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Structural basis of haem-iron acquisition by fungal pathogens

Nature Microbiology volume 1, Article number: 16156 (2016) Cite this article

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Abstract

Pathogenic microorganisms must cope with extremely low free-iron concentrations in the host's tissues. Some fungal pathogens rely on secreted haemophores that belong to the Common in Fungal Extracellular Membrane (CFEM) protein family, to extract haem from haemoglobin and to transfer it to the cell's interior, where it can serve as a source of iron. Here we report the first three-dimensional structure of a CFEM protein, the haemophore Csa2 secreted by Candida albicans. The CFEM domain adopts a novel helical-basket fold that consists of six α-helices, and is uniquely stabilized by four disulfide bonds formed by its eight signature cysteines. The planar haem molecule is bound between a flat hydrophobic platform located on top of the helical basket and a peripheral N-terminal ‘handle’ extension. Exceptionally, an aspartic residue serves as the CFEM axial ligand, and so confers coordination of Fe3+ haem, but not of Fe2+ haem. Histidine substitution mutants of this conserved Asp acquired Fe2+ haem binding and retained the capacity to extract haem from haemoglobin. However, His-substituted CFEM proteins were not functional in vivo and showed disturbed haem exchange in vitro, which suggests a role for the oxidation-state-specific Asp coordination in haem acquisition by CFEM proteins.

The fourth most-abundant transition metal on the earth's crust, iron, is also an essential nutrient for nearly all organisms. Its ability to accept and donate electrons readily makes it highly reactive, and toxic if unconstrained. Yet its reduction potential, mainly between the Fe2+/Fe3+ oxidation states, is valuable for many key biological processes which include, for example, oxygen transport, enzyme catalysis and cellular respiration. However, iron scarcity is the prevalent condition for most organisms that live in aerobic environments because of the extremely low solubility of its oxidized form at a physiological pH. Animals possess high-affinity iron-chelation mechanisms that additionally limit its bioavailability for invading microorganisms, and are thus considered an important element of the host nutritional immunity1. In humans, ∼70% of the iron is bound by the oxygen carrier haemoglobin as part of its haem prosthetic group2. Many pathogenic microorganisms, bacterial as well as fungal, have consequently evolved mechanisms to exploit haem and haemoglobin as sources of iron3–5. Some of these microorganisms utilize secreted haemophores as part of their haem-iron utilization systems, such as HasA of Serratia marcescens6, IsdX of Bacillus anthracis7 and Cig1 of Cryptococcus neoformans8.

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Figure 1: C. albicans Csa2 is a secreted haemophore.
Figure 2: Csa2 and rCsa2 exchange haem with Rbt5 and Pga7.
Figure 3: The crystal structure of the Csa2 CFEM protein reveals a novel helical-basket fold.
Figure 4: CFEM has a unique binding site for haem and an exceptional iron coordination.
Figure 5: Histidine-substituted CFEM mutants bind haem and extract haem from haemoglobin, but are inactive and defective in haem exchange.
Figure 6: Schematic pathway of haem-iron acquisition in Candidaceae.

References

  1. Weinberg, E. D. Nutritional immunity. Host's attempt to withhold iron from microbial invaders. J. Am. Med. Assoc. 231, 39–41 (1975).

    Article  CAS  Google Scholar 

  2. Andrews, N. C. Iron homeostasis: insights from genetics and animal models. Nat. Rev. Genet. 1, 208–217 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Almeida, R. S., Wilson, D. & Hube, B. Candida albicans iron acquisition within the host. FEMS Yeast Res. 9, 1000–1012 (2009).

    Article  CAS  PubMed  Google Scholar 

  4. Cassat, J. E. & Skaar, E. P. Iron in infection and immunity. Cell Host Microbe 13, 509–519 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Caza, M. & Kronstad, J. W. Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. Front. Cell Infect. Microbiol. 3, 80 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Letoffe, S., Ghigo, J. M. & Wandersman, C. Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein. Proc. Natl Acad. Sci. USA 91, 9876–9880 (1994).

    Article  CAS  PubMed  Google Scholar 

  7. Ekworomadu, M. T. et al. Differential function of lip residues in the mechanism and biology of an anthrax hemophore. PLoS Pathog. 8, e1002559 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cadieux, B. et al. The mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans. J. Infect. Dis. 207, 1339–1347 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kulkarni, R. D., Kelkar, H. S. & Dean, R. A. An eight-cysteine-containing CFEM domain unique to a group of fungal membrane proteins. Trends Biochem. Sci. 28, 118–121 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Mitchell, A. et al. The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res. 43, D213–D221 (2015).

    Article  PubMed  Google Scholar 

  11. Ding, C. et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans. PLoS One 6, e28151 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kuznets, G. et al. A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin. PLoS Pathog. 10, e1004407 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Weissman, Z. & Kornitzer, D. A family of Candida cell surface haem-binding proteins involved in haemin and haemoglobin-iron utilization. Mol. Microbiol. 53, 1209–1220 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Bailao, E. F. et al. Hemoglobin uptake by Paracoccidioides spp. is receptor-mediated. PLoS Negl. Trop. Dis. 8, e2856 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Amorim-Vaz, S. et al. RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans. mBio 6, e00942–15 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Mochon, A. B. et al. Serological profiling of a Candida albicans protein microarray reveals permanent host–pathogen interplay and stage-specific responses during candidemia. PLoS Pathog. 6, e1000827 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Sorgo, A. G. et al. Mass spectrometric analysis of the secretome of Candida albicans. Yeast 27, 661–672 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, T., Bonkovsky, H. L. & Guo, J. T. Structural analysis of heme proteins: implications for design and prediction. BMC Struct. Biol. 11, 13 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Outten, F. W. & Theil, E. C. Iron-based redox switches in biology. Antioxid. Redox Signal 11, 1029–1046 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dixon, H. B. & McIntosh, R. Reduction of methaemoglobin in haemoglobin samples using gel filtration for continuous removal of reaction products. Nature 213, 399–400 (1967).

