SPECIAL FOCUS y Targeting Antibiotic Resistance in M.

tuberculosis
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Review

WhiB7, a transcriptional activator that coordinates physiology with intrinsic drug resistance in Mycobacterium tuberculosis
Expert Rev. Anti Infect. Ther. 10(9), 10371047 (2012)

Jn Burian1,2, Santiago Ramn-Garca1,2, Charles G Howes1 and Charles J Thompson*1,2

Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 2 The Centre for Tuberculosis Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada *Author for correspondence: Tel.: +1 604 822 2501 Fax: +1 604 822 6041 charles.thompson@ubc.ca
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Current tuberculosis treatment regimens are notoriously limited, lengthy and becoming increasingly ineffective due to the emergence of drug-resistant mutant strains of Mycobacterium tuberculosis. The intrinsic resistance of M. tuberculosis to the majority of available drugs relies both on the impermeability of its cell envelope, and its ability to activate specific genes and physiological states. WhiB7 is a transcriptional regulatory protein underlying this adaptive process. Transcription of the whiB7 gene is upregulated in response to a variety of antibiotics having different structures and targets, as well as in response to metabolic signals. The whiB7 regulon activates various systems of intrinsic drug resistance involving antibiotic export, antibiotic inactivation (by chemical modifications of the drug or its target) and significant changes to thiol redox balance. Drugs have been identified that inactivate resistance determinants in the whiB7 regulon, thereby potentiating the activities of diverse antibiotics against M. tuberculosis.
Keywords: antibiotic resistance combinational therapy Mycobacterium tuberculosis redox tuberculosis
WhiB

Intrinsic antibiotic resistance of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), continues to be the leading cause of death due to bacterial infection worldwide. There are almost nine million cases and 1.5 million deaths due to TB every year [1] . The intrinsic drug resistance of M. tuberculosis has limited treatment options to a handful of drugs, with the standard regime consisting of four antibiotics (rifampicin, isoniazid, ethambutol and pyrazinamide) administered in combination for a minimum of 6 months. Unfortunately, the combined effects of antibiotic shortages, improper treatment and patient noncompliance have led to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of M.tuberculosis that are essentially untreatable. A concentrated effort is required not only to find new drugs active against M. tuberculosis, but also to generate new ideas that might allow the effective use of existing antibiotics for TB treatment.
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The cell structure and physiology of M. tuberculosis make it intrinsically resistant to many antibiotics commonly used to treat other bacterial infections. Understanding intrinsic resistance and finding compounds that inhibit it might allow the use of previously ineffective, clinically approved drugs to treat TB. Intrinsic resistance is dependent on the unique mycobacterial cell envelope acting in combination with systems that inactivate drugs in the cytoplasm. The cell envelope limits the rate of antibiotic penetration into the cytoplasm. This provides time for the activation of systems, including efflux pumps, drug degrading or modifying enzymes and target modifying enzymes [2] that reduce the toxicity of internalized drugs. As in other bacteria (described below), the M.tuberculosis genome contains genes for resistance to various antibiotics [3] ; some of these genes have alternative physiological functions [2] . In recent years, three concepts have emerged as foundations for understanding intrinsic
ISSN 1478-7210

Charles J Thompson

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resistance. The first concept is that intrinsic antibiotic resistance is dependent on hundreds of proteins having homology not only to conventional resistance genes (transporters and modifying enzymes), but also to genes that provide a broad range of cellular functions involving metabolism, cell envelope structure and redox balance. This concept arose from gene disruption experiments or overexpression of genomic libraries in various bacteria, including Escherichia coli [4] , Pseudomonas aeruginosa [5,6] , Acinetobacter baylyi [7] or Mycobacterium [2,8,9] , consistently identifying multiple genes that can contribute to resistance. The second concept is that many antibiotics activate expression of a broad array of genes having no direct relationship to conventional drug resistance genes [1014] . For example, ribosome targeting antibiotics trigger heat or cold shock responses in E. coli [15] ; in Salmonella enterica serovar Typhimurium, antibiotic treatment generates large perturbations in cell metabolism by activating transcription of up to 5% of its genes [10] ; in Streptomyces, sub-MIC concentrations of the antibiotics erythromycin, pristinamycin or thiostrepton, induce major changes in gene expression [11,12,14] . The third concept is that redox stress underlies the toxicity of many antibiotics. Oxidative stress is believed to be the cause of bacterial death generated by some bactericidal antibiotics in E. coli, Staphylococcus aureus and Enterococcus faecalis (but not in Listeria monocytogenes ) [1618] . Biosynthesis of H2S has been shown to mitigate the toxic effect of oxidative stress and provide resistance to many structurally and functionally diverse antibiotics [19] . Our studies have revealed a transcriptional regulator (WhiB7) that coordinates expression of intrinsic drug resistance genes with antibiotic-induced changes in redox physiology. In this review, the authors present evidence that intrinsic resistance in M. tuberculosis is dependent on metabolic genes that are activated by the multidrug-inducible transcriptional regulator WhiB7. The authors also analyze the appealing concept of using chemical inactivation of intrinsic drug resistance systems as a new therapeutic approach for the treatment of TB. Compounds that inactivate the unique family of seven whiBlike genes (referred to as whiB1-7 ), especially whiB7 (or proteins in its regulon), might be effective anti-TB drugs, either acting alone or in combination, to potentiate the activities of various antibiotics.
whiB genes play essential roles in morphological differentiation, metabolism & antibiotic resistance

