Citation: Potrykus J, Ballou ER, Childers DS, Brown AJP (2014) Conflicting Interests in the Pathogen–Host Tug of War: Fungal Micronutrient Scavenging Versus Mammalian Nutritional Immunity. PLoS Pathog 10(3): e1003910. doi:10.1371/journal.ppat.1003910
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Published: March 13, 2014
Copyright: © 2014 Potrykus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors are supported by the European Research Council (STRIFE project funded on grant number ERC-2009-AdG-249793, http://erc.europa.eu). AJPB is also supported by the Wellcome Trust (grant numbers 080088, 097377, www.wellcome.ac.uk) and the UK Biotechnology and Biological Sciences Research Council (grant number BB/F00513X/1, www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Strife concerning the accessibility of essential trace elements, such as transition metals, represents an important aspect of the dynamic interaction between a pathogenic fungus and its mammalian host. The host defends itself against infection by sequestering these essential micronutrients away from the invading pathogen via a phenomenon termed “nutritional immunity” . In turn, the fungus employs an array of tactics (scavenging and storage) to hoard micronutrients and support growth when these resources are scarce. In addition, micronutrient limitation triggers the expression of virulence determinants that can aggravate disease –.
Half of all enzymes and a third of all proteins require metals for their functionality , yet many transition metals are insoluble and potentially toxic at neutral pH and in an oxygenated, aqueous milieu. For example, iron and copper are redox active and capable of generating free radicals via Fenton chemistry under these conditions . Furthermore, toxic excess of a non-native metal can displace the native metals from metalloproteins or inhibit the function of non-metalloproteins. Therefore, the levels of iron, copper, and zinc and their partitioning within the body must be tightly regulated to maintain cellular homeostasis whilst avoiding cellular damage.
In the mammalian host, iron is the most abundant transition metal (3–4 g/adult human), followed by zinc (1.4–2.3 g/adult human), with copper trailing behind (~100 mg/adult human) . These metals have roles in signalling, immune responses, physiology, and development , and both metal deficiency and overload are detrimental to the host. Iron deficiency, the most common dietary deficiency in the world, weakens the immune system, whilst copper deficiency renders the host more susceptible to infection , . On the other hand, iron overload is a significant risk factor for some fungal diseases, especially for mucormycosis . Furthermore, individuals with acute myeloid leukemia, characterised by increased serum iron, have a higher risk of Candida and Aspergillus infections . Iron overload also exacerbates meningoencephalitis in a mouse model of cryptococcosis , . Conversely, fungal pathogens depend on effective metal acquisition mechanisms for full virulence , –.
The Many Faces of Nutritional Immunity
The mammalian host exploits both “constitutive” and “inducible” nutritional immunity to sequester transition metals away from microbial pathogens. The host regulates its interchangeable metal pools by sequestering metals via protein carriers or by partitioning them between intracellular stores , . This essentially imposes constitutive nutritional immunity by depleting the extracellular milieu of essential metals and creating a micronutrient-limiting environment for the pathogen. For example, the estimated serum concentration of free ferric iron is 10−24 M, many orders of magnitude lower than that expected based on its solubility (10−9 M) . Likewise, the intracellular “free pool” of copper is less than one copper atom per cell . Pathological disruption of this constitutively low metal ion environment is one factor that predisposes patients to fungal infection , .
Upon infection, the host imposes “inducible” nutritional immunity by readjusting its global micronutrient homeostasis to further limit microbial access to endogenous metal ion pools. This response is achieved both on systemic and local levels. Systemic metal readjustments are observed, such as the hypoferraemia associated with disseminated infections. In this case, regulation is achieved via the secreted hepatic hormone hepcidin, which itself is responsive to various regulators, such as IL-6 . Indeed, disseminated C. albicans infections are accompanied by increases in hepcidin and decreases in the degree of iron saturation of serum transferrin . At the local level, inducible mechanisms include the secretion by macrophages and neutrophils of siderocalins (such as lipocalin-2), which bind siderophores  (vide infra). The neutrophil copper-binding protein calgranulin C might also contribute to nutritional immunity .
