The crucial role of iron uptake in Aspergillus fumigatus virulence

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The crucial role of iron uptake in Aspergillus fumigatus virulence Margo M Moore Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised individuals. Siderophore-mediated iron acquisition has been shown to be essential for virulence. New studies have revealed that enzymes involved in siderophore biosynthesis and uptake are compartmentalized in peroxisomes and endosome-like vesicles, respectively. Gene and protein expression studies have revealed coordinated regulation of siderophore and sterol metabolism linked to the common precursor mevalonate. Several A. fumigatus transcription factors have been identified that are unexpectedly involved in the regulation of iron homeostasis. New diagnostic and drug treatments are being developed that exploit the requirement of A. fumigatus for extracellular siderophores. Addresses Department of Biological Sciences, Simon Fraser University, Burnaby, BC V5A 1S6, Canada Corresponding author: Moore, Margo M ([email protected])

Current Opinion in Microbiology 2013, 16:692–699 This review comes from a themed issue on Growth and development: eukaryotes Edited by James W Kronstad For a complete overview see the Issue and the Editorial Available online 17th August 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.07.012

Introduction

[6–9]. Polymorphisms in genes involved in host iron homeostasis have revealed that SNPs may alter susceptibility of individuals to Mycobacterium tuberculosis [10] and it would be interesting to determine whether SNPs in host iron homeostasis genes affect susceptibility to invasive mould infections. Over the past decade, a number of studies have shown that iron acquisition is essential for A. fumigatus virulence. Recent research in this area will be the focus of this review.

Mechanisms of iron uptake in A. fumigatus Micromolar levels of free iron are essential for fungal growth but in the human host, sequestration by ironbinding proteins reduces free iron to 1018 M [11]. To acquire iron, A. fumigatus possesses two high affinity iron uptake mechanisms: reductive iron assimilation (RIA) and siderophore-mediated iron acquisition. Reductive iron assimilation

RIA in A. fumigatus is accomplished by a membranebound ferric reductase associated with a ferroxidase and an iron permease, Ftr. Inactivation of ftr in A. fumigatus did not affect virulence [12]. Although freB was recently identified as the ferric reductase involved in RIA, growth defects were not observed in the DfreB strain unless siderophore biosynthesis was also knocked out [13]. A low affinity iron uptake system likely also exists because mutants defective in both RIA and siderophore biosynthesis are still able to grow in elevated concentrations of FeSO4 [14].

A. fumigatus and invasive aspergillosis

Aspergillus fumigatus is the most common cause of airborne fungal infections worldwide [1]. The most serious disease caused by A. fumigatus is invasive aspergillosis (IA). IA occurs most commonly when susceptible individuals inhale conidia. These may germinate in the lung resulting in hyphal outgrowth that penetrates epithelial and endothelial barriers [1,2]. The infection can spread hematogenously resulting in foci of fungal growth in other organs, particularly the brain. Although some improvement in patient outcomes has been achieved in the past decade [3], mortality rates still average 50% [1] and possibly higher with drug-resistant strains [4]. Immunosuppressed patients are at greatest risk to develop IA, particularly those with profound and prolonged neutropenia [2]. Iron overload in patients is an independent risk factor for IA [5]. There is evidence that polymorphisms in innate immune system genes influence host susceptibility to IA Current Opinion in Microbiology 2013, 16:692–699

Siderophore-mediated iron uptake

Siderophores are ferric iron chelators secreted by a diverse range of bacteria and fungi. With iron formation constants of 1020–1050 [15], desferri-siderophores are able to sequester iron from iron-binding proteins of the host such as transferrin [16,17]. A. fumigatus produces four hydroxamate siderophores all based on N5-hydroxy-N5acyl-L-ornithine [18]: ferricrocin and hydroxyferricrocin are predominantly intracellular siderophores found in hyphae and conidia, respectively, and fusarinine C (FSC) and its N-acetylated derivative, N0 , N00 , N000 -triacetylfusarinine C (TAFC) are secreted [19]. Ferrisiderophore uptake is mediated by specific membrane transporters. In the cell, the trilactone ring of TAFC is hydrolyzed by EstB generating a complex with decreased entropy and therefore, decreased stability [20]. Deficiency of a putative vacuolar iron transporter cccA resulted in the accumulation of TAFC and FSC breakdown products in www.sciencedirect.com

