Our data suggests that MTB senses the host inflammatory status at least in part by measuring and responding to the levels of CO in the macrophage. We propose that as MTB establishes its latent infection, it initiates a host immune response that includes both HO-1 and NOS2 production as well as granuloma formation, thus resulting in exposure of MTB to CO, NO and diminished O2
. These three gases may then act sequentially, additively or synergistically to activate the dormancy regulon via the sensors DosS and DosT (Fig. S7
). In this study, we show that mouse macrophages robustly produce HO-1 during MTB infection both ex vivo
and in vivo
. Whether the stimulus for inducing HO-1 by MTB is directly due to a mycobacterial product, or secondarily due to production of proinflammatory cytokines is unknown. However, since macrophages produce TNF within hours of MTB infection, it seems likely that HO-1 is induced by TNF. Some studies have shown that NOS2 activity is required for HO-1 induction (Alcaraz et al., 2001
; Vicente et al., 2001
), but these studies were done using cell lines rather than primary cells. We confirmed that in RAW cells (a mouse macrophage cell line), NO production is necessary for HO-1 induction by LPS (data not shown). In contrast, we found that in primary bone marrow derived macrophages, MTB infection induced HO-1 independent of NOS2 activity ().
Although macrophages are likely the major source of CO production during infection, HO-1 induction is not limited to this cell type. Other cells that are associated with MTB infection that have been shown in other systems to produce HO-1 include pulmonary epithelial cells (Zhou et al., 2004
), dendritic cells (Chauveau et al., 2005
) and T-cells (Pae et al., 2003
). In fact, in the infected mouse lung HO-1 staining was also visualized in pulmonary epithelial cells (). Thus, CO could be produced during infection by infected macrophages and dendritic cells as well as bystander cells within the granuloma (such as T-cells), thus resulting in higher local concentrations. Likewise, while HO is thought to be the major source of CO in vivo
(Wu and Wang, 2005
), a recent study demonstrated that lipid peroxidation induces a cytochrome P450 dependent activity that produces CO (Archakov et al., 2002
). Unfortunately, no studies have been performed on the CO concentration in exhaled air from individuals with tuberculosis, and the actual concentration of carbon monoxide within macrophages and granuloma is unknown. However, in olfactory neurons, the effective concentration within the cell has been determined to be 10-30 μM (Ingi et al., 1996
), which would correspond to a headspace CO concentration of ~20,000 ppm (16 μM). CO concentrations of 10-30 μM may overestimate the true in vivo
concentration, but even if the concentration is ten times lower inside macrophages or granuloma (i.e. 1-3 μM), that would still be in the range of ~2000 ppm (1.6 μM).
Systems for sensing carbon monoxide have been described in both prokaryotes and eukaryotes (Ascenzi et al., 2004
; Roberts et al., 2004b
; Ryter et al., 2004
). For example, in eukaryotes the transcription factor NPAS2, implicated in regulating circadian rhythm, was shown to bind CO resulting in decreased DNA binding activity (Dioum et al., 2002
). Likewise, the purple photosynthetic bacterium Rhodospirillum rubrum
expresses a CO-binding transcription factor, CooA, that stimulates production of a CO oxidation system (Aono et al., 2000
; Roberts et al., 2001
; Youn et al., 2004
). How is CO sensed? A common feature shared by CO sensors is the presence of a protein-associated heme moiety, which is not surprising given the propensity of CO to bind heme (Roberts et al., 2004b
). However, there is great diversity in how the heme is protein-bound and under what conditions CO can bind heme.
