through some indirect means, we studied the interaction of recombinant Rv0023 with the upstream region of ndh (MSMEG_3261) gene. However, under the experimental conditions mentioned, we did not observe any such positive interaction ( Figure 2C).

Rv0023 Negatively Regulates whiB5
Rv0023 is in operon with Rv0024 and is transcribed divergently from whiB5 (Figure 3A). Rv0023 and whiB5 are absent from the non-pathogenic, fast-growing Msmeg but are present in Mtb. So, we tried to find the co-occurrence of these two genes in other mycobacterial species. Synteny analysis of Rv0023 and whiB5 was performed for important species of the Mycobacterium genus using the "SynTax" web server ( Figure 3B). It was observed that whiB5 is in synteny with Rv0023 and that both are present only in pathogenic species of mycobacteria (Supplementary Table 1).
The syntenic relationship of the two regulators prompted us to probe the regulation of the whiB5-Rv0023 locus. To identify the regulatory elements at this locus, upstream regions of whiB5 and Rv0023 were cloned in the promoter-less vector pEJ414 and named pEJwhiB5 and pEJ0023, respectively. The coding region of Rv0023 was cloned in the pVV16 vector to obtain pVV0023. Msmeg was transformed with pEJwhiB5 or pEJ0023. The strains were again transformed with pVV023 to obtain MsmegpEJwhiB5-pVV0023 and MsmegpEJ0023-pVV0023 double transformants. Promoter activities were measured in double transformants (MsmegpEJwhiB5-pVV0023, MsmegpEJwhiB5-pVV16, MsmegpEJ0023-pVV0023, and MsmegpEJ0023-pVV16). Overexpression of Rv0023 caused approximately fivefold decrease in whiB5 promoter activity (Figure 3C), whereas no significant change in the promoter activity of Rv0023 was noted ( Figure 3D). Rv0494, a FadR transcriptional regulator, was used as a negative control, which did not affect the promoter activities of any of these genes. These data suggest that Rv0023 negatively regulates the expression of whiB5, but it is not auto-regulatory.

Rv0023 Binds to whiB5 in vitro
As we have observed that Rv0023 expression represses the promoter activity of whiB5 in Msmeg, it prompted us to further test whether Rv0023 interacts with whiB5 promoter or not. Five hundred base pairs upstream and 50 bp downstream of whiB5 gene containing the promoter were radiolabeled and EMSA was performed with the recombinant Rv0023.  The radiolabeled upstream region of whiB5 interacted with the purified recombinant Rv0023 in a dose-dependent manner.
No interaction of Rv0023 was observed with non-specific DNA ( Figure 4A). Both EMSA and β-galactosidase assays confirmed that Rv0023 interacts with whiB5 upstream and negatively regulates whiB5 expression. In order to find the exact binding site of Rv0023, the 550 bp upstream of whiB5 was divided into two fragments: UF1 of 340 bp and UF2 of 250 bp ( Figure 4B). We observed that only the UF2 fragment binds to Rv0023 (Supplementary Figure 3).
The UF2 fragment was further divided into six overlapping fragments, F1-F6 ( Figure 4B and Table 7). Purified Rv0023 binds to fragments F1 and F2, whereas no binding was observed with other fragments (Figure 4C). So, we looked for a binding site at the overlapping region of F1 and F2. We generated new oligonucleotides (OL1-OL5) covering the F1-F2 overlapping region ( Figure 5A and Table 8). EMSA with these radiolabeled fragments showed that Rv0023 binds to OL1 and OL3 fragments whereas no binding was observed with other fragments (Figure 5B). Close examination of these fragments revealed the presence of an imperfect palindrome present in both sequences, TATAcgtTTTT in OL1 and TATAcgcTTTT in OL3 ( Figure 5C). To confirm the binding sites identified, we mutated TATAcgtTTTT to TGTGcgtTGTG in OL1 and TATAcgcTTTT to TGTGcgcTGTG in OL3 to obtain OL1M and OL3M, respectively ( Table 8). Binding studies have shown that Rv0023 binds to the native OL1 and OL3 fragments, but not to the mutant fragments, OL1M and OL3M (Figure 5C). We conclude that there are two binding sites (TATAcgtTTTT and TATAcgcTTTT) of Rv0023 in the upstream region of whiB5 comprising an 11-bp imperfect palindromic sequence with a 3-bp spacer region.

