Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

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.

were normalized to obtain accurate expression levels. For comparisons between wt Mtb and Mtb∆whiB3, the induction ratio for each gene was normalized to Mtb 16s rRNA expression.

Overexpression and purification of Mtb WhiB3
Mtb WhiB3 ORF (Rv3416) was PCR amplified using complementary oligonucleotides harboring BglII and XhoI restriction enzymes sites at their 5` and 3` ends. PCR fragments was treated with BglII and XhoI and ligated into a similarly modified E. coli expression vector, pETDuet (Novagen) to generate pETDuet-W3. The pETDuet-W3 was transformed into E. coli Rossetta (Novagen) cells. Transformants were grown at 37°C in LB broth containing ampicillin to an A 600 nm of 0.6 and expression was induced with 0.8 mM of IPTG for 5 h at 37°C. Mtb WhiB3 expressed under these growth conditions were found to be in inclusion bodies. WhiB3 was purified from inclusion bodies by washing twice with 50 ml of sodium phosphate buffer, pH 7.5, 300 mM NaCl and 0.1 x Triton followed by three washes with 50 ml of sodium phosphate buffer, pH 7.5 and 300 mM NaCl. Inclusion bodies were treated with 5M urea, 3M NaCl in 50 mM sodium phosphate buffer, pH 7.5 for ~2h at 37°C with slow stirring to completely remove genomic DNA associated with WhiB3. Inclusion bodies were pelleted by centrifugation at 18,000 rpm for 30 min, and further solubilized in buffer A (8 M urea, 50 mM sodium phosphate, pH 7.5, 300 mM NaCl and 5 mM DTT) for 16 h at 37°C. Denatured WhiB3 was renatured by step-wise dialysis against buffer B (4 M urea, 50 mM sodium phosphate, pH 7.5, 300 mM NaCl and 5 mM DTT) for 6 h, followed by buffer C (2 M urea, 50 mM sodium 1 phosphate, pH 7.5, 300 mM NaCl and 5 mM DTT) for 6 h, and finally in buffer D (50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 5 mM DTT and 10 % glycerol). Refolded WhiB3 was concentrated using iCON concentrators (PIERCE) and stored at −20°C.
WhiB3 was also purified from soluble fraction as a SUMO-fusion as described previously (1). WhiB3 purity (~90%) was judged by Coomassie Blue staining of an SDS-PAGE gel and by MALDI-TOF mass spectrometry. Our UV-visible spectroscopic results showed no difference in the redox sensing properties between purified WhiB3 fused with S-or SUMO-tag. Subsequently, because of its small size, the WhiB3 S-tagged protein was used in all studies.

Preparation of apo-WhiB3
The 4Fe-4S cluster in holo-WhiB3 was removed by incubating protein with EDTA and potassium ferricyanide in a molar ratio of protein:EDTA:ferricyanide in 1:50:20 at room temperature. Loss of 4Fe-4S cluster was monitored over time by UV-vis spectroscopy.
Finally, apo-WhiB3 was subjected to gel-exclusion chromatography to remove low molecular weight materials. Purified apo -WhiB3 was used for in vitro-thiol trapping and DNA binding experiments.

In vitro-thiol trapping and MALDI-TOF analysis
Apo-WhiB3 (10 µM) was first treated with 2 mM of DTT for 1 h at room temperature followed by labeling of free thiols by treating with 20 mM IAM for 1 h in dark at room temperature. The diamide oxidized apo-WhiB3 were similarly treated with IAM and taken as a control. All the samples were precipitated with 10 % trichloroacetic acid (TCA) for 30 min on ice, washed with acetone, air dried. Samples were analyzed in the positive mode on a Voyager Elite mass spectrometer with delayed extraction technology (PerSeptive Biosystems, Framingham MA). The acceleration voltage was set at 25kV and 10-50 laser shots were summed. Sinapinic acid (Aldrich, D13, 460-0) dissolved in acetonitrile: 0.1% TFA (1:1) was the matrix used. The mass spectrometer was calibrated with apomyoglobin. Samples were diluted 1:10 with matrix, and 1 ul was pipetted on to a smooth plate.

SI note 1.
Currently, there is no known method for effectively measuring intrabacterial redox homeostasis (via NAD/NADH and/or NADP/NADPH) in infected macrophages.
Nonetheless, Boshoff et al (3) developed an "in vivo" method that utilizes Mtb cells derived from macrophages, or mouse lungs, followed by a 24 h incubation step in which [C 14 ] nicotinamide is incorporated into NAD/NADH and/or NADP/NADPH. It is wellknown that the Mtb NAD salvage pathway is not efficient during in vitro growth (3). However, the method developed by Boshoff et al., clearly demonstrated that the NAD salvage pathway is switched on in vivo and remains functional in Mtb cells derived from macrophages even when cultured for 24 h in vitro. As a result, [C 14 ] nicotinamide incorporation into NAD, followed by endogenous reduction to generate NADH, is an effective indicator of the intrabacterial redox poise.