Mycobacterium tuberculosis senses host-derived carbon monoxide during macrophage infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.     

DosS responds to a reduced electron transport system to induce the Mycobacterium tuberculosis DosR regulon

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.     

Blockage of the channel to heme by the E87 side chain in the GAF domain of Mycobacterium tuberculosis DosS confers the unique sensitivity of DosS to oxygen

Two sensor kinases, DosS and DosT, are responsible for recognition of hypoxia in Mycobacterium tuberculosis. Both proteins are structurally similar to each other, but DosS is a redox sensor while DosT binds oxygen. The primary difference between the two proteins is the channel to the heme present in their GAF domains. DosS has a channel that is blocked by E87 while DosT has an open channel. Absorption spectra of DosS mutants with an open channel show that they bind oxygen as DosT does when they are exposed to air, while DosT G85E mutant is oxidized similarly to DosS without formation of an oxy-ferrous form. This suggests that oxygen accessibility to heme is the primary factor governing the oxygen-binding properties of these proteins.     

Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor

A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.     

Protein-protein interactions between histidine kinases and response regulators of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a two-component system with the DosR RR, have different cross-interaction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the α2-helices of their dimerization domains.     

Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.Oxygen sensing is important for facultative anaerobes to adapt to changes in metabolic necessities during the transition between aerobic and anaerobic conditions. Although Mycobacterium tuberculosis (MTB) is an obligate aerobe, a gradual depletion of O₂ from its culture is known to lead to a drastic change in gene expression (8, 21, 24, 28, 34, 37, 39). Approximately 48 genes of M. tuberculosis were reported to be induced under early hypoxic conditions, which is mediated by the DosSR (DevSR) two-component system (16, 24, 34). The induction of the DosR regulon is important for survival of M. tuberculosis under hypoxic conditions and for it to enter the nonreplicating dormant state (2, 19). The DosSR two-component system consists of the DosS histidine kinase (HK) and its cognate DosR response regulator (RR) (24, 26, 29). The DosT HK, which shares high sequence similarity to DosS over the length of their primary structures, was also found to cross talk with DosR (26, 30). The N-terminal domains of DosS and DosT contain two tandem GAF domains (GAF-A and GAF-B from their N termini), and the three-dimensional structure of the GAF-A and GAF-B domains was determined (5, 25). A b-type heme is embedded in the GAF-A domain, composed of one five-stranded antiparallel β-sheet and four α-helices (5, 14, 25, 32). The heme is positioned nearly perpendicular to the β-sheet, and H149 and H147 of the polypeptides serve as the proximal axial ligands for DosS and DosT, respectively (5, 25). The ligand-binding state at the distal axial position of heme and the redox state of the heme iron modulate the autokinase activity of DosS and DosT. The O₂-bound (oxyferrous) and ferric forms of the HKs are inactive, whereas the unliganded ferrous (deoxyferrous) form as well as NO- and CO-bound forms are active (17, 36). The heme iron of DosT is stable against autooxidation of Fe²⁺ to Fe³⁺ in the presence of O₂, indicating that its conversion between deoxyferrous and oxyferrous forms is the mechanism by which DosT recognizes O₂ (17). However, the autooxidation property of oxyferrous DosS remains controversial. Kumar et al. (17) and Cho et al. (5) reported that DosS undergoes autooxidation on exposure to O₂, while other research groups demonstrated that the oxyferrous form of DosS is stable against autooxidation (13, 14, 36). Recently, different roles of DosS and DosT in O₂ sensing by M. tuberculosis were suggested. DosT plays a more important role in the early phase of hypoxic conditions than DosS when the growth of M. tuberculosis is transferred from aerobic to hypoxic conditions (11).Mycobacterium smegmatis possesses a single DevS HK that phosphorylates the DevR RR (20). The DevSR two-component system is also implemented in hypoxic adaptation of this bacterium (20). Like DosT of M. tuberculosis, the autokinase activity of M. smegmatis DevS was shown to be controlled by the ligand-binding state of its heme (18). Regarding the autooxidation property, DevS of M. smegmatis was suggested to be similar to DosT rather than DosS; i.e., the heme iron in DevS is resistant to autooxidation from an oxyferrous to a ferric state in the presence of O₂ (18).In this paper we report several lines of evidence for the functional difference between DosS and DosT in the hypoxic adaptation of mycobacteria and discuss the implications of these findings.     

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.     

Nitric oxide dioxygenation reaction in DevS and the initial response to nitric oxide in Mycobacterium tuberculosis

DevS and DosT from Mycobacterium tuberculosis (MTB) are paralogous heme-based sensor kinases that respond to hypoxia and to low concentrations of nitric oxide (NO). Both proteins work with the response regulator DevR as a two-component regulatory system to induce the dormancy regulon in MTB. While DevS and DosT are inactive when dioxygen is bound to the heme Fe(II) at their sensor domain, autokinase activity is observed in their heme Fe(II)-NO counterparts. To date, the conversion between active and inactive states and the reactivity of the heme-oxy complex toward NO have not been investigated. Here, we use stopped-flow UV-vis spectroscopy and rapid freeze quench resonance Raman spectroscopy to probe these reactions in DevS. Our data reveal that the heme-O(2) complex of DevS reacts efficiently with NO to produce nitrate and the oxidized Fe(III) heme through an NO dioxygenation reaction that parallels the catalytic reactions of bacterial flavohemoglobin and truncated hemoglobins. Autophosphorylation activity assays show that the Fe(III) heme state of DevS remains inactive but exhibits a high affinity for NO and forms an Fe(III)-NO complex that is readily reduced by ascorbate, a mild reducing agent. On the basis of these results, we conclude that upon exposure to low NO concentrations, the inactive oxy-heme complex of DevS is rapidly converted to the Fe(II)-NO complex in the reducing environment of living cells and triggers the initiation of dormancy.     