Iron uptake in the virulence of Aspergillus fumigatus Moore 693

the cytosol indicating that iron is transferred to intracellular stores before the siderophores are recycled [14]. A siderophore-like ferric iron-binding molecule called hexadehydroastechrome (HAS) was recently isolated from A. fumigatus using a combination of microarray and comparative metabolomics [21]. HAS is a product of a non-ribosomal peptide synthetase (NRPS) pathway regulated by LaeA, a global regulator of secondary metabolism. Interestingly, knockout of HAS biosynthesis did not decrease virulence in a mouse model of IA but overexpression increased virulence accompanied by the accumulation of the HAS intermediate, terezine. The function of HAS is still unknown; however, the authors postulate that due to its structural similarity and requirement for iron, the HAS and siderophore pathways may interact [21].

Siderophore biosynthesis is essential for A. fumigatus virulence Elimination of hydroxamate siderophore production resulted in an avirulent strain [12,22] whereas deficiency in either intracellular or extracellular siderophores attenuated virulence [23]. The survival of A. fumigatus conidia in macrophages is also dependent on siderophore production [24]. Moreover, in a recent study of gene expression in A. fumigatus internalized by respiratory epithelial cells, sidA was amongst the genes showing the greatest increase in expression [25]. Hence, uptake of A. fumigatus by both professional and non-professional phagocytes imposes an iron limitation that triggers siderophore biosynthesis. Intracellular survival could provide a source of infection even in the presence of an immune response. The siderophore biosynthetic pathway in A. fumigatus has been elucidated by Haas and colleagues [19] and a schematic is shown in Figure 1a. Recent developments related to specific gene products in this pathway and their regulation are summarized below.

SidL is involved in FC acetylation The first step in ferricrocin synthesis is by acetylation of N5-hydroxyornithine. Blatzer et al. [26] have shown a GNAT-type acetyltransferase encoded by sidL catalyzes this step under iron-sufficient conditions, perhaps to ensure sufficient iron storage capacity. However, the lack of regulation by SreA as well as residual ferricrocin synthesis in the DsidL mutant suggests that an uncharacterized iron-regulated acetyltransferase is required when iron is limiting [26].

sidI, sidH and sidF are imported into peroxisomes The biosynthesis of TAFC poses a problem in that the presence of the desferri-siderophore in the cytoplasm has the potential to chelate ferric iron released from imported ferrated TAFC, thereby creating a futile cycle. Recent www.sciencedirect.com

work by Grundlinger et al. has revealed one solution to this problem: at least some TAFC biosynthetic enzymes are sequestered in peroxisomes, specifically, SidI, SidH (see below and Figure 1) and Sid F [27]. GFP-tagged proteins were found in punctate bodies throughout the cytoplasm of wild type A. fumigatus cells. In addition, A. nidulans mutants in peroxisomal biogenesis ( pex) showed decreased growth and conidiation on iron-limited media. Peroxisomal targeting signals (PTS1 [28] and PTS2) were identified in the SidI, SidH and SidF as well as in orthologues in other fungal species suggesting that peroxisomes may be the site of siderophore biosynthesis in many fungi [27].

MirB siderophore transporter In A. nidulans, the SIT transporters MirA and MirB are specific for enterobactin and TAFC uptake, respectively [29]. A study of A. fumigatus MirB showed that it transported TAFC, ferricrocin and coprogen but not ferrichrysin suggesting that MirB recognizes the L-cis structure around the iron centre and not amino acid linkages in the siderophores [30]. As with other fungal siderophore transporters, the 7th loop Tyr was required for function. MirB was found in vesicles that were concentrated in hyphal tips (Figure 2) and localization was influenced by the level of stored iron rather than by TAFC [30]. Because these receptors are absent from the human host, siderophore-antibiotic complexes may target fungal growth by a ‘Trojan Horse’ mechanism [31]. Chemical synthesis of the fusarinine C scaffold will facilitate the generation of novel hydroxamate siderophore derivatives [32].