Most data on nutritional immunity relate to bacterial infections. However, there is evidence for localised, induced nutritional immunity during systemic fungal disease. During disseminated candidiasis, infiltrating neutrophils appear to seal off the developing fungal lesions from the rest of the tissue to create a metal-deprived environment . Macrophages parasitized by Histoplasma capsulatum are reprogrammed to exclude zinc and, probably, iron . Neutrophil extracellular traps (NETs), which can control fungal growth via either cidal (C. albicans, Cryptococcus neoformans) or static mechanisms (Aspergillus fumigatus), are decorated with proteins such as the zinc-scavenging calprotectin . Furthermore, the release of the polymorphonuclear leukocyte protein lactoferrin at sites of infection inhibits the growth of C. albicans and A. fumigatus, presumably by inducing localized iron deprivation .
Metal deprivation is not the only means by which the host manipulates micronutrient levels to contain an infection. A contrasting mechanism, involving the “poisoning” of microbes with excess metal ions, is gaining attention in the literature . An elegant example is the case of copper poisoning during Cr. neoformans infection, where copper redistribution in alveolar macrophages is countered by expression of fungal metallothioneins sequestering the toxic excess of metal .
Overcoming Host Nutritional Defences—Many Tricks up the Fungal Sleeve
Fungi exploit many mechanisms to scavenge metal ions from different substrates, adjusting their metal acquisition machinery as required . The molecular mechanics that underlie metal sensing remain unclear , and the regulatory networks that control metal acquisition systems are complex, responding not just to micronutrient availability but also to environmental cues such as carbon source, hypoxia, and pH. These are reviewed elsewhere (e.g., , , ). Here, we summarise the fungal micronutrient acquisition pathways themselves and discuss how they respond to nutritional immunity.
Multiple acquisition pathways
Low and high affinity fungal transporters have been characterised for copper and zinc, but little is known about the acquisition of these metals in vivo. Notable exceptions include the Pra1/Zrt1-based zinc uptake system in C. albicans  and copper acquisition by Cr. neoformans via CTR1 and CTR4 , . In contrast, iron acquisition is relatively well researched.
Complex iron homeostasis within the host involves a plethora of storage proteins (e.g., ferritin), carrier proteins (e.g., transferrin, lactoferrin), and the machinery for senescent red blood cell recycling . The continual transfer of iron between various body compartments provides potential targets for fungal interference. To access iron, fungi employ: (1) a reductive iron acquisition (RIA) pathway, comprising a permease and a multicopper oxidase for high-affinity ferric import; (2) siderophore transporters for endogenous or exogenous siderophores; (3) secreted and cell surface reductants; and (4) the endocytic pathway for haem internalisation, in conjunction with intracellular haem oxygenase-1 (Hmx1) for haem iron extraction (Figure 1). Not all of these pathways are present in all fungi. For example, A. fumigatus mainly exploits endogenous siderophores for iron scavenging and also possesses the RIA system (FtrA/FetC), but only the former pathway is required for virulence . C. albicans utilizes RIA (Ftr1/Fet3 and additional Ftr1/ferroxidase-homologue combinations), xenosiderophore transport (Sit1 transporter), and haem acquisition (Rbt5 and other receptors) . Cr. neoformans relies on RIA (Cft1/Cfo1), a xenosiderophore transporter (Sit1), secreted and extracellular reductants (3-hydroxyanthranilic acid, melanin), and haem acquisition (Cig1) . H. capsulatum employs RIA (Ftr1/Fet3), produces and internalises siderophores, secretes gamma-glutamyltransferase and glutathione-dependent ferric reductase, and utilizes haem (Figure 1) , .