The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis

Heme oxygenase is the rate limiting enzyme in heme degradation to carbon monoxide (CO), iron and bilirubin. The inducible isoform of the protein, heme oxygenase-1 (HO-1), is susceptible to up-regulation by a diverse variety of conditions and agents in mammalian tissue, leading to the common conception that HO-1 is a stress related enzyme. However, as attempts are made to unravel the mechanisms by which HO-1 is induced and as we discover that CO, iron and bilirubin may be important effector molecules, we are learning to appreciate that heme oxygenases may be central to the regulation of many physiological and pathophysiological processes besides their established function in heme catabolism. One such process may be closely linked to nitric oxide (NO). It has been demonstrated that NO and NO donors are capable of inducing HO-1 protein expression, in a mechanism depending on the de novo synthesis of RNA and protein. Thus, it is postulated that NO may serve as a signaling molecule in the modulation of the tissue stress response. This review will highlight the current ideas on the role of CO-heme oxygenase and NO-nitric oxide synthase in cell signaling and discuss how the two systems are interrelated.     

A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins

The heme-PAS is a specialized domain with which a broad class of signal-transducing heme proteins detect physiological heme ligands. Such domains exhibit a wide range of ligand binding parameters, yet they are all expected to feature an alpha-beta heme binding fold and a predominantly hydrophobic heme distal pocket without a distal histidine. We have compared, for the first time, the resonance Raman spectra of several heme-PASs: the heme-binding domains of Bradyrhizobium japonicum FixL, Escherichia coli Dos, Acetobacter xylinum PDEA1, and Methanobacterium thermoautotrophicum Dos. In all cases, the nu(Fe)-(CO) and nu(C-O) values of the carbonmonoxy forms were consistent with coordination of the heme iron to histidine on the proximal side and binding of the CO without electrostatic interaction with the heme distal pocket. EcDos was unusual in having predominantly hexacoordinate heme iron in the deoxy and met forms. Despite an evident lack of CO interaction with the EcDos heme pocket, relatively low Fe-O(2) (562 cm(-1)) and N-O (1576 cm(-1)) stretching frequencies indicated that strong polar interactions with that heme distal pocket are possible for highly bent ligands such as O(2) or NO. None of the newly studied NO adducts exhibited evidence of the Fe-His rupture and pentacoordination previously noted for Sinorhizobium meliloti FixL. A low Fe-His stretching frequency, formerly interpreted as a strained Fe-His bond, and the slow association of O(2) with S. meliloti FixL failed to correlate with the newly studied proteins having low association rate or low equilibrium association constants for binding of O(2). We conclude that although heme-PASs share some features, they represent distinct signal transduction mechanisms.     

Cognate sensor kinase‐independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti‐mycobacterial drug design

The DevRS/DosT two‐component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under ‘non‐inducing’ aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase‐phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate‐grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild‐type DevS and phosphorylation‐defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous‐like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate‐dependent phosphorylation can occur in the absence of DevR’s cognate kinases. Based on our findings, we conclude that anti‐mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.     

Dynamic changes in expression of heme oxygenases in mouse heart and liver during hypoxia

Heme oxygenase cleaves heme to form biliverdin, carbon monoxide (CO), and iron, and consists of two structurally related isozymes, HO-1 and HO-2. HO-2 is also known as a potential oxygen sensor. Here we show that the relative CO content in arterial blood, which reflects the total amount of endogenous heme degradation, dynamically changes in mice during acclimatization to normobaric hypoxia (10% O2), with the two peaks at 1 day and 21 days of hypoxia. The expression levels of HO-1 and HO-2 proteins were decreased by 20% and 40%, respectively, in the mouse liver at 7 days of hypoxia, which returned to the basal levels at 14 days. On the other hand, HO-1 and HO-2 proteins were increased 2-fold and 1.3-fold, respectively, in the heart at 28 days of hypoxia. Thus, hypoxia induces or represses the expression of HO-1 and HO-2 in vivo, depending on cellular microenvironments.     

Carbon monoxide actuates O(2)-limited heme degradation in the rat brain

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.     

Carbon monoxide-induced stomatal closure in Vicia faba is dependent on nitric oxide synthesis

Recently, in animals, carbon monoxide (CO), like nitric oxide (NO), was implicated as another important physiological messenger or bioactive molecule. Previous researches indicate that heme oxygenase (HO)-1 (EC 1.14.99.3) catalyzes the oxidative conversion of heme to CO and biliverdin IXa (BV) with the concomitant release of iron. However, little is known about the physiological roles of CO in plant, especially in stomatal movement of guard cells. In the present paper, the regulatory role of CO during stomatal movement in Vicia faba was surveyed. Results indicated that, like sodium nitroprusside (SNP), CO donor hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proved by the addition of gaseous CO aqueous solution with different concentrations, showing for the first time that CO and NO exhibit similar regulation role in the stomatal movement. Moreover, our data showed that 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)/N^G-nitro- l -arginine-methyl ester ( l -NAME) not only reversed stomatal closure by CO, but also suppressed the NO fluorescence induced by CO, implying that CO-induced stomatal closure probably involves NO/nitric oxide synthase (NOS) signal system. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX), NO scavenger cPTIO and NOS inhibitor l -NAME reversed the darkness-induced stomatal closure and NO fluorescence. These results show that, maybe like NO, the levels of CO in guard cells of V.   faba is higher in dark than that in light, HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness, respectively, and that CO being from HO-1 mediates darkness-induced NO synthesis in guard cells' stomatal closure of V.   faba .