Transcriptional regulation of siderophoremediated iron acquisition Not only does obtaining adequate iron present a challenge to pathogens, iron metabolism must be highly regulated because free ferrous iron in the cytosol has the potential to damage cellular macromolecules via Fenton/Haber-Weiss-mediated generation of oxygen radicals [33]. The following section highlights recent work on transcription factors that have a direct or indirect effect on iron metabolism in A. fumigatus (Figure 1B). HapX and SreA

The GATA factor SreA is a transcriptional repressor of iron acquisition genes in A. fumigatus [34] and in A. nidulans, SreA and the CCAAT-binding transcription factor, HapX have been shown to regulate iron homeostasis via a negative feedback loop [19,35,36]. HapX represses iron utilization pathways to spare iron in low iron conditions. A hapX deletion strain was avirulent in two mouse infection models of IA [35] (whereas DsreA mutants retain virulence [34]). Interestingly, wild type levels of fusarinine C were produced by DhapX though TAFC levels were greatly reduced [35] suggesting that fusarinine C cannot substitute for TAFC in vivo as a source of iron, perhaps because of its lower stability in Current Opinion in Microbiology 2013, 16:692–699

694 Growth and development: eukaryotes

Figure 1

(a)

Nucleus

ER

SreA

Golgi

HapX Srb A

*SrbA HacA

Ire A

MpkA AcuM

?

MirB

*hacA mRNA HacA-independent mevalonate pathway

PrtT

Hmg1

Ergosterol acetyl CoA

mevalonate

C N5-acetyl− N5-hydroxyornithine

mevalonate I

Peroxisome

TAFC

Ferricrocin

mevalonyl CoA H anhydromevalonyl CoA

?

L

Fe-TAFC

Fe3+ MirB

Fe-TAFC

acetyl CoA TAFC

N5-hydroxyornithine

F N5-anhydromevalonyl CoA-N5-hydroxyornithine

A Fusarinine C

L-ORNITHINE

Fe-TAFC

D

Fusarinine C G

Low iron

SreA off

-

Fe-TAFC

?

?

(b)

MirB

TAFC

Low oxygen

HapX on

+

SrbA on

+

-

? PrtT on

AcuM Siderophore Synthesis/Uptake on

Iron utilization pathways off

IreA ± HacA on

iron replete

UPR

MpkA on Low iron Current Opinion in Microbiology

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Iron uptake in the virulence of Aspergillus fumigatus Moore 695

body fluids [37]. HapX was shown to regulate amino acid biosynthetic genes thereby ensuring adequate L-ornithine levels for siderophore biosynthesis under iron limitation: L-ornithine levels declined 10-fold in a DhapX mutant [35]. In the SreA regulon, Yasmin et al. identified two novel genes required for TAFC (but not ferricrocin) biosynthesis: sidI and sidH encode the enzymes that together catalyze the conversion of mevalonate to anhydromevalonyl CoA [38]. This report also linked siderophore and ergosterol biosynthesis by showing that blocking mevalonate synthesis with lovastatin (which inhibits 3-hydroxy-3-methyl-glutaryl-CoA reductase, Hmg1) caused a 40% decline in TAFC levels. Iron limitation resulted in up-regulation of hmg1; however, ergosterol levels decreased because of the increased diversion of mevalonate to TAFC biosynthesis. sidI or sidH knockout strains were avirulent in a mouse IA model. These results indicate that statins may be useful antifungal agents as they inhibit both TAFC and ergosterol synthesis by blocking the production of the key intermediate, mevalonate [38]. Transcriptomic and proteomic analyses of A. fumigatus grown in a chemostat under oxygen limitation confirmed that hypoxia resulted in the up-regulation of genes involved in both ergosterol metabolism and iron metabolism [39] consistent with the requirement for iron in sterol synthesis. Transcription factors that act on or in conjunction with HapX and SreA SrbA

How are iron uptake and sterol metabolism co-regulated in A. fumigatus? In hypoxia, decreased ergosterol is sensed by a sterol responsive element binding protein (SREBP) in the ER membrane; SREBPs are activated by proteolytic cleavage to release the N-terminus that acts as the transcription factor. In A. fumigatus, the SREBP is SrbA and it is required for hypoxia adaptation and virulence [40]. Blatzer et al. found that in addition to genes involved in glycolysis and ribosome biogenesis, SrbA upregulated ergosterol biosynthesis and iron acquisition genes in hypoxia. Moreover, DsrbA mutants had only 10% of wild type TAFC levels and deletion of sreA did not restore TAFC levels [41]. Linde et al. [42] used a systems biology approach to generate a model of iron regulation that predicted SrbA binding targets such as hapX. Using surface plasmon resonance, they confirmed binding of purified SrbA to hapX, hemA and srbA promoters [42].

