Heme and hemoglobin utilization by Mycobacterium tuberculosis

Abstract

Iron is essential for growth of Mycobacterium tuberculosis (Mtb), but most iron in the human body is stored in heme within hemoglobin. Here, we demonstrate that the substrate-binding protein DppA of the inner membrane Dpp transporter is required for heme and hemoglobin utilization by Mtb. The 1.27 Å crystal structure of DppA shows a tetrapeptide bound in the protein core and a large solvent-exposed crevice for heme binding. Mutation of arginine 179 in this cleft eliminates heme binding to DppA and prevents heme utilization by Mtb. The outer membrane proteins PPE36 and PPE62 are also required for heme and hemoglobin utilization, indicating that these pathways converge at the cell surface of Mtb. Albumin, the most abundant blood protein, binds heme specifically and bypasses the requirements for PPE36, PPE62 and Dpp. Thus, our study reveals albumin-dependent and-independent heme uptake pathways, highlighting the importance of iron acquisition from heme for Mtb.

Comprehensive analysis of iron utilization by Mycobacterium tuberculosis

“…The genes required for utilization of hemin and hemoglobin as iron sources are very similar (Fig 3B,3C and S1 Fig). This is consistent with our previous finding that hemoglobin and heme acquisition pathways overlap [36]. In the presence of hemin or hemoglobin as the sole iron source, Mtb exhibits increased requirements for genes involved in lipid metabolism, PDIM biosynthesis, ESX-3 secretion system, leucine biosynthesis, and decreased requirements for genes involved in heme/porphyrin biosynthesis, glycine metabolism, inorganic phosphate transporters, RND efflux pumps and Mce4 family proteins the ΔmbtD::hyg r mutant (B) in self-made low-iron 7H9 medium supplemented with: 20 μM ammonium ferric citrate; 10 μM human holotransferrin; 10 μM human holo-lactoferrin; 5 μM human hemoglobin; 20 μM hemin; 0.2 μM mycobactin (MBT) and 0.2 μM carboxymycobactin (cMBT), respectively.…”

“…We measured growth of the avirulent parent strain Mtb mc 2 6230 (H37Rv ΔRD1, ΔpanCD; S1 Table) and of Mtb ML1600 in self-made low-iron Middlebrook 7H9 medium using 20 μM ammonium ferric citrate (control), 10 μM human holo-transferrin, 10 μM human holo-lactoferrin, 5 μM human hemoglobin, 20 μM hemin, 0.2 μM mycobactin and 0.2 μM carboxymycobactin as sole iron sources, respectively. These experiments clearly showed that Mtb utilizes iron from mycobactin, carboxymycobactin, hemin and hemoglobin consistent with previous results [23,24,27,36]. In contrast, iron salts, transferrin and lactoferrin were only used in the presence of siderophores (Fig 1).…”

“…This is consistent with our previous observation that both pathways appear to converge on the cell surface of Mtb [26]. However, the TnSeq analysis did not identify any gene required for this pathway such as the ppe36 and dpp genes [26,36]. This might be a consequence of a second, previously unrecognized pathway for heme utilization which depends on albumin.…”

“…An interesting observation was that a few Mtb genes such as mmpS5, mmpL5, rv2047c and rv0455c were essential for growth in medium with high concentrations of MBT in the presence of hemin, although the same genes were not required for growth in medium with hemin or hemoglobin alone. This phenotype cannot be explained by the dependency of Mtb growth on a particular iron source since hemin can rescue mutants with defects in siderophore utilization and vice versa [23,26,36]. This phenotype, however, mimics the self-poisoning phenotype observed for the ΔmmpS4/ΔmmpS5 siderophore secretion mutant [13] and for the ΔmmpL4/ ΔmmpL5 in this study.…”

“…This might be a consequence of a second, previously unrecognized pathway for heme utilization which depends on albumin. Serum albumin binds heme with high affinity [62,63] and enables heme uptake independent of the ppe36 and dpp genes, although these genes are essential for growth of Mtb in medium with heme or hemoglobin as sole iron sources in the absence of albumin [36]. Since Mtb was grown on agar plates containing albumin in our TnSeq experiments, albumin-dependent heme uptake might explain the lack of requirement for the ppe36 and dpp genes.…”