Pathogenic fungi employ different combinations of evolutionarily related strategies to acquire iron in the limiting microenvironments of the host. The figure focuses on those fungal species for which data are currently available. See text for details. Hb: haemoglobin, RIA: reductive iron acquisition, Afum: A. fumigatus, Calb: C. albicans, Cneo: Cr. neoformans, Hcap: H. capsulatum, Rory: R. oryzae.
Certain substrates require specialised surface receptors. For example, C. albicans uses the protein Als3 for ferritin iron and the Rbt5 receptor (and other CFEM-type receptors) for haemoglobin and haem . Als3 also acts as an adhesin and invasin. It is expressed during hyphal development, promoting the adhesion and host endocytosis of C. albicans cells. This suggests interesting interconnections between micronutrient availability and acquisition and other types of fungus–host interaction during disease progression. Haem is also readily utilised by Cr. neoformans via an uptake pathway that involves the secreted haemophore Cig1 . In Rhizopus oryzae, the ferric reductase Ftr1 has been implicated in haem iron acquisition . No specific uptake systems for haem, ferritin, or transferrin have been described for A. fumigatus, although, like H. capsulatum, this fungus uses secreted siderophores to chelate ferric iron from transferrin , .
The various iron acquisition mechanisms seem to become prominent at different stages of infection. Accordingly, C. albicans requires Sit1 for epithelial escape, RIA for the establishment of disseminated bloodstream infections, and Hmx1 for sustaining deep-seated organ infections , . In contrast, H. capsulatum requires RIA during early infection stages and siderophore biosynthesis later in the infection process .
Siderophores are small organic molecules with ferric iron binding affinities of ~1030 M at neutral pH. Other than the carboxylate siderophore rhizoferrin found in zygomycetes (R. oryzae), all other known fungal siderophores are hydroxamates . In H. capsulatum and A. fumigatus, the SID1/SIDA gene is required for the first dedicated step in siderophore biosynthesis. This gene plays a role in the later stages of H. capsulatum infection and is required for A. fumigatus virulence , , . Furthermore, A. fumigatus produces secreted siderophores, as well as intracellular siderophores for hyphal and conidial iron storage . Although C. albicans and Cr. neoformans do not produce their own siderophores, they can exploit hydroxamate-type xenosiderophores produced by other microorganisms , .
It is important to emphasise that iron is not the only transition metal limiting to the fungus within the host environment. This is evident from the discovery that fungi also take advantage of induced secretory mechanisms to obtain non-ferric metal ions. For example, C. albicans possesses a dedicated zinc scavenging system, comprised of the secreted Pra1 “zincophore” and the Zrt1 transporter . Additionally, methanobactin-like compounds, i.e., secreted scavengers of copper, have been discovered in Saccharomyces cerevisiae , . However, the prevalence of such molecules among fungal pathogens of mammals remains unknown.
Storage and conservation
In fungi, the intracellular storage of transition metals is not limited to vacuoles . A. fumigatus produces intracellular siderophores for hyphal and conidial iron storage, and both intra- and extracellular siderophores are required for full virulence . Ferritin-like genes have been identified in A. fumigatus and R. oryzae, but not in basidiomycetous genomes . Copper hyperaccumulation by Cr. neoformans suggests the existence of copper storage mechanisms in this pathogen, which might promote fungal survival inside the macrophages (see above) , .
When micronutrients are scarce, some fungi fine-tune their metabolism towards micronutrient conservation. For example, Cr. gattii and C. albicans respond to zinc restriction by down-regulating zinc-dependent alcohol dehydrogenases to increase zinc availability for other metalloproteins , . Similarly, in A. fumigatus, C. albicans, and Cr. neoformans, iron-conserving mechanisms operate via the transcription factor HapX to down-regulate genes in iron-consuming pathways, such as amino acid metabolism, tricarboxylic acid cycle, respiration, and haem biosynthesis .