Analysis of Rv0023 Binding Site
The importance of each residue within the Rv0023 binding site was determined by generating 39 bp probes (M0-M12)  containing specific point mutations on each half of the imperfect palindrome and in the spacer region ( Figure 6A). The probes were radiolabeled and were used in binding studies with recombinant Rv0023. The binding was observed with probes M0 (unaltered), M1, M4, and M5, and significant loss of binding was observed with M2, M3, M6, M7, and M8 fragments, suggesting the importance of these residues in Rv0023 binding. Changing the residues in the spacer region (M9-M10) did not affect Rv0023 binding; however, changing the length of spacer region by one base lead to significant loss of Rv0023 binding (M11-M12) ( Figure 6B). As mentioned earlier, the organization of Rv0023 and whiB5 is conserved across pathogenic mycobacterial species. Therefore, we looked into the conservation of identified Rv0023 binding sites across the conserved genomes. For this, we aligned the upstream region of whiB5 orthologs from different pathogenic mycobacterial species strains and found that the two binding sites of Rv0023 are conserved across the analyzed mycobacterial species (Figure 6C). These results highlight the important residues for Rv0023 binding and its conservation across mycobacterial species.

Rv0023 Binding Site Overlaps With the whiB5 Promoter Region
In order to understand the mechanism of Rv0023-mediated repression of whiB5, the TSSs of whiB5 and Rv0023 were mapped. The putative −10 region of whiB5 was identified earlier by RACE (Casonato et al., 2012) (Figure 7A). To test the functionality of the putative −10 region, we cloned the 250 bp upstream of whiB5 into lacZ reporter vector (pEJwhiB5WT). The promoter activity was measured using the  β-galactosidase assay in Msmeg. Mutations in the putative −10 region of whiB5 (atacgctt to gcacgcgg) abolished the promoter activity, confirming the −10 region of whiB5 ( Figure 7B).
To identify the promoter region of Rv0023, primer extension was performed. We identified the TSS at 228 bp upstream of Rv0023 start codon ( Figure 7C). A −10-like hexamer sequence TCATAG was identified upstream of TSS. To validate the functionality of the −10 region of Rv0023, 400 bp upstream of Rv0023 was cloned into a lacZ reporter vector (pEJ0023WT). β-Galactosidase assay with WT promoter and mutated promoter (pEJ0023MUT) indicated significant decrease in the promoter activity of the mutant strain compared to WT (Figure 7D).
Mapping the promoters of whiB5 and Rv0023 with Rv0023 binding site revealed that Rv0023 binding site overlaps with the promoter of whiB5 but not of the Rv0023 promoter region ( Figure 7E).