whi genes (whi A-I) were initially identified in studies of sporulation in Streptomyces coelicolor [20,21] . Mutations were easily recognized as white colonies covered with filamentous aerial mycelia that are unable to divide and mature into gray spores. Later, the whiB locus was recognized as a novel transcription factor not necessarily linked to sporulation, likely playing related roles in cell division throughout the actinomycete taxon [22,23] . The whiB gene from S. coelicolor has served as a prototype for the WhiB-family of putative transcriptional regulators. Actinomycetes (and at least one actinophage), including mycobacteria, all have multiple whiB paralogs in their genomes (Figur e 1A) [23,24] . These whiB paralogs also
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play important roles in functions unrelated to cell division, such as antibiotic biosynthesis and redox balance. Most studies have focused on the seven whiB homologs found in M. tuberculosis. WhiB1 is an essential transcriptional regulatory protein that is nitric oxide sensitive and behaves as a transcriptional repressor of its own promoter as well as that of the chaperonin groEL2 [25,26] . It is constitutively expressed throughout growth and can be upregulated by Rv3676 , encoding a cAMP-dependent t ranscriptional activator protein [27,28] . cAMP promotes whiB1 transcription at low concentrations and inhibits transcription at high concentrations [29] . Interestingly, M. tuberculosis produces a burst of cAMP upon entry into macrophages that may promote survival within the antibacterial environment of phagosomes. Rv3676 also positively regulates rpfA [28] , a resuscitation-promoting factor that serves to stimulate growth in stationary phase cultures of M. tuberculosis [30] . These observations suggest that, in addition to its essential function during normal growth, whiB1 may be coregulated with genes involved in both survival and reactivation of dormant M. tuberculosis in macrophages. whiB2 is the ortholog of the S. coelicolor whiB gene. It is essential in M. tuberculosis and plays roles in cellular septation as well as regulating its own expression [3133] . Transcription of whiB2 is increased as cultures enter stationary phase, and is stimulated during nutrient starvation or after treatment with cell envelope inhibitors [34] . Interestingly, the mycobacterial phage TM4 uses an N-terminally truncated homolog of WhiB2 as a negative regulator of the host whiB2 to prevent superinfection [33] . WhiB3 has an important role in pathogenesis, attributed to its redox-sensitive activity as a regulator of transcription [35] . Its transcriptional regulatory function is thought to be dependent on various redox forms and on its ability to associate with two partners, that is, DNA sequences in its target promoters [36,37] and SigA, the vegetative sigma factor of M. tuberculosis [35] . The WhiB3 apoprotein binds DNA in its oxidized, but not reduced, form; the WhiB3 holoprotein (containing an ironsulfur cluster) may bind DNA weakly and independently of its redox state [36,37] . The effect of redox on SigA binding has not been reported. Steyn and colleagues proposed a reductive stress model to explain its role in the host. Inhibition of respiration by nitric oxide or hypoxia, together with fatty acid oxidation during macrophage infection, are believed to generate a reducing environment that can be reversed by synthesis of polyketides and storage lipids that serve as redox sinks [36,37] . The precise role of WhiB3 as an activator or repressor of transcription of target genes has not been documented biochemically; however, a screen of potential WhiB3 binding sites suggests that it transcriptionally activates genes involved in fatty acid metabolism and stress responses [38] . The whiB3 gene is also highly upregulated in liquid cultures during late stationary phase and upon acid stress [34] . whiB4 is expressed throughout growth [34] and may be within a regulon under the control of an alternate sigma factor (SigF). SigF activates gene expression in response to various stress inducers [39] . whiB5 is expressed throughout growth, down-regulated during late stationary phase, and repressed in response to membrane
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WhiB3