Autophagy, a major pathway for the bulk degradation of cytosolic components during nutrient starvation, might represent a non-conventional mechanism for recycling and conserving essential micronutrients to sustain hyphal growth in A. fumigatus, although its role in vivo is unverified. Other fungi, such as C. albicans, also undergo autophagy, but unlike in A. fumigatus, this phenomenon is not suppressed by zinc supplementation .
Impact on virulence factors
As already stated, low metal concentrations induce the expression of fungal metal acquisition genes, which themselves are indispensable for host colonisation, dissemination, and virulence for many fungal pathogens , , –. Furthermore, pathogenic fungi can activate other virulence determinants in response to micronutrient limitation. For example, iron depletion induces capsule formation in Cryptococci, which allows the pathogens to escape recognition by the immune system , . As another example, iron deprivation of A. fumigatus results in the expression of the major surface allergen ribotoxin AspF1, which could potentiate allergic bronchopulmonary aspergillosis . Thus, in the act of depriving fungal pathogens of nutrients to limit their growth, the host can trigger virulence factors that may exacerbate fungal disease.
The metal tug of war between pathogen and host is a complex and dynamic process, with each party striving to procure and retain essential micronutrients. The great complexity of this interaction is only now being realised. It is becoming apparent that the host responds to infection by redirecting micronutrients via niche- and perhaps infection-stage–specific mechanisms to various ends, e.g., micronutrient limitation versus micronutrient poisoning. On the other hand, the apparent redundancy of some fungal metal acquisition mechanisms needs to be explored, and their essentiality to the different stages of pathogenesis remains to be established. Also, before we can start drawing global comparisons of metal homeostasis in fungal and bacterial infectious agents, more information is required about micronutrient warfare in fungal pathogens outside the small handful of established model organisms. The ultimate challenge will be to integrate the responses of host and pathogen into a holistic model that describes how the host modulates micronutrient homeostasis during infection and how the pathogen responds to these changes, spatially and temporally. New technologies, such as live-animal imaging of fungal gene expression , MALDI (matrix-assisted laser desorption/ionization) mass spectrometry imaging of proteins, and 2-D element mapping directly from biological specimens , are beginning to illuminate the pathogen–host tug-of-war over micronutrients. We are confident that the future holds many exciting discoveries in this field.
Numerous colleagues have published fascinating work in this area. We apologise to those we have been unable to cite because of space constraints.
- 1. Wang L, Cherayil BJ (2009) Ironing out the wrinkles in the host defence: interactions between iron homeostasis and innate immunity. J Innate Immun 1: 455–464. doi: 10.1159/000210016
- 2. Schrettl M, Beckmann N, Varga J, Heinekamp T, Jacobsen ID, et al. (2010) HapX-mediated adaptation to iron starvation is crucial for virulence of Aspergillus fumigatus. PLOS Pathog 6: e1001124 doi:10.1371/journal.ppat.1001124.
- 3. Schneider Rde O, Fogaça Nde S, Kmetzsch L, Schrank A, Vainstein MH, et al. (2012) Zap1 regulates zinc homeostasis and modulates virulence in Cryptococcus gattii. PLOS ONE 7: e43773 doi:10.1371/journal.pone.0043773.