Vodisch et al. [43] compared the proteome of A. fumigatus in hypoxia compared to normoxia. Cells grown in an oxygen-controlled, glucose-limited chemostat accumulated heme, iron and mitochondrial respiratory proteins to compensate for the low oxygen levels. In addition, the heme may also provide the necessary cofactor for flavohemoprotein, the protein found to be most strongly upregulated protein in hypoxic cells. The authors suggest that flavohemoprotein may detoxify reactive nitrogen intermediates such as nitric oxide, produced under hypoxic conditions [43–45]. AcuM

Liu et al. created deletion mutants of 11 candidate A. fumigatus transcription factor genes and evaluated these for altered virulence in Galleria mellonella larvae [46]. The zinc cluster transcription factor AcuM was required for full virulence in both G. mellonella and a murine infection model, and expression studies showed that in addition to gluconeogenesis, AcuM regulated genes involved in iron acquisition, including RIA and siderophore genes. Up-regulation occurred via repression of sreA though the nature of the interaction between AcuM and sreA is presently unknown [46]. PrtT

PrtT is a C6 zinc finger transcription factor that regulates protease expression in A. fumigatus. Studies of a DprtT mutant revealed that PrtT has other targets including genes involved in iron uptake, sterol biosynthesis and secondary metabolite biosynthesis [47]. In the DprtT mutant, the 30-fold decreased expression of genes involved in siderophore biosynthesis and uptake (sidA, mirB and sit1) was much greater than the observed downregulation of secreted protease genes (2–14 fold). Unexpectedly, the prtT mutant did not have greater sensitivity to iron limitation compared to wild type; siderophore secretion and virulence were maintained. qPCR analysis showed up-regulation of hapX and a concomitant downregulation of sreA in the prtT mutant indicating that compensatory mechanisms were activated upon iron limitation. The authors concluded that PrtT operates to increase iron acquisition under iron sufficiency and that it affects hapX and sreA expression independently of AcuM and SrbA [47]. Transcription factors that appear to work independently from HapX and SreA HacA/IreA

Accumulation of unfolded protein response during ER stress triggers the unfolded protein response (UPR).

Figure Legend 1 (a) Schematic illustrating the location of the proteins involved in siderophore biosynthesis and uptake in hyphae of A. fumigatus. Siderophore biosynthetic enzymes are denoted by letters inside block white arrows. Transcription factors implicated in siderophore regulation are listed in the nucleus. * indicates the active form of the molecule and ‘?’ indicates interactions that have not been experimentally confirmed. (b) Transcription factors shown to be involved in regulating siderophore-mediated iron acquisition in A. fumigatus. ‘+’ and ‘’ indicate activation and repression, respectively. Effects on ergosterol biosynthesis not indicated in this figure but are described in the text. UPR is Unfolded Protein Response. www.sciencedirect.com

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Figure 2

possibly to maintain polyamine levels. Thus, MpkA may oppose the effect of HapX and SreA to limit the diversion of cellular resources to siderophore biosynthesis.

(a)

To expand the list of genes regulated by MpkA, a paired end mRNA-seq study was undertaken by Muller et al. and the results compared to published proteome and transcriptome studies [51]. Their data confirmed that in A. fumigatus, deletion of the mpkA gene resulted in upregulation of genes involved in iron acquisition. The RNA-seq methodology yielded 185 new MpkA-regulated transcripts of which 50% were conserved in other Aspergillus species [51].

(b)

(c)

Future directions Current Opinion in Microbiology

The ferrisiderophore transporter MirB is localized to punctate bodies in the hyphal tips of A. fumigatus. Micrographs of conidia (a), a germling (b) and hyphus (c) of A. fumigatus. From left to right are the DIC, red channel showing MirB immunofluoresence and merged images. Images were obtained from cells grown in iron-limited medium as described in [30].

