The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence. Currently, approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB). A central question in TB research is to identify the mechanisms that allow the organism to persist for long periods of time in humans. The mycobacterial cell wall has a high lipid content and contains several important lipid groups, including poly- and di-acyltrehaloses (PAT/DAT), sulfolipids (SL-1), and phthiocerol dimycocerosates (PDIM). These lipids are produced and actively secreted during infection to subvert the host immune system, eventually leading to Mtb persistence. We have discovered that the regulatory protein WhiB3 controls the differential production of PAT, DAT, SL-1, and PDIM and the storage lipid triacylglycerol (TAG) in response to fluctuations in the intracellular redox environment. We demonstrated that WhiB3 directly regulates lipid production by binding to the promoter regions of lipid biosynthetic genes in a redox-dependent manner. We also discovered that through this regulatory process, WhiB3 controls fatty acid metabolism and maintains intracellular redox homeostasis by channeling toxic reducing equivalents into lipid anabolism. Thus, our results suggest that Mtb lipid anabolism functions as a reductant sink to neutralize the reductive stress associated with the catabolism of host lipids during infection. These findings may serve as a model foundation for how pathogens adjust their metabolism to cope with stresses encountered during infection.

Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (E_MSH) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial E_MSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of E_MSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in E_MSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in E_MSH, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied E_MSH during infection and show that subpopulation with higher E_MSH are susceptible to clinically relevant antibiotics, whereas lower E_MSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence. Approximately 30% of the global population is infected with Mycobacterium tuberculosis (Mtb). Persistence of Mtb in host phagocytes depends on its ability to resist oxidant-mediated antibacterial responses. Mycothiol (MSH) is the main antioxidant that provides an abundant source of reducing equivalent, which protects Mtb from oxidative stress encountered during infection. The majority of research into redox signaling in Mtb has relied on chemical analysis of MSH in whole cell extract, which creates oxidation artifacts and prohibits dynamic imaging of MSH redox state during infection. We have successfully developed a novel and noninvasive tool based on genetically encoded redox sensitive fluorescent probes to perform real-time measurement of mycothiol redox potential (E_MSH) in Mtb during infection. For the first time we reveal the E_MSH of virulent and avirulent mycobacterial strains, including drug-resistant clinical isolates. We used this technology and came to the surprising conclusion that within a single infected macrophage there is heterogeneity in the redox signature of individual Mtb bacilli. Importantly, we show that anti-TB drugs accelerate oxidative stress in Mtb within infected macrophages and redox heterogeneity can contribute to emergence of drug tolerant population. These findings have implications for mycobacterial persistence following treatment with anti-TB drugs.

A fundamental challenge in the redox biology of Mycobacterium tuberculosis ( Mtb ) is to understand the mechanisms involved in sensing redox signals such as oxygen (O ₂ ), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that Mtb WhiB3 responds to the dormancy signals NO and O ₂ through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the Mtb cysteine desulfurase (IscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in Mtb . EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [4Fe-4S] ²⁺ to EPR visible [4Fe-4S] ⁺ . Atmospheric O ₂ gradually degrades the WhiB3 [4Fe-4S] ²⁺ cluster to generate a [3Fe-4S] ⁺ intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl–iron–dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly, Mtb Δ whiB3 shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and O ₂ through its [4Fe-4S] cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism.

The sudden increase in information derived from the completed Mycobacterium tuberculosis ( Mtb ) genome sequences has revealed the need for approaches capable of converting raw genome sequence data into functional information. To date, an experimental system for studying protein–protein association in mycobacteria is not available. We have developed a simple system, termed mycobacterial protein fragment complementation (M-PFC), that is based upon the functional reconstitution of two small murine dihydrofolate reductase domains independently fused to two interacting proteins. Using M-PFC, we have successfully demonstrated dimerization of yeast GCN4, interaction between Mtb KdpD and KdpE, and association between Esat-6 and Cfp-10. We established the association between the sensor kinase, DevS, and response regulator, DevR, thereby demonstrating the potential of M-PFC to study protein associations in the mycobacterial membrane. To validate our system, we screened an Mtb library for proteins that associate with the secreted antigen Cfp-10 and consistently identified Esat-6 in our screens. Additional proteins that specifically associate with Cfp-10 include Rv0686 and Rv2151c (FtsQ), a component and substrate, respectively, of the evolutionary conserved signal recognition pathway; and Rv3596c (ClpC1), an AAA-ATPase chaperone involved in protein translocation and quality control. Our results provide empirical evidence that directly links the Mtb specialized secretion pathway with the evolutionary conserved signal recognition and SecA/SecYEG pathways, suggesting they share secretory components. We anticipate that M-PFC will be a major contributor to the systematic assembly of mycobacterial protein interaction maps that will lead to the development of better strategies for the control of tuberculosis.

Summary: Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host–pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70‐fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E_MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E_MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E_MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.