- 4. Kronstad JW, Hu G, Jung WH (2013) An encapsulation of iron homeostasis and virulence in Cryptococcus neoformans. Trends Microbiol 21: 457–465. doi: 10.1016/j.tim.2013.05.007
- 5. Waldron KJ, Rutherford JC, Ford D, Robinson NJ (2009) Metalloproteins and metal sensing. Nature 460: 823–830. doi: 10.1038/nature08300
- 6. Ehrensberger KM, Bird AJ (2011) Hammering out details: regulating metal levels in eukaryotes. Trends Biochem Sci 36: 524–531. doi: 10.1016/j.tibs.2011.07.002
- 7. Bleackley MR, Macgillivray RT (2011) Transition metal homeostasis: from yeast to human disease. Biometals 24: 785–809. doi: 10.1007/s10534-011-9451-4
- 8. Samanovic MI, Ding C, Thiele DJ, Darwin KH (2012) Copper in microbial pathogenesis: meddling with the metal. Cell Host Microbe 11: 106–115. doi: 10.1016/j.chom.2012.01.009
- 9. Ibrahim AS (2011) Host cell invasion in mucormycosis: role of iron. Curr Opin Microbiol 14: 406–411. doi: 10.1016/j.mib.2011.07.004
- 10. Iglesias-Osma C, Gonzalez-Villaron L, San Miguel JF, Caballero MD, Vazquez L, et al. (1995) Iron metabolism and fungal infections in patients with haematological malignancies. J Clin Pathol 48: 223–225. doi: 10.1136/jcp.48.3.223
- 11. Jung WH, Kronstad JW (2008) Iron and fungal pathogenesis: a case study with Cryptococcus neoformans. Cell Microbiol 10: 277–284. doi: 10.1111/j.1462-5822.2007.01077.x
- 12. Barluzzi R, Saleppico S, Nocentini A, Boelaert JR, Neglia R, et al. (2002) Iron overload exacerbates experimental meningoencephalitis by Cryptococcus neoformans. J Neuroimmunol 132: 140–146. doi: 10.1016/s0165-5728(02)00324-7
- 13. Ramanan N, Wang Y (2000) A high-affinity iron permease essential for Candida albicans virulence. Science 288: 1062–1064. doi: 10.1126/science.288.5468.1062
- 14. Schrettl M, Bignell E, Kragl C, Sabiha Y, Loss O, et al. (2007) Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection. PLOS Pathog 3: e128 doi:10.1371/journal.ppat.0030128.
- 15. Hilty J, George Smulian A, Newman SL (2011) Histoplasma capsulatum utilizes siderophores for intracellular iron acquisition in macrophages. Med Mycol 49: 633–642. doi: 10.3109/13693786.2011.558930
- 16. Ding C, Festa RA, Chen YL, Espart A, Palacios Ò, et al. (2013) Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence. Cell Host Microbe 13: 265–276. doi: 10.1016/j.chom.2013.02.002
- 17. Raymond KN, Dertz EA, Kim SS (2003) Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci U S A 100: 3584–3588. doi: 10.1073/pnas.0630018100
- 18. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O'Halloran TV (1999) Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284: 805–808. doi: 10.1126/science.284.5415.805
- 19. Drakesmith H, Prentice AM (2012) Hepcidin and the iron-infection axis. Science 338: 768–772. doi: 10.1126/science.1224577
- 20. Armitage AE, Eddowes LA, Gileadi U, Cole S, Spottiswoode N, et al. (2011) Hepcidin regulation by innate immune and infectious stimuli. Blood 118: 4129–4139. doi: 10.1182/blood-2011-04-351957
- 21. Silva MG, Schrank A, Bailão EF, Bailão AM, Borges CL, et al. (2011) The homeostasis of iron, copper, and zinc in Paracoccidioides brasiliensis, Cryptococcus neoformans var. Grubii, and Cryptococcus gattii: a comparative analysis. Front Microbiol 2: 49 doi:10.3389/fmicb.2011.00049.
- 22. Potrykus J, Stead D, MacCallum DM, Urgast DS, Raab A, et al. (2013) Fungal Iron Availability during Deep Seated Candidiasis Is Defined by a Complex Interplay Involving Systemic and Local Events. PLOS Pathog 9: e1003676 doi:10.1371/journal.ppat.1003676.
- 23. Winters MS, Chan Q, Caruso JA, Deepe GS Jr (2010) Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses. J Infect Dis 202: 1136–1145. doi: 10.1086/656191
- 24. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, et al. (2009) Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLOS Pathog 5: e1000639 doi:10.1371/journal.ppat.1000639.
