Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon

Abstract

The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR. We have demonstrated previously that O(2), nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O(2) and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1 (HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1(+/+) and HO-1(-/-) bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally, we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O(2), NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously unrecognized host factor, is a physiologically relevant Mtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence.

FIGURE 1.

Up-regulation of the complete Mtb Dos regulon upon exposure to CO. Microarray expression analysis was performed using RNA isolated from wild-type Mtb and MtbΔdosR cells treated/untreated with 50 μm CO for 3 h. Arrows indicate the direction of transcription according to TubercuList. CHP, conserved hypothetical protein; HP, hypothetical protein.

FIGURE 2.

Regulation of the Mtb Dos regulon in response to CO. A, using Q-PCR, fdxA expression was examined after exposing Mtb cells to different concentrations of CO. B, Mtb DosS is the preferred sensor of CO. Wild-type Mtb, MtbΔdosS, MtbΔdosT, and MtbΔdosSΔdosT cells were independently exposed to CO followed by dosR, hspX, and fdxA expression analysis using Q-PCR. Results are expressed as mean ± S.D. (n = 3 in triplicate).

FIGURE 3.

Induction of the Mtb Dos regulon in macrophages is modulated by HO-1. A, HO-1 expression in HO-1^+/+ and HO-1^-/- BMM. BMM were collected from HO-1^-/- and HO-1^+/+ littermates and infected with Mtb. HO-1 protein levels were analyzed using Western blotting. Note that levels of HO-2, the constitutive isoform of HO, are unchanged. U, uninfected; I, infected. B, HO-1-generated CO induces the Mtb Dos regulon. HO-1^-/- and HO-1^+/+ BMM were independently infected with Mtb and RNA isolated from intracellular bacilli. Q-PCR was used to analyze the expression of dosR, hspX, and fdxA (an established “fingerprint” of the Dos regulon). Results are expressed as mean ± S.D. (n = 3 in triplicate).

FIGURE 4.

Induction of HO-1 in response to Mtb infection is independent of the NO signaling pathway. A, increase in HO-1 mRNA in response to Mtb infection. RAW cells and BMM from iNOS2^-/- mice were infected with Mtb, and HO-1 expression was analyzed using Q-PCR. Results are expressed as mean ± S.D. (n = 3 in triplicate). B, increase in HO-1 protein levels in response to Mtb infection. HO-1 protein is increased in RAW cells 24 h post infection and in the presence and absence of 1400W. Note that HO-1 can migrate as two bands (see “Results” for details).

FIGURE 5.

HO enzymatic activity is increased in response to Mtb infection. Biliverdin is a precise indicator of the amount of released CO (see “Results” for details). HO enzymatic activity was measured in RAW 264.7 (A), RAW 264.7 γ-NO(-) (B), and RAW 264.7 (C) cells with 1400W 24 h post-infection with Mtb. U, uninfected; I, infected. Results are expressed as mean ± S.E.^*, p <0.001 (n = 3–6/gp).

FIGURE 6.

HO-1 is significantly induced in the lungs of Mtb-infected mice. A, relative HO-1 mRNA abundance measured in the lungs of uninfected (U) or Mtb-infected (I) (4 weeks post infection) C3HeB/FeJ mice. Results are expressed as mean ± S.D. (n = 4 in triplicate). B, immunohistochemistry of Mtb-infected lungs (4 weeks post infection). Expression of HO-1 protein within TB lung lesions was demonstrated using HO-1-specific antibodies. Panel I, staining with HO-1-specific antibodies; panel II, hematoxylin and eosin staining. TB lung lesions of C3HeB/FeJ mice (panels I and II) contained numerous HO-1-positive cells (brown color in panel I and inset) within cell wall surrounding areas of necrosis.

FIGURE 7.

Hypothetical model depicting a role for CO in Mtb pathogenesis. Because numerous studies reported striking differences in local O₂, NO, and CO concentrations in organs, tissues, and single cells (see supplemental note 3), the model predicts that gradients of O₂, NO, or CO are sensed in conjunction by DosS and DosT, rather than independently. DosS is the preferred sensor of CO (thick blue arrow), whereas DosT is less capable (thin blue arrow) of inducing the Dos regulon. This contrasts with O₂, which inhibits expression of the Dos regulon (blocked arrow), albeit not during hypoxia. The model predicts that a combination of microenvironmental O₂, NO, or CO (overlapping circles) is crucial in modulating the state of the disease. Although modeling the above events in the context of chronic TB is technically difficult, the well established role of HO-1 in modulating apoptosis/necrosis, its anti-inflammatory properties (specifically the effect on tumor necrosis factor α), and the release of Fe(II) have significant implications for understanding the mechanism of Mtb persistence.