WhiB2

www.expert-reviews.com WhiB1 WhiB4 WhiB7_SC WhiB7_RH WhiB7 WhiB7_SM WhiB5 WhiB6 Variable N-terminus Core sequence Ironsulfur cluster-binding cysteines Unique turn AT-hook motif

WhiB7 WhiB7_SM WhiB7_RH WhiB7_SC

MSVLTVPRQTPRQRLPVLPC HVGDPDLWFADTPAGLEVAKTLC VSCPIRRQC LAAALQRAEPWGVWGGEIFDQGSIVSHKRPRGRPRKDAVA MTAPTTGVAPMTCETRLPAVPC HVGDPDLWFAENPGDLERAKALC AGCPIRVQC LTAALERQEPWGVWGGEILDRGSIVARKRPRGRPRKDSGGNPAAA MSTVTCRGVSETSTATSGFVQIVSARGDLP C RVDDPDLWFADSPTELEQAKALC ASCPIRSRC LDAALDRGEPWGVWGGEIFDQGVVIARKRPRGRPRKTQTLVCA MQLEAHAPSVPPSDTIPKPCSTEDSTLTPLTALTALDDAIENLGVPVP C RSYDPEVFFAESPADVEYAKSLC RTCPLIEAC LAGAKERREPWGVWGGELFVQGVVVARKRPRGRPRKNPVSA

WhiB7 regulates intrinisic antibiotic resistance in Mycobacterium tuberculosis

Fully conserved Strong group conserved

Review

Figure 1. Conserved sequence and structural features of WhiB7 and its paralogs in WhiB phylogeny. (A) Phylogenetic tree of seven proteins encoded by whiB paralogs found in Mycobacterium tuberculosis, including whiB7 homologs from SM, RH and SC. (B) Sequence comparison of WhiB7 proteins from (A). Conserved structural features predicted by the sequence are highlighted; four conserved cysteines in yellow, WhiB-specific tryptophan-containing motif predicting a turn in green, AT-hook motif in red, other fully conserved residues in blue and strong group conservations in purple. The alignment and tree were generated by ClustalX2 [76] . The tree was visualized using Tree view. Protein sequences used were as indicated by the Kyoto Encyclopedia of Genes and Genomes. RH: Rhodococcus jostii; SC: Streptomyces coelicolor ; SM: Mycobacterium smegmatis.

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stress [34] . Recently, whiB5 has been implicated in M. tuberculosis virulence and reactivation, influencing transcriptional activation of 58 genes and possibly repressing its own promoter [40] . whiB6 is highly upregulated under membrane stress (SDS and ethanol treatment, heat shock) [34] . It may play a role in the activation of the ESX-1, a secretion system important for pathogenesis and m acrophage escape [41] . whiB7, the primary focus of this review, is critical for the activation of several intrinsic antibiotic resistance systems, and is one of a handful of genes (including whiB3 ) that is globally upregulated in the M. tuberculosis complex within resting or activated murine macrophages [42,43] . whiB7 is of special interest as it provides high or low resistance (464-fold) corresponding to diverse structural classes of antibiotics. whiB7 mutants are more sensitive to a variety of antibiotics including macrolides, tetracyclines, lincosamides and some aminoglycosides. This suggests that the chemical inactivation of intrinsic resistance systems, including genes in the whiB7 regulon, might allow effective use of numerous currently available antibacterial drugs for TB treatment.
Aminoglycosides
c Ma rol id es

Activation of whiB7 transcription by compounds with diverse structures & targets

Flu

oro

qui

no l

one

nes cycli Tetra

Figure 2. Transcription of whiB7 is activated by a subset of antibiotics with diverse structures and targets. Compounds that induce whiB7 expression can be compared and grouped using factors that reflect chemical structure using Tanimoto structural clustering algorithms [101]. The analysis is presented as a tree similar to those used to compare gene or protein homologies. It allows visualization of the structural heterogeneity of the compounds that induce whiB7 transcription (highlighted in red). Branches that occur within the pink circle are structurally dissimilar (Tanimoto score <0.7; this includes all compounds in the upper half of the circle) and are likely to have different bioactivities. Macrolides, aminoglycoside and tetracyclines inhibit protein synthesis by targeting different sites in the ribosome. Fluoroquinolones inhibit DNA synthesis by targeting DNA gyrase or topoisomerase II. Adapted with permission from [45].