    Article  CAS  PubMed  Google Scholar 

  22. Contreras, H., Chim, N., Credali, A. & Goulding, C. W. Heme uptake in bacterial pathogens. Curr. Opin. Chem. Biol. 19, 34–41 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Arnoux, P. et al. The crystal structure of HasA, a hemophore secreted by Serratia marcescens. Nat. Struct. Biol. 6, 516–520 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Mazmanian, S. K. et al. Passage of heme-iron across the envelope of Staphylococcus aureus. Science 299, 906–909 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Grigg, J. C., Ukpabi, G., Gaudin, C. F. & Murphy, M. E. Structural biology of heme binding in the Staphylococcus aureus Isd system. J. Inorg. Biochem. 104, 341–348 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Grigg, J. C., Vermeiren, C. L., Heinrichs, D. E. & Murphy, M. E. Haem recognition by a Staphylococcus aureus NEAT domain. Mol. Microbiol. 63, 139–149 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Bodanszky, M., Klausner, Y. S. & Said, S. I. Biological activities of synthetic peptides corresponding to fragments of and to the entire sequence of the vasoactive intestinal peptide. Proc. Natl Acad. Sci. USA 70, 382–384 (1973).

    Article  CAS  PubMed  Google Scholar 

  28. Weissman, Z., Shemer, R. & Kornitzer, D. Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans. Mol. Microbiol. 44, 1551–1560 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Atir-Lande, A., Gildor, T. & Kornitzer, D. Role for the SCFCDC4 ubiquitin ligase in Candida albicans morphogenesis. Mol. Biol. Cell 16, 2772–2785 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Feng, Q., Summers, E., Guo, B. & Fink, G. Ras signaling is required for serum-induced hyphal differentiation in Candida albicans. J. Bacteriol. 181, 6339–6346 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000 the integration of data reduction and structure solution—from diffraction images to an initial model in minutes. Acta Crystallogr. D 62, 859–866 (2006).

    Article  PubMed  Google Scholar 

  32. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002).

    Article  PubMed  Google Scholar 

  33. Otwinowski, Z. in Proc. CCP4 Study Weekend. Isomorphous Replacement and Anomalous Scattering (eds Wolf, W., Evans, P. R. & Leslie, A. G. W. ) 80–85 (Daresbury Laboratory, 1991).

    Google Scholar 

  34. Cowtan, K. D. & Zhang, K. Y. Density modification for macromolecular phase improvement. Progr. Biophys. Mol. Biol. 72, 245–270 (1999).

    Article  CAS  Google Scholar 

  35. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).

    Article  PubMed  Google Scholar 

  36. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002).

    Google Scholar 

  39. Weinstein, J. D. & Beale, S. I. Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. J. Biol. Chem. 258, 6799–6807 (1983).

    CAS  PubMed  Google Scholar 

  40. Schneider, S., Marles-Wright, J., Sharp, K. H. & Paoli, M. Diversity and conservation of interactions for binding heme in b-type heme proteins. Nat. Prod. Rep. 24, 621–630 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Zacharias, N. & Dougherty, D. A. Cation–pi interactions in ligand recognition and catalysis. Trends Pharmacol. Sci. 23, 281–287 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Hargrove, M. S., Whitaker, T., Olson, J. S., Vali, R. J. & Mathews, A. J. Quaternary structure regulates hemin dissociation from human hemoglobin. J. Biol. Chem. 272, 17385–9 (1997).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to D. Hiya (Technion Center for Structural Biology) for help with protein crystallization, T. Ziv (Smoler Proteomics Center) for mass spectroscopy analysis, G. Kuznets and Y. Gorelik for constructing plasmids KB2366 and KB2221/2, U. Roy for producing Csa2 cysteine mutants, M. Lebendiker (Hebrew University of Jerusalem) for advice with SEC-MALS, R. Zarivach (Ben-Gurion University) for the Origami B strain and O. Lewinson, I. Silman, N. Adir, N. Levanon, A. Haber and S. Selig for discussions and critical reading of the manuscript. This research was supported by grants from the Israel Science Foundation and the Ministry of Health's Chief Scientist Office to D.K. H.D. thanks the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 330879-MC-CHOLESTRUCTURE for financial support.

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Authors and Affiliations

  1. B. Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, and the Rappaport Institute for Research in the Medical Sciences, 31096, Haifa, Israel

    Lena Nasser, Ziva Weissman, Mariel Pinsky & Daniel Kornitzer

  2. Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem, Jerusalem, Israel

    Hadar Amartely

  3. Technion Center for Structural Biology, Technion – Israel Institute of Technology, 32000, Haifa, Israel

    Hay Dvir

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  1. Lena Nasser
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Contributions

L.N., Z.W., M.P. and D.K. constructed the plasmids and strains, purified the proteins and performed experiments, H.A. performed the SEC-MALS analysis, H.D. crystallized the protein and determined its structure, and H.D. and D.K. developed the project, interpreted the data and wrote the paper.

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Correspondence to Hay Dvir or Daniel Kornitzer.

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Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References (PDF 4321 kb)

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Nasser, L., Weissman, Z., Pinsky, M. et al. Structural basis of haem-iron acquisition by fungal pathogens. Nat Microbiol 1, 16156 (2016). https://doi.org/10.1038/nmicrobiol.2016.156

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