Mycobacterium tuberculosis heme-degrading protein MhuD degrades heme to mycobilin isomers and iron, while its closest homologues from Staphylococcus aureus, IsdG and IsdI, degrade heme to staphylobilin isomers, formaldehyde, and iron. Superposition of the structures of the heme-bound complexes reveals that the heme molecule in the MhuD active site is rotated ∼90° about the tetrapyrrole plane with respect to IsdG and IsdI active site heme molecules. Therefore, the variation in IsdG/IsdI and MhuD chromophore products may be attributed to the different heme orientations. In MhuD, two arginines, Arg22 and Arg26, stabilize the heme propionates and may account for the heme orientation. Herein, we demonstrate that the MhuD-R26S variant alters the resulting chromophore product from mycobilin to biliverdin IXα (α-BV), whereas the R22S variant does not. Surprisingly, unlike canonical heme oxygenase (HO) that also degrades heme to α-BV, the MhuD-R26S variant produces the C1 product formaldehyde rather than carbon monoxide as observed for HO. The MhuD-R26S variant is an important tool for further probing the mechanism of action of MhuD and for studying the fate of the MhuD product in mycobacterium.

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.

MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 μM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, in both sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes, including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein-product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD's high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.

Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1β (Interleukin)-1β was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

Heme oxygenase-1 (HO-1) is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function.

Importance

There is no reliable vaccine against tuberculosis (TB), and conventional antibiotic therapy is administered over at least 6 months. This prolonged treatment period can lead to noncompliance resulting in relapsed infection as well as the emergence of multidrug resistance. Thus, there is an urgent need for improved therapeutic regimens that can more rapidly and efficiently control M. tuberculosis in infected patients. Here, we describe a potential strategy for treating TB based on pharmacological inhibition of the host heme-degrading enzyme HO-1. This approach results in significantly reduced bacterial burdens in mice, and when administered in conjunction with conventional antibiotic therapy, leads to faster, more effective pathogen clearance without detectable direct effects on the mycobacteria themselves. Interestingly, the effects of HO-1 inhibition on M. tuberculosis infection in vivo are dependent on the presence of an intact host immune system. These observations establish mammalian HO-1 as a potential target for host-directed therapy of TB.