An M. tuberculosis whiB7 mutant is more sensitive to ribosome-targeting antibiotics of various structural classes (macrolides, lincosamides, tetracyclines and aminoglycosides), and whiB7 transcription is highly upregulated by these drugs [43] . Independent microarray analysis further supported this conclusion and also showed that the ionophore valinomycin and ofloxacin (a fluoroquinolone) increased whiB7 transcription [Burian J, Howes CG, Thompson CJ, Unpublished Data] [44] . This led to a comprehensive study of whiB7 inducers. A plasmid carrying the whiB7 promoter fused to a green fluorescent protein was used as a reporter to screen our library of approximately 600 drugs, including the majority of available antibiotics (approximately 500), as well as other biologically active compounds. In total, 86 inducers of the whiB7 promoter were identified. They belonged to 25 different structural groups and had a broad range of targets not limited to the ribosome (Figure 2) [45] . Importantly, not all of the activators were more effective against the whiB7 mutant. These results demonstrated that the ability of the compounds to induce the whiB7 promoter was independent of their structure, target or efficacy against a whiB7 mutant [45] . whiB7 transcription apparently responds to a common downstream metabolic change elicited by these different antibiotics. The observation that compounds targeting cell wall synthesis do not induce whiB7 transcription argues against cell wall defects as a trigger of whiB7 transcription. whiB7 is also induced in response to a variety of physiological stresses, including entry into stationary phase, iron starvation and heat shock (but not acidic conditions [pH 4.5] or treatment with SDS or peroxide) [34] . Therefore, functions of the stress responsive systems coordinated by WhiB7 may not be limited to antibiotic resistance but also provide adaptive responses during stationary growth phase or within host cells. For example, tap is within the whiB7 regulon and is required for both stationary phase survival and antibiotic resistance [46] . The fact that strong induction of whiB7 occurs shortly after engulfment by macrophages suggests possible roles in virulence and bacterial survival within the host [42] . Although the effects of antibiotics were classically perceived to reflect inhibition of a single target, they are now known to have additional, far reaching consequences on gene expression and cell metabolism as described above. Of particular interest is the whiB7 inducer acivicin. Acivicin is a glutamine amidotransferase inhibitor triggering a stress response described as metabolic mayhem in E. coli [47] . In Mycobacterium smegmatis, while acivicin has no detectable growth inhibitory activity under the conditions tested, it strongly activates whiB7 transcription [45] . In addition, the level of whiB7 transcription responds to the amino acid content of the media, again underlining the influence of cell metabolism on WhiB7 activity [45] .
WhiB7 responses to redox stress & regulates cell metabolism

Our current understanding of how WhiB7 responds to a diverse repertoire of inducers and then activates transcription of a variety
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Review

of intrinsic resistance genes has emerged primarily from studies of its own autoregulated promoter. As described above, inducers of the whiB7 promoter include antibiotics with different structures and targets, as well as certain physiological stresses. Transcriptional responses to stress are typically mediated by specialized sigma factors that activate general or stress-specific responses in other bacteria. However, the sequences of whiB7 promoter hexamer motifs, conserved upstream of all representative actinomycete homologs, suggest that whiB7 is transcribed by the primary vegetative bacterial sigma factor (SigA) (Figure 3A) [45] . Similarly, the promoters of genes in the whiB7 regulon (eis, erm and tap) include this promoter sequence motif. These promoters all contain a 5bp AT-rich sequence motif located 3bp upstream of the -35 hexamer. WhiB7 contains a C-terminal AT-hook motif predicted to bind this sequence. By analogy to the WhiB3 protein [35] , WhiB7 may also interact with SigA, thereby catalyzing transcription. All of these promoters are nearly inactive in a whiB7 mutant, reflecting their strong dependence on WhiB7 [43,45] . Nevertheless, it is important to note that antibiotic treatment slightly activates the whiB7 promoter in a whiB7 mutant, suggesting the participation of other activators that can independently sense antibiotic-induced stress signals (Figure 3B) [45] . WhiB7, WhiB3 and perhaps other WhiB proteins, may alter SigA activity to upregulate genes within their respective regulons. This would adapt the vegetative sigma factor, allowing it to react to WhiB-specific metabolic stress signals without the participation of specialized sigma factors. Our studies of mycobacteria emphasize the important role of thiol reductive changes that occur shortly after antibiotic treatment [45] . Indeed, oxidative and reductive systems must involve complex and poorly understood fluxes of electrons between pools of NADH, FADH and thiol redox compounds. Reductive stress associated with accumulation of reduced NADH or FADH may have numerous metabolic consequences on enzymes that use them as cofactors, and also result in electron leakage that can induce oxidative stress [48] . In the case of oxidative stress, it is reasonable to assume that protective cellular responses would include systems able to generate reducing potential to eliminate oxidizing agents and repair damaged proteins. Mycothiol, the primary thiol reducing agent in actinomycetes, may play a central role in this process. Importantly, mycobacterial mycothiol metabolic enzymes are known to provide antibiotic resistance [49] . This is due in part to a spontaneous thiol cross-linking reaction that bonds mycothiol to reactive groups within some antibiotics, leading to drug inactivation. Furthermore, macrolide treatment results in a major reductive shift in thiols within the M. smegmatis cytoplasm, as reflected by the increased ratio of reduced to oxidized mycothiol [45] . Evidence presented below suggests that this reductive shift may also increase antibiotic resistance by promoting WhiB7 activation of its intrinsic resistance regulon. Biochemical studies demonstrate that binding of WhiB1, WhiB2 and WhiB3 to DNA is dependent on redox conditions [25,33,36] . Indeed, WhiB7s activity as a transcriptional activator in vivo may be increased by a more reduced cytoplasm that occurs in response to antibiotic treatment or physiological changes that occur in various
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environments. The whiB7 promoter response to a minimal concentration of erythromycin can be drastically amplified if reducing power is provided by a reductive thiol (dithiothreitol) in the medium [45] . Conversely, the presence of a thiol oxidant (diamide) inhibits induction, underlining the dominant role of reduction. However, it is also important to note that the dithiothreitol alone is only able to generate a low level of whiB7 induction. Therefore, in the authors current working model, the initial activation of whiB7 transcription by antibiotics may occur via a whiB7-independent mechanism. WhiB7 activity might subsequently be stimulated by reducing conditions to amplify transcription of its own promoter and regulon (Figure 3) . M. tuberculosis may generate excess reducing equivalents during growth on fatty acids in lung macrophages, thereby generating a stress response [36,50] . The increase in reducing potential may explain the global activation of whiB7 in the M.tuberculosis complex after macrophage entry [42] or in response to lipids in the growth media [43] . Furthermore, glutathione, a major thiol in the eukaryotes, is toxic to M. tuberculosis and used by macrophages to combat infection [51] . Glutathione encountered in the macrophage may be another reductant that promotes WhiB7 activity. Like whiB7, whiB3 is also globally upregulated upon macrophage entry [42] . However, the fact that apo-WhiB3 DNA binding is promoted by oxidizing conditions [36] may suggest additional metabolic complexity. Interestingly, although accumulation of NADH leads to reductive stress, it can, counter-intuitively, also lead to oxidative stress by the production of reactive oxygen species. This has been described in studies of aging in mitochondria [52] and also occurs spontaneously by auto-oxidation of NADH [53] . Together, this suggests that the activity of WhiB proteins may be promoted under either reducing or oxidizing conditions, but also hints that subtle redox changes may lead to important signaling and adaptation cascades. The fact that the thiol reductant dithiothreitol (presumably mimicking mycothiol), synergizes with antibiotics to activate whiB7 transcription in vitro suggests that mycothiol reduction is needed to promote WhiB7 activity (Figure 3) . However, WhiB7 is also required to maintain mycothiol concentration and promote its reduction (in the presence or absence of antibiotic) [45] . These results imply that the reduction of WhiB7 and mycothiol are linked, suggesting a coordinated partnership.
The whiB7 regulon & its roles in drug resistance

whiB7 is a multidrug resistant determinant in M. tuberculosis, Mycobacterium bovis BCG, M. smegmatis and Streptomyces lividans [43] . Microarray analysis of M. tuberculosis identified a regulon including known antibiotic resistance genes and suggested that WhiB7 is autoregulatory [43] . The regulon includes eis (Rv2416c), tap (Rv1258c), ermMT (Rv1988 ) as well as other genes with functions seemingly unrelated to antibiotic resistance. Transcriptional analyses discussed above provide additional evidence that WhiB7 is autoregulatory and that the promoter motifs required for it to activate transcription are found upstream of whiB7, eis, tap and ermMT [45] . Of note, proteogenomic analysis of M. tuberculosis in exponential phase liquid cultures identified proteins corresponding to about 80% of its predicted genes [54] . The fact that no WhiB
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WhiB7 binding site

-35

-10

GCAGGTAGAAAATAGGTTGTGCGATTCAGCTGCTAGGCTCTAGCCTCAGAAAGTCAACTC Wild-type Fluorescence/A600 nm Fluorescence/A600 nm 12 12 Ery Iso Untreated whiB7

