Extracellular Heme Uptake and the Challenges of Bacterial Cell Membranes

Smith, Aaron D.; Wilks, Angela

doi:10.1016/b978-0-12-394390-3.00013-6

Cited by 27 publications

(33 citation statements)

References 176 publications

(219 reference statements)

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“…Here, we ascertain that the heme binding domain in HmbR is natively folded, as deemed by the following: 1) The circular dichroism spectrum in Fig. 2 reveals that HmbR possesses an overall native β-barrel fold after purification, 2) the spectral signature of this spectrum is consistent with previous reports of the behavior of other TonB-dependent hemin transport proteins in the detergents we have used [7,40,41], and 3) the electronic absorption, 4) EPR, and 5) rR analysis all show conformational heterogeneity of the heme that co-purified with HmbR. Taken together, we deem that the heme environment analyzed in this study is biologically relevant, and is not due to promiscuous interactions with HmbR, even if other regions of the protein outside the heme binding domain were altered.…”

Section: Discussionsupporting

confidence: 85%

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Mokry

Nadia-Albete

Johnson

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood. Methods An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques. Results HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand. Conclusion In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter. General Significance A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.

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Section: Discussionsupporting

confidence: 85%

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Mokry

Nadia-Albete

Johnson

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Mechanisms of heme uptake have been well characterized in pathogenic bacteria and involve the recognition of heme or heme-loaded hemophores by surface receptors before delivery to membrane transporters and subsequent internalization (55). Although less is known about the use of heme in parasites and pathogenic fungi, Leishmania amazonensis was recently found to have a heme receptor that was required for heme utilization, and the pathogenic fungi C. albicans and Histoplasma capsulatum utilize heme and/or hemoglobin via cell surface receptors (9,10,56,57).…”

Section: Discussionmentioning

confidence: 99%

Role of Ferric Reductases in Iron Acquisition and Virulence in the Fungal Pathogen Cryptococcus neoformans

Saikia

Oliveira

et al. 2014

Infect Immun

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Iron acquisition is critical for the ability of the pathogenic yeast Cryptococcus neoformans to cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors. C. neoformans acquires iron by multiple mechanisms, including a ferroxidase-permease high-affinity system, siderophore uptake, and utilization of both heme and transferrin. In this study, we examined the expression of eight candidate ferric reductase genes and their contributions to iron acquisition as well as to ferric and cupric reductase activities. We found that loss of the FRE4 gene resulted in a defect in production of the virulence factor melanin and increased susceptibility to azole antifungal drugs. In addition, the FRE2 gene was important for growth on the iron sources heme and transferrin, which are relevant for proliferation in the host. Fre2 may participate with the ferroxidase Cfo1 of the high-affinity uptake system for growth on heme, because a mutant lacking both genes showed a more pronounced growth defect than the fre2 single mutant. A role for Fre2 in iron acquisition is consistent with the attenuation of virulence observed for the fre2 mutant. This mutant also was defective in accumulation in the brains of infected mice, a phenotype previously observed for mutants with defects in high-affinity iron uptake (e.g., the cfo1 mutant). Overall, this study provides a more detailed view of the iron acquisition components required for C. neoformans to cause cryptococcosis.

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“…Bis-histidine motifs are found in HmuY from Porphyromonas gingivalis (45) as well as in membrane-spanning heme receptors (7). Other motifs in the heme receptor class are also known; HmuR from Neisseria meningitidis has a five-coordinate tyrosine ligation (46) while PhuR from P. aeruginosa has been shown to have His/Tyr ligation (47).…”

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confidence: 99%

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment

et al. 2015

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The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicate that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen-bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen-bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

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Extracellular Heme Uptake and the Challenges of Bacterial Cell Membranes

Cited by 27 publications

References 176 publications

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Role of Ferric Reductases in Iron Acquisition and Virulence in the Fungal Pathogen Cryptococcus neoformans

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment

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