Rather than use a single CO-binding transcription factor, our data shows that MTB sense CO via a two component system comprised of two sensors, DosS and DosT, and a single response regulator, DosR. In this study, we demonstrate genetically that dosR
is required for CO signaling, and that either dosS
can transmit the dormancy signal mediated by CO or NO at high concentrations. Kumar et al. also reported that MTB can induce hspX
by CO, but their single in vitro
experiment was done using a high concentration of a chemical CO donor, CORM-3. Further, the precise amount of CO delivered by the CO donor was unclear, in contrast to the use of pure CO gas in our study. Moreover, they did not assess whether the CO sensing was dependent on dosS
(Kumar et al., 2007
). In contrast, we show that CO sensing occurs in vitro
at physiologically relevant CO concentrations (as low as 20 ppm/16 nM of CO) and that both DosS and DosT can transmit the CO signal at high concentrations of CO. By carefully assessing the CO response of individual mutants in dosS
, we also show that the sensors can differentially regulate the dormancy regulon at physiologically relevant concentrations. DosS has a 25 fold lower Kd for CO than DosT (0.036 μM versus 0.9 μM) while its Kd for NO is 4 fold higher (0.020 μM versus 0.005 μM) (Sousa et al., 2007
). Interestingly, despite its higher Kd for CO, DosT undergoes more rapid autophosphorylation in the presence of CO than DosS (Sousa et al., 2007
). However, neither the affinities of DosS or DosT for DosR nor the kinetics of phosphorylation and activation of DosR by active DosS or DosT are known. Thus, in wild type cells where both DosS and DosT are present, we propose that at low CO concentrations, DosT could bind DosR and maintain it in an inactive state. This initial interaction of phosphorylated DosT with DosR might attenuate the dormancy response by preventing DosS from binding to and activating DosR. Thus DosT could behave as a “low affinity” receptor for CO and an inhibitor of the CO-induced response at physiologic CO concentrations. What happens in the absence of DosT? Since the activity of DosS is maximal with CO as its ligand (Sousa et al., 2007
), and since DosS is autoinduced as part of the dormancy regulon, a feedback loop could be rapidly established where unopposed binding of CO to DosS results in increased expression of both DosS and DosR.
We also observed that the response to CO is both dose and time dependent. Interestingly, the CO response differs from the response to NO in that the effect of CO is long-lasting (i.e. persists for >24h), while the NO effect is short-lived (Voskuil et al., 2003
). Despite the fact that NO binding to the sensors DosS and DosT is more avid than CO binding (Sousa et al., 2007
), the activation of the Dos regulon by NO is more transient, likely owing to its greater chemical reactivity (i.e. NO that dissociates from the heme of the Dos sensor can react with other molecules) or its chemical conversion to nitrate over time. Conversely, the persistent effect of CO suggests that MTB lacks the ability to eradicate or remove CO once it is bound to the heme group.
While our in vitro
experiments demonstrate the feasibility of CO sensing by MTB, our data from macrophage infections demonstrates that CO sensing actually occurs inside macrophages. Using naïve mouse macrophages or a mouse macrophage cell line, we show that genetic ablation or chemical inhibition of macrophage heme oxygenase reduces CO-dependent gene induction during infection. Interestingly, despite the presence of low amounts of NO, DosS and DosT signaling was lost. Conversely, in NOS2-deficient macrophages, heme oxygenase activity alone was insufficient to trigger the dormancy response. It is unclear why this would occur, but one possibility is that lack of NOS2 activity reduces HO-1 activity, either through a decrease in available heme or by increasing the association of HO-1 with its negative regulator, caveolin-1 (Kim et al., 2004
). An alternative possibility is that in vivo
, the short-acting gas NO “triggers” the activation of the dosS/T/R
system while the longer-acting gas CO “maintains” the activation state. In sum, it appears that both NO and CO have redundant roles in stimulating the MTB dormancy response during infection of naïve macrophages.
Thus, a plausible biochemical and physiologic mechanism is that during infection, exposure of DosS and/or DosT to CO, NO and hypoxia either sequentially or in combination stimulates histidine kinase activity and activation of DosR (Figure S7
). Active DosR then converts the signal(s) into a transcriptional response. Notably, other pulmonary pathogens including Pseudomonas aeruginosa, Bacillus anthracis, Bordetella
spp. and Nocardia
spp. have histidine kinases with GAF domains. This suggests that the ability to integrate several gaseous signals, as illustrated by the combined activities of DosS and DosT, may be a prevalent mechanism by which pathogens adapt and respond to changes in the host immune status.