Mycobacterium tuberculosis

Review

SPECIAL FOCUS y Targeting Antibiotic Resistance in M. tuberculosis

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WhiB7, a transcriptional activator that coordinates physiology with intrinsic drug resistance in Mycobacterium tuberculosis Expert Rev. Anti Infect. Ther. 10(9), 1037–1047 (2012)

Ján Burian1,2, Santiago Ramón-García1,2, 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 [email protected] 1

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. www.expert-reviews.com

10.1586/ERI.12.90

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

© Charles J Thompson

ISSN 1478-7210

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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 [10–14] . 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) [16–18] . 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 (whiA-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 1038

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 [31–33] . 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 iron–sulfur 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 Expert Rev. Anti Infect. Ther. 10(9), (2012)

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Fully conserved Strong group conserved

Iron–sulfur cluster-binding cysteines

Unique turn

AT-hook motif

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

Core sequence

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.

WhiB7 WhiB7_SM WhiB7_RH WhiB7_SC

Variable N-terminus

WhiB6

WhiB5

WhiB7_SM

WhiB7

WhiB7_RH

WhiB7_SC

WhiB4

WhiB1

WhiB2

WhiB3


WhiB7 regulates intrinisic antibiotic resistance in Mycobacterium tuberculosis

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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 (4–64-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 id

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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