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. 2007 Jul 10;104(28):11562-7.
doi: 10.1073/pnas.0700490104. Epub 2007 Jul 3.

Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival

Amit Singh  1 , Loni Guidry, K V Narasimhulu, Deborah Mai, John Trombley, Kevin E Redding, Gregory I Giles, Jack R Lancaster Jr, Adrie J C Steyn
Affiliations

Mycobacterium tuberculosis WhiB3 responds to O2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival

Amit Singh et al. Proc Natl Acad Sci U S A. .

Abstract

A fundamental challenge in the redox biology of Mycobacterium tuberculosis (Mtb) is to understand the mechanisms involved in sensing redox signals such as oxygen (O2), 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 O2 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 O2 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 DeltawhiB3 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 O2 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mtb WhiB3 is an Fe-S cluster protein. (A) WhiB3 Fe-S cluster reconstitution catalyzed by IscS over time. At various time intervals (10, 30, 60, 120, and 180 min), aliquots from the reconstitution reactions were removed and analyzed by native PAGE followed by autoradiography. (B and C) Reconstitution of the WhiB3 Fe-S cluster was carried out for 1 h in the presence of 50, 100, 250, and 500 μM Fe2+ (B) or 10, 25, 50, and 100 μM Fe2+ chelator (2,2-dipyridyl) (C). (D) The four conserved cysteine residues in WhiB3 are essential for Fe-S cluster reconstitution. Fe-S cluster reconstitution was carried out with WhiB3 and WhiB3ΔCys-1–4 as described earlier and analyzed by native PAGE.
Fig. 2.
Fig. 2.
Spectroscopic characterization of WhiB3. UV-visible spectroscopy of WhiB3 Fe-S cluster reconstitution is shown. (A) Reconstitution was carried out inside an anaerobic glove box as described in Experimental Procedures. Time points at which samples were scanned by a UV-visible spectrophotometer are indicated. Note the time-dependent increase in the characteristic 4Fe-4S absorption peak at 413 nm. After completion of the Fe-S cluster reassembly, the Fe-S cluster was reduced by adding 5 mM DTH. (Inset) Development of a greenish-brown color during the WhiB3 Fe-S cluster reconstitution before (left cuvette) and after (right cuvette) the reconstitution reaction. (B) The four conserved cysteine residues (C23-C53-C56-C62) in WhiB3 are required for Fe-S cluster assembly. Fe-S cluster reconstitution in WhiB3 and WhiB3ΔCys-1–4 was carried out as described in Experimental Procedures and analyzed by UV-visible spectroscopy. (C and D) EDFS EPR spectra of IscS- (C) and NifS- (D) reconstituted WhiB3 after reduction with DTH. EPR spectra were obtained at 12 K, the EPR experimental conditions; π/2 and π pulses are 16 and 32 ns, τ = 180 ns. The spectra were acquired with 60 shots with a two-step phase cycle at a repetition rate of 1 kHz. Microwave frequency = 9.8046 GHz.
Fig. 3.
Fig. 3.
The Mtb WhiB3 Fe-S cluster responds to O2 and NO. (A) Anaerobically reconstituted WhiB3 was purified by size-exclusion column and exposed to O2 by bubbling air through the sample for 2 min. UV-visible spectra were acquired before and after air exposure at various time intervals as indicated. (Inset) Rate of the 4Fe-4S cluster loss was determined by calculating the percent loss of absorbance at 413 nm upon exposure to air at various time intervals. (B) The O2-treated sample of reconstituted WhiB3 was withdrawn immediately (5 min) after exposure to O2 (red line) and 60 min after exposure (blue line) and analyzed by EPR. EDFS EPR spectra were measured at 12 K; the π/2 and π pulses used were 16 and 32 ns with, τ = 180 ns. The spectrum was acquired with 60 shots with a two-step phase cycling at repetition rate of 1 kHz. Microwave frequency = 9.806 GHz. The appearance of a sharp signal at g = 2.01 indicates a [3Fe-4S]+ cluster. (C) Loss of the 4Fe-4S cluster in WhiB3 was also confirmed by exposing 35S-labeled WhiB3 to air as described before. At various time intervals (0, 30, 60, 90, and 120 min), samples were removed and analyzed by native PAGE followed by autoradiography (Upper) and SDS/PAGE followed by Coomassie blue staining to rule out degradation of WhiB3 upon air exposure (Lower). (D) Effect of NO on WhiB3. Reconstituted WhiB3 was exposed to various concentrations of proline NONOate (WhiB3:NO) as indicated and analyzed by EPR. (Inset) Aerobically purified WhiB3 was deoxygenated and exposed to a 10-fold molar excess of proline NONOate. EPR data were acquired by using a continuous-wave EPR spectrometer at a microwave frequency of 9.669 GHz operating at 100-kHz field modulation and 150 K. A modulation amplitude of 0.5 mT and microwave power of 2 mW were used. The peak at 2.036 g is consistent with the formation of monomeric DNIC (14).
Fig. 4.
Fig. 4.
Growth phenotype of MtbΔwhiB3. Identical volumes (30 μl) and equal number of cells (3 × 105, 3 × 104) of WT Mtb and MtbΔwhiB3 were spotted on Dubos–agar medium either in the absence [basal (A)] or in the presence of 50 mM glucose (B), 50 mM succinate (C) or 25 mM acetate (D) as sole carbon substrate. Growth was analyzed every week after inoculation, and photographs were taken at 4–6 weeks after inoculation at ×7 magnification by using a Zeiss stereo microscope. (Left) WT Mtb. (Right) MtbΔwhiB3.
Fig. 5.
Fig. 5.
Hypothetical model depicting WhiB3 as an intracellular redox sensor. In an oxygen-rich environment, the WhiB3 [4Fe-4S]+ cluster is oxidized to [4Fe-4S]2+ and undergoes sequential conversion, yielding [3Fe-4S]+ with the concomitant production of H2O2 and [2Fe-2S]2+ intermediates and eventually complete loss of the cluster. Mtb IscS can activate apo-WhiB3 in vivo to generate WhiB3 [4Fe-4S]2+. The [4Fe-4S]2+ cluster can undergo reduction by thioredoxin and mycothiols to generate [4Fe-4S]+. In vivo growth of Mtb is accompanied by carbon source and O2 depletion thereby causing fluctuations in the intracellular redox state. WhiB3 senses these changes in the redox state through its [4Fe-4S] cluster to regulate catabolic [glycolysis, tricarboxylic acid (TCA) cycle, glyoxylate cycle] and anabolic (polyketides biosynthesis) metabolism for maintaining redox balance and persistence. The O2- and NO-sensing properties of WhiB3 and the growth defect exhibited by MtbΔwhiB3 on various carbon sources suggest a role as redox sensor responsible for integrating environmental signals to core intermediary metabolism.
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