Ery Iso Untreated

-2 0 300 600 Time (min) 900

-2 0 300 600 Time (min) 900

WhiB7 RNA polymerase SigA whiB7

AT-hook

whiB7

Figure 3. Transcription of the whiB7 promoter is induced by antibiotic treatment, with WhiB7 required for maximal induction. (A) Sequence of the Mycobacterium smegmatis whiB7 promoter [45] . A conserved AT-rich region likely serving as the WhiB7 binding site is highlighted in red. The -10 and -35 hexamers matching the consensus sequence for SigA-targeted promoters are highlighted in gray. The transcriptional start site is boxed. (B) GFP transcriptional fusion was used to monitor whiB7 promoter activity in M. smegmatis wild type (left) or whiB7 (right) either untreated (red), treated with Ery (blue) or treated with Iso (green). The promoter is induced by Ery but not by Iso. The whiB7 strain has a reduced response to Ery relative to wild-type cells, implying WhiB7 is required for maximal induction. (C) Stress conditions initiate whiB7 transcription, leading to low-level synthesis of WhiB7 protein. WhiB7 then functions to generate a highly reduced cytoplasm that promotes its activity (light blue to dark blue). The initial production of WhiB7 promotes whiB7 transcription by binding to an AT-rich region located 3bp upstream of the -35 hexamer, likely interacting with SigA. This leads to a dramatic increase in transcriptional activity. (B) This research was originally published in the Journal of Biological Chemistry [45] . The American Society for Biochemistry and Molecular Biology. Ery: Erythromycin; GFP: Green fluorescent protein; Iso: Isoniazid.

proteins were identified supported the concept that they require specific stress conditions for upregulation. Curiously, eis, but not tap or ermMT, was detected, suggesting that genes within the WhiB7 regulon may be subject to multiple regulatory pathways. eis was first described as a gene that promotes mycobacterial survival within macrophages [55] . It acetylates the host protein DUSP16/MKP-7, thereby suppressing the immune response [56] . Surprisingly, Eis also provides antibiotic resistance by acetylating aminoglycosides [56,57] . In mycobacteria, eis is transcribed from a
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promoter recognized by SigA, which contains an AT-rich region located 3bp upstream of the -35 hexamer [45,58] . This promoter has homology with generic vegetative promoters in Mycobacterium as well as E. coli, and the minimal promoter, including -10 and -35 hexamer sequences, has been identified and genetically dissected. Mutations in corresponding promoters of aminoglycoside-resistant M. tuberculosis clinical isolates dramatically increased transcription of eis and conferred kanamycin resistance [58] . In addition, the levels of SigA correlated with an increase in eis activation [59] .
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WhiB7 regulates intrinisic antibiotic resistance in Mycobacterium tuberculosis

Review

The authors suggest that there are yet unidentified transcriptional activators (that) contribute to eis expression, which is likely to be WhiB7 [43,45] . Tap is an efflux pump that provides resistance to tetracycline, spectinomycin and several other antibiotics [46,60,61] . Importantly, tap also has physiological roles required for repeated passage in liquid cultures and detoxification of cytoplasm upon entry into stationary phase [46] . Overexpression of an efflux pump may contribute to the multidrug resistance phenotype, not only in mycobacteria but other pathogens as well [62,63] . The clinical significance of this has recently been demonstrated by Louw et al., who showed that rifampicin induced multiple efflux pumps in rifampicin-resistant M. tuberculosis, resulting in cross-resistance to the fluoroquinolone ofloxacin [64] . Erm is a ribosomal RNA methyltransferase that provides resistance to macrolides [65] . Treatment of M. smegmatis with subinhibitory concentrations of macrolides significantly increases erm-dependent and whiB7-dependent macrolide resistance [45,66] . As in M. tuberculosis, the erm promoter is dependent on WhiB7. Consequently, preincubation with acivicin, a nonmacrolide inducer of whiB7, generates increased macrolide resistance [45] . The fact that acivicin, a drug that disrupts metabolism without inhibiting growth, increases macrolide resistance, is further evidence that metabolic state plays a role in determining intrinsic drug resistance.
WhiB7 structures & functions

humans. In eukaryotic cells, iron-sulfur clusters supply many critical redox functions [69] , and the AT-hook motif is found on high-mobility group proteins that participate in a variety of protein/genome interactions in eukaryotes [70] . The predicted helical turn is unique, likely to be exposed, and may interact with a conserved part of the transcription machinery [22,23] . Furthermore, drugs that bind to and inhibit its function may also inhibit other essential WhiB paralogs in M. tuberculosis. Interestingly, WhiB3 binds to SigA [35] within a region (4.2) known to interact with other transcriptional regulators [71] . SigA containing a mutation in region 4.2 (Arg515His) is unable to bind WhiB3. M. tuberculosis strains with either whiB3 or SigA (Arg515His) mutations show a similar decrease in virulence [36,37,72] . It is also important to note that the Arg515His mutant is viable, implying that essential WhiB1 or WhiB2 functions are not severely disrupted. This may mean that not all WhiB proteins interact with transcription machinery in the same way, perhaps involving different sigma factors. In addition to their activities as transcriptional regulators, WhiB proteins may have alternate roles as enzymes. All M. tuberculosis reduced apo-WhiB proteins, with the exception of WhiB2, can reduce insulin disulfide at various efficiencies [67] . The relevance of a redox reaction dependent on dithiothreitol and an artificial substrate has been a concern. However, thioreductase activity of WhiB1 acting on the M. tuberculosis protein GlgB has also been reported [73] .
Discovery of compounds that make M. tuberculosis drug sensitive by inhibiting genes in the whiB7 regulon