- 25. Zarember KA, Sugui JA, Chang YC, Kwon-Chung KJ, Gallin JI (2007) Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion. J Immunol 178: 6367–6373. doi: 10.4049/jimmunol.178.10.6367
- 26. Canessa P, Larrondo LF (2012) Environmental responses and the control of iron homeostasis in fungal systems. Appl Microbiol Biotechnol 97: 939–955. doi: 10.1007/s00253-012-4615-x
- 27. Citiulo F, Jacobsen ID, Miramón P, Schild L, Brunke S, et al. (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLOS Pathog 8: e1002777 doi:10.1371/journal.ppat.1002777.
- 28. Waterman SR, Park YD, Raja M, Qiu J, Hammoud DA, et al. (2012) Role of CTR4 in the virulence of Cryptococcus neoformans. MBio 3: e00285–12 doi:10.1128/mBio.00285-12.
- 29. Hissen AH, Wan AN, Warwas ML, Pinto LJ, Moore MM (2005) The Aspergillus fumigatus siderophore biosynthetic gene SIDA, encoding 1-ornithine N5- oxygenase, is required for virulence. Infect Immun 73: 5493–5503. doi: 10.1128/iai.73.9.5493-5503.2005
- 30. Almeida RS, Wilson D, Hube B (2009) Candida albicans iron acquisition within the host. FEMS Yeast Res 9: 1000–1012. doi: 10.1111/j.1567-1364.2009.00570.x
- 31. Cadieux B, Lian T, Hu G, Wang J, Biondo C, et al. (2013) The mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans. J Infect Dis 207: 1339–1347. doi: 10.1093/infdis/jit029
- 32. Ibrahim AS, Gebremariam T, Lin L, Luo G, Husseiny MI, et al. (2010) The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis. Mol Microbiol 77: 587–604. doi: 10.1111/j.1365-2958.2010.07234.x
- 33. Seifert M, Nairz M, Schroll A, Schrettl M, Haas H, et al. (2008) Effects of the Aspergillus fumigatus siderophore systems on the regulation of macrophage immune effector pathways and iron homeostasis. Immunobiology 213: 767–778. doi: 10.1016/j.imbio.2008.07.010
- 34. Hwang LH, Mayfield JA, Rine J, Sil A (2008) Histoplasma requires SID1, a member of an iron-regulated siderophore gene cluster, for host colonization. PLOS Pathog 4: e1000044 doi:10.1371/journal.ppat.1000044.
- 35. Cobine PA, Pierrel F, Bestwick ML, Winge DR (2006) Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase. J Biol Chem 281: 36552–36559. doi: 10.1074/jbc.m606839200
- 36. Balasubramanian R, Rosenzweig AC (2008) Copper methanobactin: a molecule whose time has come. Curr Opin Chem Biol 12: 245–249. doi: 10.1016/j.cbpa.2008.01.043
- 37. Raja MR, Waterman SR, Qiu J, Bleher R, Williamson PR, et al. (2013) A copper hyperaccumulation phenotype correlates with pathogenesis in Cryptococcus neoformans. Metallomics 5: 363–371. doi: 10.1039/c3mt20220h
- 38. Nobile CJ, Nett JE, Hernday AD, Homann OR, Deneault JS, et al. (2009) Biofilm matrix regulation by Candida albicans Zap1. PLOS Biol 7: e1000133 doi:10.1371/journal.pbio.1000133.
- 39. Richie DL, Fuller KK, Fortwendel J, Miley MD, McCarthy JW, et al. (2007) Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus. Eukaryot Cell 6: 2437–2447. doi: 10.1128/ec.00224-07
- 40. Crestani J, Carvalho PC, Han X, Seixas A, Broetto L, et al. (2012) Proteomic profiling of the influence of iron availability on Cryptococcus gattii. J Proteome Res 11: 189–205. doi: 10.1021/pr2005296