Virulence factor SenX3 is the oxygen-controlled replication switch of Mycobacterium tuberculosis

Abstract

Aim: Morphogenetic switching between the replicating and nonreplicating states of Mycobacterium tuberculosis is regulated by oxygen, nitric oxide, and carbon monoxide levels. The mechanisms by which M. tuberculosis senses these diatomic gases remain poorly understood. In this study, we have examined whether virulence factor SenX3 plays any role in oxygen sensing.

Results: In this study, we demonstrate that the virulence factor SenX3 is a heme protein that acts as a three-way sensor with three levels of activity. The oxidation of SenX3 heme by oxygen leads to the activation of its kinase activity, whereas the deoxy-ferrous state confers a moderate kinase activity. The binding of nitric oxide and carbon monoxide inhibits kinase activity. Consistent with these biochemical properties, the SenX3 mutant of M. tuberculosis is capable of attaining a nonreplicating persistent state in response to hypoxic stress, but its regrowth on the restoration of ambient oxygen levels is significantly attenuated compared with the wild-type and the complemented mutant strains. Furthermore, the presence of signaling concentrations of nitric oxide and carbon monoxide was able to inhibit the regrowth of M. tuberculosis in response to ambient oxygen levels.

Innovation and conclusions: Evidence presented in this study delineates a plausible mechanism explaining the oxygen-induced reactivation of tuberculosis diseases in humans after many years of latent infection. Furthermore, this study implicates nitric oxide and carbon monoxide in the inhibition of mycobacterial growth from the nonreplicating state.

FIG. 1.

SenX3 binds heme and is responsive to O₂, NO, and CO. (A) SenX3 reacts reversibly with O₂. UV-visible absorption spectra of native SenX3 (3 μM in 50 mM sodium phosphate, 200 mM NaCl, and pH 8.0) indicate that a hexa-coordinated heme bound to the protein. Treatment of native SenX3 with a 100-fold molar excess of DTH followed by re-exposure to air leads to reversible spectral changes, as observed with classical hypoxia/O₂ sensors. (B, C) NO and CO are ligands of SenX3. DTH-treated SenX3 was exposed to a 50-fold molar excess of NO donor ProliNONOate in an anaerobic glove box, and absorption spectra were recorded (B). DTH-treated SenX3 was exposed to a 100-fold molar excess of CO donor CORM-2, and the absorption spectra were recorded (C). Insets display enlarged images of the 500–600 nm regions. Numbers in parentheses indicate the absorption maxima in nanometers. CO, carbon monoxide; CORM-2, carbon monoxide-releasing molecule-2; DTH, sodium dithionite; NO, nitric oxide; O₂, oxygen. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

SenX3 is oxidized by O₂. (A) SenX3 protein was exposed to air for 2 min after DTH treatment. The air-exposed SenX3 was then treated with a 100-fold molar excess of KCN, which produced a peak at 540 nm, indicating the presence of a met-CN⁻ complex. Insets display enlarged images of the 500–600 nm regions. (B) DTH-treated SenX3 does not react with KCN. Exposure of DTH-treated SenX3 to a 100-fold molar excess of KCN does not result in any spectral changes, but it only reacts with KCN after treatment with a 500-fold molar excess of the chemical oxidant Fe(CN)₆³⁻ (C). (D) Air-exposed SenX3 does not react with CO. Air-exposed SenX3 was treated with a 100-fold molar excess of CO donor CORM-2. It is very well known that CO does not react with met-heme proteins. In agreement with this, we did not observe any spectral changes in the air-exposed SenX3 on CO treatment. Numbers in parentheses denote absorption maximum in nanometers. KCN, potassium cyanide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Oxygen-catalyzed oxidation of SenX3 heme activates its autokinase activity. (A) Purified native SenX3 was exposed to DTH (Fe²⁺ form) and analyzed for autokinase activity. The treatment of 5 μM native protein with the single electron donor DTH (100-fold molar excess) generates deoxy-ferrous heme ligated to SenX3. This change in the redox state of heme resulted in a significant inhibition of kinase activity. (B) The autokinase activity of DTH-reduced SenX3 (5 μM in 50 mM sodium phosphate, 200 mM NaCl, and pH 8.0) was compared with that of the protein exposed to air for 2 min. The activity of air-exposed SenX3 was found to be significantly enhanced compared with ferrous SenX3. (C) A 3 μM solution of DTH-reduced SenX3 in 50 mM sodium phosphate, 200 mM NaCl, pH 8.0 was exposed to a 100-fold molar excess of Fe(CN)₆³⁻ to generate met-SenX3 in the absence of O₂. The chemical oxidation by Fe(CN)₆³⁻ in the absence of O₂ enhanced the autokinase activity of SenX3, similar to the air-oxidized protein. Numbers on each lane indicate the incubation time at 37°C in minutes. (D–E) A 6 μM solution of SenX3 in 50 mM sodium phosphate, 200 mM NaCl, pH 8.0 was exposed to a 100-fold molar excess of DTH to create the deoxy-ferrous form (Fe²⁺), followed by treatment with a 50-fold molar excess of NO donor ProliNONOate (D) and a 100-fold molar access of CO donor CORM-2 (E). Both NO and CO inhibited the autophosphorylation of SenX3. The numbers on each lane indicate the incubation time at 37°C in minutes.