WhiB proteins contain several conserved structural motifs. WhiB7, like all other WhiBs, contains four conserved cysteines that bind a redox-sensitive iron-sulfur cluster as well as a characteristic tryptophan-containing G(V/I)WGG turn (Figure 1B) [24,43,67] . All WhiB proteins have basic amino acid residues near their C-termini that are most likely responsible for DNA binding [24] . In the case of WhiB7, this sequence includes an AT-hook, a motif commonly found in eukaryotic transcriptional factors that binds the minor groove of AT-rich DNA [43,68] . Structural studies of other WhiB proteins suggest that all three features are essential for WhiB7 function [22,32] . Studies of WhiB proteins have been complicated by the fact that the cysteines can form disulfide bonds or load ironsulfur clusters, resulting in many different redox states that have structural and functional importance [37] . In principle, the four cysteine residues in WhiB apoproteins can be fully reduced or oxidized, generating various folded or unfolded structures. Alternatively, these four cysteines can bind an ironsulfur cluster. The ironsulfur cluster can exist in one of at least four redox states. Such a variety of potentially unstable redox states make it difficult to generate one homogeneous form of the protein for in vitro studies of structure and function. Similarly in vivo, WhiB proteins may adopt different structures and functions in response to changing redox metabolism. One of the three conserved structural motifs, a predicted helical turn unique to WhiB proteins, may be the most attractive target for a clinically useful WhiB7 inhibitor. Drug compounds inhibiting one of the other two conserved structures (iron-sulfur binding cysteines and AT-hook) are likely to be toxic for
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Spectinomycin, an antibiotic with limited efficacy in vitro against M. tuberculosis , but within the whiB7 susceptibility spectrum, is both more potent against a M. smegmatis whiB7 mutant [R amn-Garca S, Thompson CJ, Unpublished Data] and is a strong inducer of the whiB7 promoter [45] . Interesting results were obtained when spectinomycin was used in a novel high-throughput screen to find compounds that potentiate its activity [74] . This screen identified more than 20 compounds that acted synergistically with spectinomycin in M. smegmatis. These pairwise drug combinations were confirmed in M. tuberculosis and proved to be synergistically effective in a macrophage model of infection. This provided initial proof of principle that chemical inactivation of intrinsic resistance systems could sensitize M. tuberculosis to clinically approved antibacterial drugs and make them more effective agents for treating TB [74] . The spectinomycin sensitizers each generated a unique sensitivityinducing profile when tested in combinations with a variety of antibiotics. However, they did not increase the activities of all other antibiotics in the whiB7 sensitivity spectrum (clarithromycin, clindamycin, streptomycin and tetracycline were tested). This implies that the sensitizers do not inhibit WhiB7 directly, but may inactivate WhiB7-dependent resistance systems or cause other metabolic changes that can decrease antibiotic resistance [75] . The fact that none of these sensitizers acted in synergy with
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spectinomycin in a whiB7 mutant provides evidence that their targets are in the whiB7 regulon [Ramn-Garca S, Thompson CJ, Unpublished Data] . A better understanding of WhiB7 and its regulon may provide vital information for the synthesis of rationally designed inhibitors.
Expert commentary & five-year view

Future studies of the WhiB family of proteins in M. tuberculosis will not only increase our understanding of the fundamental physiological processes underlying intrinsic antibiotic resistance, redox balance, virulence and cell division, but may also provide new TB treatment strategies. Many intriguing questions remain to be addressed. How does metabolism affect intrinsic resistance and what is the role of the WhiB7 protein in regulating metabolism? WhiB7 not only activates antibiotic resistance systems, but also influences redox metabolism under both normal growth conditions and stress. How are these two functions related? What are the common metabolic or redox signals generated by the diverse array of compounds that activate whiB7 transcription? While its promoter reacts to thiol redox balance, this is not sufficient for full induction. What are the other metabolites that serve as essential partners in the general response? The introduction of metabolomic approaches should provide important insights into the role of WhiB7 in physiology, complementing expression microarrays that have provided insights into its genetic regulon. Can compounds be developed to inactivate WhiB7 directly or block metabolic activating signals? Inhibiting specific antibiotic resistance determinants is a proven means of increasing antibiotic efficacy. While no direct inhibitor of WhiB7 has yet been identified, structural information may allow rational design. What are the structures of WhiB proteins in different redox states? The multiple redox states of cysteine residues and ironsulfur clusters in WhiB proteins makes them extremely difficult to study. While producing and characterizing WhiB proteins in various redox states is a challenging task, it could provide valuable information on how these different structures underlie their function(s). Key issues

How do WhiB proteins interact with promoters and RNA polymerase holoenzymes to activate or repress transcription? To date, only WhiB1 has been shown to have activity in vitro, acting as a repressor in a biochemical assay. Mutant phenotypes and microarrays support the idea that many WhiB proteins function as transcriptional activators but this has not been directly demonstrated. Developing in vitro assays for transcriptional activity may provide essential tools not only to analyze WhiB activity but also as a platform to screen small molecule inhibitors. Are the thioreductase activities of WhiB proteins relevant to their functions in vivo ? Even though WhiB proteins may function as essential transcriptional regulators of specific promoters, they may have additional functions. The specificity of thioreductase activity on putative target proteins and metabolic consequences need to be clarified. The fact that core amino acid motifs are conserved in all WhiB proteins and apparently required for their activity, evokes the question of how individual WhiBs react specifically to different stresses and activate specific promoters? WhiB proteins vary primarily in their C-termini, the likely determinants of their DNA binding specificities. A simplistic model based on their sequence homology, suggests that all should be activated by the same stress signal, and then that each activates transcription of its respective regulon. However, studies in liquid cultures show that each whiB gene is regulated by a unique repertoire of inducers and stress conditions [34] . Is their specificity derived from variation at the N-terminus or perhaps subtle changes around the core motifs? whiB7 transcriptional induction seems partially controlled by another antibiotic-responsive transcriptional activator (Figure 3B) . Likewise, in the case of other whiB genes, is the specificity of induction dependent on additional regulators? Can clinically relevant WhiB7 inhibitors be identified and used in combination for TB treatment? Understanding the downstream effectors of WhiB7, as well as other WhiB proteins, may lead to the identification of novel targets to treat TB or increase the efficacy of currently available antibiotics. Compounds that inhibit several WhiB target proteins would likely be bactericidal, inactivating multiple systems essential for growth, virulence or

Tuberculosis is a global health problem that is notoriously difficult to treat, in part due to its intrinsic resistance to most available antibiotics. The few drugs that are used to treat tuberculosis have been rendered ineffective by mutations that make some strains multidrug resistant and extensively drug resistant. Intrinsic resistance to several structural classes of antibiotics (macrolides, lincosamides, tetracyclines and aminoglycosides) is dependent on whiB7, encoding a putative redox sensitive transcriptional regulator. WhiB7 activates intrinsic resistance systems in response to many antibiotics that do not have common structures or targets, and in response to physiological stresses. WhiB7 mediates resistance to many, but not all, antibiotic inducers. There is a link between WhiB7 and the main mycobacterial thiol antioxidant, mycothiol, underlying the importance of redox metabolism in antibiotic resistance. There are seven members of the Mycobacterium tuberculosis WhiB family of regulators, playing critical roles in antibiotic resistance, pathogenesis, redox balance, cellular septation and survival within macrophages. Understanding how WhiB7 responds to antibiotic stress and upregulates transcription to achieve intrinsic resistance would allow the design of inhibitory compounds. Such studies could be expanded to other WhiB-like proteins. Several inhibitors of enzymes in the WhiB7 regulon have already been isolated, providing promising lead compounds that are able to sensitize Mycobacterium tuberculosis to clinically available drugs.

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intrinsic antibiotic resistance in M. tuberculosis. However, the focus of M.tuberculosis research must expand beyond screens for antimycobacterial compounds. While identifying novel inhibitors is an essential task, it should be a part of a diversified research program. Understanding the fundamental ways M. tuberculosis controls essential aspects of its metabolism and intrinsic resistance may lead to novel approaches for TB therapy.
Acknowledgements

the review; S Ramn-Garca made important contributions in developing concepts; CG Howes contributed data analysis.
Financial & competing interests disclosure

The authors would like to thank Kien Nguyen and Gaye Sweet for their constructive comments on the review. J Burian and CJ Thompson wrote

This work was supported by grants from the Canadian Institute of Health Research (MOP-82855) and the Canadian Lung Association. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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