PPE37 Is Essential for Mycobacterium tuberculosis Heme-Iron Acquisition (HIA), and a Defective PPE37 in Mycobacterium bovis BCG Prevents HIA

Abstract

Mycobacterium tuberculosis, one of the world’s leading causes of death, must acquire nutrients, such as iron, from the host to multiply and cause disease. Iron is an essential metal and M. tuberculosis possesses two different systems to acquire iron from its environment: siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA), involving uptake and degradation of heme to release ferrous iron. We have discovered that Mycobacterium bovis BCG, the tuberculosis vaccine strain, is severely deficient in HIA, and we exploited this phenotypic difference between BCG and M. tuberculosis to identify genes involved in HIA by complementing BCG’s defect with a fosmid library. We identified ppe37, an iron-regulated PPE family gene, as being essential for HIA. BCG complemented with M. tuberculosis ppe37 exhibits HIA as efficient as that of M. tuberculosis, achieving robust growth with <0.2 µM hemin. Conversely, deletion of ppe37 from M. tuberculosis results in a strain severely attenuated in HIA, with a phenotype nearly identical to that of BCG, requiring a 200-fold higher concentration of hemin to achieve growth equivalent to that of its parental strain. A nine-amino-acid deletion near the N terminus of BCG PPE37 (amino acids 31 to 39 of the M. tuberculosis PPE37 protein) underlies BCG’s profound defect in HIA. Significant genetic variability exists in ppe37 genes across different M. tuberculosis strains, with more than 60% of sequences from completely sequenced M. tuberculosis genomes having mutations that result in altered PPE37 proteins; furthermore, these altered PPE37 proteins are nonfunctional in HIA. Our findings should allow delineation of the relative roles of HIA and SMIA in M. tuberculosis pathogenesis.

Comprehensive analysis of iron utilization by Mycobacterium tuberculosis

“…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).…”

“…Among those genes, we found that Mtb exhibits significantly stronger requirements for ppe37 and rv1085c encoding a hemolysin-like protein (Figs 3D and 8A; S7 and S9 Tables). The growth defect of Mtb mutants with insertions in ppe37 is consistent with previous findings that PPE37 is involved in heme utilization in the Mtb Erdman strain [27]. However, the requirement for Rv1085c for growth of Mtb in vitro in the absence of erythrocytes indicates that the molecular function of Rv1085c is probably different from that of hemolysins [49].…”

“…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. By contrast, ppe37 which was identified in Mtb Erdman as required for heme utilization in the presence of albumin [27] was classified as "growth defect" in our TnSeq experiments. These divergent results might be caused also by significant strain differences between the Mtb H37Rv derivative ML1600 (ΔRD1 ΔpanCD ΔmbtD::hyg R ) used in our study (S1 Table) and the Mtb Erdman strain used in the previous study [27].…”

“…This pathway is important because 70% of the available iron in the human host is stored in heme [25], mostly in hemoglobin, which is inaccessible to siderophores. Several proteins have been implicated in heme utilization by Mtb, such as the heme-binding protein Rv0203 [24], the RND efflux pumps MmpL11 and MmpL3 [24] and the cell surface proteins PPE36, PPE62 [26] and PPE37 [27]. However, the molecular mechanisms of how these proteins contribute to heme uptake by Mtb remain unclear.…”

New insights into the mycobacterial PE and PPE proteins provide a framework for future research

“…The PPE‐PPW PPE20 and its cognate partner PE15 were experimentally identified as ESX‐3 substrates (Tufariello et al , ). Two other PPW proteins, PPE36 and PPE37, are also described to play a role in iron homeostasis through functions in heme‐iron acquisition (Mitra et al , ; Tullius et al , ). Together with sequence analysis of the EspG‐binding domain, these data render the hypothesis that PPE‐PPW proteins may be generally dependent on ESX‐3, rather than ESX‐5 for their translocation, increasingly likely (Abdallah et al , ; Korotkova et al , ; Tufariello et al , ).…”