FIG. 4.

The carbonyl-heme and nitrosyl-heme complexes of SenX3 are stable in the presence of O₂, locking SenX3 in the inactive conformation. A 3 μM solution of SenX3 in 50 mM sodium phosphate, 200 mM NaCl, pH 8.0 was treated with a 100-fold molar excess of DTH followed by treatment with a 50-fold molar excess of NO donor ProliNONOate (A) and a 100-fold molar access of CO donor CORM-2 (B), and the absorption spectra were recorded. CO and NO form stable nitrosyl and carbonyl-heme complexes, respectively, thus locking the protein in an inactive conformation. Exposure of DTH-treated SenX3 to NO (using ProliNONOate) (C) and CO (using CORM-2) (D) locks SenX3 in the inhibited state, which remains unchanged on exposure to air. (E, F) Inhibition of SenX3 kinase activity by NO (donated by ProliNONOate) and CO (released by CORM-2) depends on reductant. A 3 μM solution of SenX3 protein in 50 mM sodium phosphate, 200 mM NaCl, pH 8.0 was treated with a 100-fold molar excess of DTH followed by exposure to air for 2 min. The air-exposed protein was then treated with a 100-fold molar excess of NO (donated by ProliNONOate) (E) or CO (released by CORM-2) (F), and the kinase activities were measured. NO and CO did not inhibit the kinase activity of air-exposed SenX3. Numbers on each lane indicate the time of incubation at 37°C in minutes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

SenX3 is required for the regrowth of Mtb in response to the restoration of ambient oxygen levels. (A) Wild-type, senX3 mutant, and senX3 complementing strains were found to have similar growth profiles in the Wayne model. (B) No significant differences in colony-forming units between wild-type, mutant, and complementing strains were observed during hypoxic adaptation after days 10, 20, 30, and 40. After 10 days (C), 20 days (D), 30 days (E), and 40 days (F) of hypoxic adaptation, the wild-type, SenX3 mutant, and SenX3 complementing strains were exposed to O₂ and allowed to resume growth. (G) The reactivation of the wild-type, ΔsenX3 mutant, and complemented strains was assessed based on the time required for the development of a detectable growth signal by the BACTEC MGIT 960 system. A consistent and statistically significant delay in regrowth after the restoration of O₂ was observed in all cultures of the ΔsenX3 mutant (at 20, 30, 40, and 130 days) compared with the wild type. However, episomal expression of SenX3 in the mutant strain led to a decrease in that attenuation. WT, SKO, and SCO indicate the wild-type, senX3 knockout Mtb, and senX3 complementing Mtb, respectively. (H, I) The results of the kinase assay were further validated in the Wayne model by exposing the hypoxic culture at day 40 to 200 μM of NO (generated by DETA NONOate) (H) and 50 μM of CO (released by CORM-2) (I) in the presence of ambient oxygen. Exposure of the wild-type and complemented strains to NO and CO leads to an arrest in regrowth in response to ambient oxygen similar to that observed in the SenX3 mutant strain. *p<0.05. Statistical significance was determined using Student's t-test. These figures (A–I) are representative of at least three independent experiments performed in triplicate. Mtb, Mycobacterium tuberculosis; SCO, Senx3 complementing strain of Mtb; SKO, SenX3 knockout strain of Mtb. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Model depicting the role of SenX3 in the reactivation of Mtb in response to O₂. The current paradigm of oxygen sensing and mycobacterial response suggests that hypoxia is sensed by the heme-based sensors DosS and DosT. These sensors relay the signal to DosR, which induces the DosR regulon on phosphorylation. The DosR regulon plays a critical role in the hypoxia-induced transition into nonreplicating persistence. This model further suggests that apart from inducing DosS and DosT, hypoxia also inhibits senX3 (as demonstrated in this study). NO and CO also induce DosS and DosT while inhibiting SenX3 kinase activity. While ambient levels of oxygen induce SenX3 kinase activity, they simultaneously inhibit the kinase activity of DosS and DosT. SenX3 relays this signal to RegX3, which regulates the replication and respiration of Mtb. This model further suggests that the presence of NO and CO could inhibit the kinase activity of SenX3, thus inhibiting replication and activating the DosR regulon through the activation of DosS and DosT. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars