Gene Expression of Mycobacterium tuberculosis Putative Transcription Factors whiB1-7 in Redox Environments

et al. (2012) Gene Expression of Mycobacterium tuberculosis Putative Transcription Factors whiB1-7 in Redox Environments. PLoS ONE 7(7): e37516. doi:10.1371/journal.pone.0037516 Gene Expression of Mycobacterium tuberculosis Putative Transcription Factors whiB1-7 in Redox Environments Christer Larsson 0 Brian Luna 0 Nicole C. Ammerman 0 Mamoudou Maiga 0 Nisheeth Agarwal 0 William R. Bishai 0 Niyaz Ahmed, University of Hyderabad, India 0 1 Center for Tuberculosis Research, Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 2 Vaccine and Infectious Disease Research Center Translational Health Science and Technology Institute, Gurgaon (Haryana), India, 3 KwaZulu-Natal Research Institute for Tuberculosis and HIV, Durban, South Africa, 4 Howard Hughes Medical Institute , Chevy Chase, Maryland , United States of America The seven WhiB proteins of Mycobacterium tuberculosis (M.tb) are widely believed to be redox-sensing transcription factors due to their binding of iron-sulfur clusters and similarities to DNA binding proteins. Here, we explored the nature of this hypothesized relationship. We exposed M.tb to physiologic conditions such as gradual hypoxia, nitric oxide (NO), cyclic AMP and in vivo conditions, and measured transcription of the whiB genes. We found whiB3 to be induced both by hypoxia and NO, whiB7 to be induced in macrophage-like cells, and whiB4 to be induced in mouse lung. Cyclic AMP induced whiB1,22, 24, 26 and 27. Our data indicate that the M.tb whiB genes are induced independently by various stimuli which may add versatility to their suggested redox-sensing properties. - Funding: This work was supported by The Swedish Research Council (VR) www.vr.se, the Swedish Society for Medical Research (SSMF) www.ssmf.se, and the National Institute of Health (NIH) www.nih.gov AI36973 and AI37856. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Mycobacterium tuberculosis (M.tb) is one of the worlds most successful pathogens. Approximately one-third of humanity is infected, but only 510% of this population develops active disease, which in 2010 accounted for 8.8 million cases of tuberculosis (TB) and 1.45 million deaths [1]. The remaining 9095% either clears the infection or carries a latent or quiescent infection remaining for extensive time, often for life. This requires an extraordinary adaptation to the various and often extreme environments the bacteria meet in the host such as nitric oxide (NO) and low pH in the phagosome, or months or years in hypoxic or even anaerobic compartments such as caseous granulomas. In this state of latency bacteria still have to be able to respond to stimuli to become active again when conditions to do so are favorable. Availability of oxygen is fundamental for M.tb metabolism and may be important for the entering into latency and triggering reactivation [2]. The most well-studied oxygen-sensing gene regulator in M.tb is the DosR/DevR two-component signaling system [3], but there are likely other systems also responding to redox changes. One group of proteins that has been suggested is the WhiB family. The WhiB-like proteins are unique to Actinomycetes. They are small (75130 amino acids) with 4 conserved cysteine residues typical for metal-coordinating DNAbinding proteins, and these proteins also have a conserved aspartate and a helix-turn-helix-like motif [4]. The 4 cysteines bind a [4Fe-4S] cluster which changes the conformation of the protein in a redox-dependent manner [5,6]. Together with the DNA binding properties of helix-turn-helix-like structures, the WhiB proteins are hypothesized to be transcription factors binding to or releasing DNA depending on redox potential in a manner similar to that of E. coli FNR [7,8]. The first protein in this family (WhiB) was discovered in Streptomyces coelicolor and found to be essential for sporulation [9]. The M.tb genome contains seven orthologs named whiB1-7, of which only whiB2 is postulated to be an essential gene [10]. Apart from the conserved motifs described above, the sequences of the seven genes are not very similar, indicating different functional properties. Further, the mycobacteriophage TM4 carries a whiB homolog similar to whiB2 binding to the whiB2 promoter region and inhibiting its synthesis [11]. In a previous study, Geiman et al. found that transcription of M.tb whiB2 was strongly down-regulated in late stationary phase compared to early log phase, while, in contrast, transcription of whiB3 was more than 20-fold induced at late stationary phase, and more than 10-fold induced at low pH [12]. whiB6 seemed to be more of a general stress responder, being induced by various in vitro stresses such as sodium dodecyl sulfonate, ethanol, cumene hydroperoxide, diamide and incubation at 42uC. whiB7 expression has been reported to be induced by antibiotics inhibiting protein synthesis such as streptomycin and kanamycin, heat shock, iron starvation and palmitic acid [12,13,14]. Interestingly, both Mycobacterial and Streptomyces whiB7 null mutants are hypersensitive to antibiotics, suggesting a role for WhiB7 in antibiotic tolerance [13]. Our objective was to mimic various physiological conditions that M.tb are likely to encounter in vivo, with emphasis on prolonged exposure (hours) rather than shock response (minutes), and to analyze the whiB response to these conditions. We studied redox environments, such as the slow, self-generated oxygen depletion of the Wayne model, mimicking the progressive hypoxia during granuloma formation, and incubation with DETA-NO, modeling the phagosomal NO burst. We also assessed whiB1-7 expression in macrophage-like cells in vitro and in caseous, hypoxic granulomas in vivo [15,16]. Finally, we evaluated the importance of cyclic AMP, a signaling molecule important in both prokaryotic and eukaryotic gene regulation, on whiB expression [17]. Our data indicate that the M.tb whiB genes respond to several different stimuli in a non-coordinate manner, suggesting unique functions of the seven genes and their cognate protein products. The WhiB proteins are believed to be redox sensing transcription factors. To determine the conditions in which WhiB1-7 are utilized, M.tb were exposed to various conditions likely to occur in physiological environments with emphasize on redox change. The whiB transcript abundance was fairly consistent and similar to the expression level of housekeeping gene sigA throughout the whiB family with the exception of whiB1 being about 100-fold higher and whiB5 being about 50-fold lower than sigA transcripts (and the rest of the whiB gene family) when grown in 7H9 broth under aerobic conditions (Figure 1). We found sigA to be most suitable as a housekeeping gene in our experiment since its expression was close to the levels of the (...truncated)