Transcriptional regulation of the UPR is accomplished by HacA, a bZip transcription factor activated by the ER membrane protein, IreA. In A. fumigatus, Feng et al. [48] found 1305 transcripts regulated by IreA and HacA of which 914 were regulated by IreA independently of HacA. DireA mutants were avirulent in a mouse IA model whereas DhacA strains retained partial virulence; both strains had decreased ergosterol content. The loss of virulence could be attributed in part to lower expression in the mutants of genes involved in siderophore biosynthesis, for example, sidA. IreA may indirectly affect iron metabolism via interaction with SrbA; the authors postulate that because both SrbA and IreA are ER membrane proteins that regulate ergosterol metabolism, there may be cross talk between these two pathways [48,49]. MpkA

A mitogen-activated protein kinase (MAP kinase) is encoded by the mpkA gene in A. fumigatus. Jain et al. studied the transcriptional response in an mpkA knockout strain and found a link between siderophore biosynthesis and MpkA [50]. Iron starvation resulted in MpkA phosphorylation and translocation to the nucleus where it affected target genes including the down-regulation of sidA. Because expression of hapX and sreA were unaffected in the DmpkA mutant, the effect on siderophore biosynthesis was deemed independent of HapX and SreA. The mechanism by which siderophore secretion is reduced by MpkA even when iron is limiting appears to be related to its effect on reducing the ornithine pool, Current Opinion in Microbiology 2013, 16:692–699

Fungal iron acquisition and the diagnosis and treatment of invasive aspergillosis 68 Ga-siderophores for diagnosis

Early diagnosis is essential in the treatment of IA and recent studies in animals have shown that siderophores complexed to 68Ga may be taken up by A. fumigatus in vivo and detected by positron emission tomography (PET) [37,52]. mPET imaging of 68Ga-TAFC and 68Ga-ferrioxamine E was able to discriminate between mild versus severe infection in rats and correlated with the CT scans of the same animals [52]. The short half-life of 68Ga (68 min) and its existing use in nuclear medicine make 68 Ga-siderophore complexes promising diagnostic tools for early detection of IA. Iron chelators in combination drug therapy

The importance of iron uptake to the survival of A. fumigatus has prompted the investigation of iron chelators for the treatment of IA. Ibrahim et al. used deferasirox, a drug approved for treatment of iron overload, in combination with liposomal amphotericin B (LAmB) in a mouse model of IA [53]. Only the mice receiving LAmB + deferasirox had a significant decrease in fungal burden compared to placebo and this was manifested as improved survival in the combination group [53]. However, triple therapy (micafungin + LAmB + deferasirox) of mice infected with A. fumigatus did not enhance survival compared to monotherapy [54]. Addition of deferasirox to micafungin actually worsened survival compared to micafungin alone [54]. Thus, although the use of iron chelators in combination with conventional antifungals shows promise, additional work is required to determine safe and effective treatment regimens. Interactions between A. fumigatus and Pseudomonas aeruginosa in vitro reveal the complexity of polymicrobial infections

In vivo work to date has been carried out by exposing experimental animals to pure cultures of a single microbe. However, patients are often colonized by more than one pathogen. For example, the lungs of patients with cystic fibrosis may contain a mixed microbial community www.sciencedirect.com

Iron uptake in the virulence of Aspergillus fumigatus Moore 697

including the bacterium Pseudomonas aeruginosa and A. fumigatus. Using a novel approach combining MALDITOF and MALDI-FT-ICR along with MS/MS network analysis, Moree et al. were able to visualize and identify the small molecules secreted by co-cultures of P. aeruginosa and A. fumigatus [55]. They showed that A. fumigatus metabolized phenazines secreted by P. aeruginosa to 1-hydroxyphenazine and that 1-hydroxyphenazine stimulated secretion of TAFC by A. fumigatus by an unknown mechanism [55]. Combining MALDI-MS and MS/MS to identify metabolites produced during interactions between A. fumigatus and other microbes in mixed communities and A. fumigatus with host cells will yield new insights into iron homeostasis in situ.

Conclusion The central role played by iron in A. fumigatus is highlighted by the expanding list of transcriptional regulators that have been shown to affect iron uptake and utilization. Future work will no doubt untangle the levels of complexity in regulation of iron metabolism and further expand our understanding of how proteins involved in iron acquisition are compartmentalized in the fungal cell. Because these pathways do not exist in the host, exciting research is underway to exploit siderophore-mediated iron uptake for development of novel diagnostic and antifungal agents.

