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 have 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.

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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, both in 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 heme substrate, no structures have been reported of IsdG-type proteins in complex with product, unlike HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXa (aBV) rather than the wild-type (WT) mycobilin isomers as product. 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 aBV as a proxy to study the IsdG-type protein/product complex. First we show that aBV has nanomolar affinity for MhuD and the R26S variant. Second we determined the MhuD-R26S-aBV complex structure to 2.5 Å, which reveals two notable features (1) two aBV molecules bound per active site and (2) a new a-helix (a3) as compared with the MhuD-heme structure. Finally, by molecular dynamics simulations we show that a3 is stable with the proximal aBV alone. MhuD's high affinity for its product and structural and electrostatic changes that accompany substrate turnover suggest that there is an unidentified protein that is responsible for product extraction from MhuD and other IsdG-type proteins.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), results in 10 million infections and 1.5 million deaths annually. Current TB treatments are typically a cocktail of up to five antibiotics that needs to be administered for a 9‐month duration; unfortunately, several of these drugs illicit severe side effects. These long, harsh treatments lead to patient non‐compliance resulting in a rise in multiple‐drug resistant Mtb strains ‐ MDR‐TB. Due to these factors new treatment strategies are needed. As host iron acquisition is essential for Mtb’s survival, elucidating key players in the Mtb iron uptake pathways may yield good drug targets. Mtb predominately acquires host iron by the siderophore‐mediated uptake pathway, in which small molecules with high affinity for iron are secreted to scavenge for host iron. We seek to shed light on the mechanism by which ferric‐siderophores are transported into the Mtb cytosol. At present there is little known about the proteins required to shuttle ferric‐siderophores across the outer membrane, cell‐wall environment, and periplasmic space to the inner membrane. There are two putative Mtb periplasmic binding proteins (PBPs), FecB and FecB2, and we hypothesize that one or both of these PBPs shuttle ferric‐siderophores across the periplasmic space. To test this, we examined the affinity of FecB and FecB2 for the Mtb secreted ferric‐siderophore, ferric‐carboxymycobactin (Fe‐cMB), by tryptophan fluorescence quenching titration assays. The affinity of FecB for Fe‐cMB was in the high nanomolar range (Kd = 355 ± 146 nM); in contrast, the affinity of FecB2 was in the high micromolar range. This result suggests that FecB is involved in transporting Fe‐cMB across the periplasmic space. To further probe the FecB residues involved in Fe‐cMB binding, we carried out a comprehensive mutational analysis. In an attempt to decipher the protein‐protein interaction network of FecB, we utilized co‐immunopreciptation (co‐IP) of FLAG‐tagged FecB in the non‐pathogenic Mtb model organism Mycobacterium smegmatis, followed by mass spectrometry analysis. The co‐IP FLAG‐FecB experiments resulted in identification of known proteins in the import of Fe‐cMB and surprisingly, also proteins thought to be involved in the export of apo‐siderophores. Together, these data clearly place FecB in the siderophore‐mediated iron‐uptake pathway, and will be discussed in more detail. Finally, this work has broadened our understanding of the Mtb iron acquisition pathway, which may lead to the identification of new therapeutic targets.