DISCUSSION
In this study, Rv0023, a member of the XRE family of transcriptional regulators, has been characterized, and its role in INH and ETH drug tolerance has been explored. The first part of our study concerns the role of Rv0023 in the physiology of mycobacteria. For our study, we used Msmeg as a surrogate model to study the effects of various stress conditions on wild type and Rv0023-expressing strains. It was observed that overexpression of Rv0023 confers higher tolerance toward INH and ETH in Msmeg. It was seen that the ectopic expression of Rv0023 alters the NADH/NAD + ratio, thereby increasing drug tolerance. In the second part of the manuscript, we identified Rv0023 as a transcriptional regulator and studied the regulatory effect at whiB5-Rv0023 locus and observed that Rv0023 negatively regulates whiB5 expression but is not auto-regulatory. Rv0023 regulates whiB5 expression by binding to specific sequences in the upstream region of whiB5. Our data indicate that there are two binding sites for Rv0023 upstream of whiB5 and one of the sites overlaps with the whiB5 promoter, thereby possibly occluding the binding of RNA polymerase. The binding site of Rv0023 is conserved across mycobacterial species as confirmed by multiple sequence alignment. Apart from this, we have also characterized the binding site of Rv0023 and found important bases for binding. While analyzing the spacer region, it was found that the length of the spacer region is important for proper binding. Isoniazid, isonicotinic acid hydrazide, is a synthetic drug and the occurrence of INH-resistant strains is significantly more frequent than other drug-resistant Mtb clinical strains (Nachega and Chaisson, 2003). ETH, 2-ethylthioisonicotinamide, is a structural analog of INH and was first synthesized in 1956 (Grumbach et al., 1956). Many genes have been found to be associated with INH and ETH resistance in clinical isolates, but the role of transcription factors regulating INH and ETH co-resistance is poorly understood. Rv0023, a transcriptional regulator, conferring higher tolerance toward INH and ETH in Msmeg, provided us a chance to explore the role of transcription factors in INH and ETH co-resistance in Mtb. To elucidate the mechanism by which Rv0023 confers drug tolerance, we investigated two main genes known to be involved in INH and ETH co-resistance, inhA (enzyme involved in the synthesis of cell wall mycolic acid) (Banerjee et al., 1994) and ndh (NADH dehydrogenase maintains the NADH/NAD + ratio) (Miesel et al., 1998). We checked their expression levels in the vector control and Rv0023-expressing Msmeg strains and differential expression was only observed in the ndh gene. The ndh gene oxidizes NADH to NAD + , which maintains the ratio of NADH/NAD + in mycobacterial cells (Jaworowski et al., 1981). Loss of ndh function leads to higher levels of NADH in the cytosol. Previous studies have shown that the higher NADH/NAD + levels interfere with activation of INH/ETH drugs by competitive inhibition with the formation of INH-NAD and ETH-NAD and thus confer resistance to INH/ETH in Msmeg and BCG (Miesel et al., 1998;Vilcheze et al., 2005). So, we further measured the NADH/NAD + ratio in vector control and Rv0023-expressing Msmeg strains. The NADH/NAD + ratio was found to be increased in the Rv0023-overexpressed Msmeg strain, suggesting that negative regulation of ndh gene by Rv0023 is probably the mechanism by which Rv0023 confers INH and ETH tolerance in Msmeg. Our results were supported by previous studies where it was shown that Rv0023 regulon is enriched for NAD reductases (Rustad et al., 2014). Rv0023 did not bind to the upstream region of ndh, indicating that the regulation occurs possibly through indirect means, which may involve multiple intermediate gene products. Further studies are required to completely decipher this mechanism.
Synteny analysis has shown that whiB5-Rv0023 locus is present mainly in pathogenic species of mycobacteria. The gene whiB5 belongs to the WhiB family of transcriptional regulators, which are exclusive to actinomycetes, such as Mycobacterium and Streptomyces spp. (Soliveri et al., 2000). Earlier studies have shown that whiB5 is a global transcriptional regulator, regulating 58 genes of diverse functions, including sigM and genes encoding for Type VII secretion systems such as esx-2 and esx-4. It was shown that whiB5 has a role in Mtb virulence and reactivation (Casonato et al., 2012). As the whole locus of whiB5-Rv0023-Rv0024 is absent in Msmeg, it is difficult to gauge the true effect of Rv0023 in the physiology of Mtb. Further investigation in Mtb may reveal important aspects of Rv0023 regulation and its effects. Hence, taking into account the earlier studies and our current data, we surmise that Rv0023 plays a significant role in modulating genes associated with NADH/NAD + levels, which further influences mycobacterial physiology.
In conclusion, our work shows, for the first time, that Rv0023 has a role in conferring INH and ETH tolerance in Msmeg. Understanding the role of transcriptional factors in the development of drug resistance will open new avenues in the field of drug discovery and may provide important insights into Mtb physiology.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

AUTHOR CONTRIBUTIONS
AR, SG, RA, and SY conceived the hypothesis and rationale of the study, analyzed the results, and wrote the manuscript. SG performed all the experiments. CR contributed in cloning of constructs and recombinant protein purification.

FUNDING
SG was supported by a Centre for DNA Fingerprinting and Diagnostics fellowship. RA was supported earlier by a Department of Biotechnology fellowship. SY was supported earlier by a Council of Scientific and Industrial Research fellowship at the Computational and Functional Genomics Group. CR was supported by a Centre for DNA Fingerprinting and Diagnostics fellowship.