Acknowledgements The financial support of the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. Thanks to L.J. Pinto and C.R. Roberts for critical review of this manuscript and to L. Pinto for obtaining the images in Figure 2. I apologize to authors whose work was not cited due to space limitations.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Dagenais TR, Keller NP: Pathogenesis of Aspergillus fumigatus in invasive Aspergillosis. Clin Microbiol Rev 2009, 22:447-465.

2.

Lass-Florl C, Roilides E, Loffler J, Wilflingseder D, Romani L: Minireview: host defence in invasive aspergillosis. Mycoses 2013.

3.

Lewis RE, Lortholary O, Spellberg B, Roilides E, Kontoyiannis DP, Walsh TJ: How does antifungal pharmacology differ for mucormycosis versus aspergillosis? Clin Infect Dis 2012, 54(Suppl. 1):S67-S72.

4. 

allogeneic hematopoietic stem cell transplantation. Cancer 2007, 110:1303-1306. 6.

Sainz J, Lupianez CB, Segura-Catena J, Vazquez L, Rios R, Oyonarte S, Hemminki K, Forsti A, Jurado M: Dectin-1 and DC-SIGN polymorphisms associated with invasive pulmonary Aspergillosis infection. PLoS ONE 2012, 7:e32273.

7.

Cunha C, Rodrigues F, Zelante T, Aversa F, Romani L, Carvalho A: Genetic susceptibility to aspergillosis in allogeneic stem-cell transplantation. Med Mycol 2011, 49(Suppl. 1):S137-S143.

8.

van der Velden WJ, Blijlevens NM, Donnelly JP: Genetic variants and the risk for invasive mould disease in immunocompromised hematology patients. Curr Opin Infect Dis 2011, 24:554-563.

9.

Thompson GR 3rd, Patterson TF: Pulmonary aspergillosis: recent advances. Semin Respir Crit Care Med 2011, 32:673-681.

10. Kasvosve I: Effect of ferroportin polymorphism on iron homeostasis and infection. Clin Chim Acta 2013, 416:20-25. 11. Bullen JJ: The significance of iron in infection. Rev Infect Dis 1981, 3:1127-1138. 12. Schrettl M, Bignell E, Kragl C, Joechl C, Rogers T, Arst HN Jr, Haynes K, Haas H: Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J Exp Med 2004, 200:1213-1219. 13. Blatzer M, Binder U, Haas H: The metalloreductase FreB is involved in adaptation of Aspergillus fumigatus to iron starvation. Fungal Genet Biol 2011, 48:1027-1033. 14. Gsaller F, Eisendle M, Lechner BE, Schrettl M, Lindner H, Mueller D, Geley S, Haas H: The interplay between vacuolar and siderophore-mediated iron storage in Aspergillus fumigatus. Metallomics 2012, 4:1262-1270. 15. Miethke M: Molecular strategies of microbial iron assimilation: from high-affinity complexes to cofactor assembly systems. Metallomics 2013, 5:15-28. 16. Hissen AH, Chow JM, Pinto LJ, Moore MM: Survival of Aspergillus fumigatus in serum involves removal of iron from transferrin: the role of siderophores. Infect Immun 2004, 72:1402-1408. 17. Hissen AH, Moore MM: Site-specific rate constants for iron acquisition from transferrin by the Aspergillus fumigatus siderophores N0 , N00 , N000 -triacetylfusarinine C and ferricrocin. J Biol Inorg Chem 2005, 10:211-220. 18. Budzikiewicz H: Siderophores from bacteria and from fungi. In Iron Uptake and Homeostasis in Microorganisms. Edited by Cornelis P, Andrews SC. 2010. 19. Haas H: Iron — a key nexus in the virulence of Aspergillus fumigatus. Front Microbiol 2012, 3:28. 20. Kragl C, Schrettl M, Abt B, Sarg B, Lindner HH, Haas H: EstBmediated hydrolysis of the siderophore triacetylfusarinine C optimizes iron uptake of Aspergillus fumigatus. Eukaryot Cell 2007, 6:1278-1285. 21. Yin WB, Baccile JA, Bok JW, Chen Y, Keller NP, Schroeder FC: A  nonribosomal peptide synthetase-derived iron(III) complex from the pathogenic fungus Aspergillus fumigatus. J Am Chem Soc 2013, 135:2064-2067. Comparative metabolomics was effectively used in combination with knockout and overexpression strains to identify novel NRPS products in A. fumigatus.

van der Linden JW, Snelders E, Kampinga GA, Rijnders BJ, Mattsson E, Debets-Ossenkopp YJ, Kuijper EJ, Van Tiel FH, Melchers WJ, Verweij PE: Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007–2009. Emerg Infect Dis 2011, 17:1846-1854. A prospective study of A. fumigatus isolates in The Netherlands found that 5% of isolates were multiazole resistant. Of concern was the elevated (almost 90%) case-fatality rate in the patients infected with resistant strains warranting further study with a larger cohort.

22. Hissen AH, Wan AN, Warwas ML, Pinto LJ, Moore MM: The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence. Infect Immun 2005, 73:5493-5503.

5.

24. Schrettl M, Ibrahim-Granet O, Droin S, Huerre M, Latge JP, Haas H: The crucial role of the Aspergillus fumigatus siderophore system in interaction with alveolar macrophages. Microbes Infect 2010, 12:1035-1041.

Kontoyiannis DP, Chamilos G, Lewis RE, Giralt S, Cortes J, Raad II, Manning JT, Han X: Increased bone marrow iron stores is an independent risk factor for invasive aspergillosis in patients with high-risk hematologic malignancies and recipients of

www.sciencedirect.com

23. Schrettl M, Bignell E, Kragl C, Sabiha Y, Loss O, Eisendle M, Wallner A, Arst HN Jr, Haynes K, Haas H: Distinct roles for intraand extracellular siderophores during Aspergillus fumigatus infection. PLoS Pathog 2007, 3:1195-1207.

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25. Oosthuizen JL, Gomez P, Ruan J, Hackett TL, Moore MM,  Knight DA, Tebbutt SJ: Dual organism transcriptomics of airway epithelial cells interacting with conidia of Aspergillus fumigatus. PLoS ONE 2011, 6:e20527. The transcriptomes of A. fumigatus conidia internalized by airway epithelial cells and the airway cells were profiled in parallel in this study, the first dual transcriptome study of A. fumigatus and host cells. Siderophore biosynthetic enzymes were upregulated in internalized A. fumigatus. 26. Blatzer M, Schrettl M, Sarg B, Lindner HH, Pfaller K, Haas H: SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin. Appl Environ Microbiol 2011, 77:4959-4966. 27. Grundlinger M, Yasmin S, Lechner BE, Geley S, Schrettl M,  Hynes M, Haas H: Fungal siderophore biosynthesis is partially localized in peroxisomes. Mol Microbiol 2013, 88:862-875. Using A. nidulans peroxisome biogenesis mutants and immunolocalization of GFP-tagged proteins, the authors provided the first report showing peroxisomal localization of siderophore biosynthetic enzymes. They also showed that this role for peroxisomes is likely widespread in fungi. 28. Beck J, Ebel F: Characterization of the major Woronin body protein HexA of the human pathogenic mold Aspergillus fumigatus. Int J Med Microbiol 2013, 303:90-97. 29. Haas H, Schoeser M, Lesuisse E, Ernst JF, Parson W, Abt B, Winkelmann G, Oberegger H: Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C. Biochem J 2003, 371:505-513. 30. Raymond-Bouchard I, Carroll CS, Nesbitt JR, Henry KA, Pinto LJ, Moinzadeh M, Scott JK, Moore MM: Structural requirements for the activity of the MirB ferrisiderophore transporter of Aspergillus fumigatus. Eukaryot Cell 2012, 11:1333-1344. 31. Wencewicz TA, Long TE, Moellmann U, Miller MJ: Trihydroxamate siderophore-fluoroquinolone conjugates are selective sideromycin antibiotics that target Staphylococcus aureus. Bioconjug Chem 2013, 24:473-486. 32. Bertrand S, Duval O, Helesbeux J, Larcher G, Richomme P: Synthesis of the trans-fusarinine scaffold. Tetrahedron Lett 2010, 51:2119-2122. 33. Halliwell B, Gutteridge JM: The importance of free radicals and catalytic metal ions in human diseases. Mol Aspects Med 1985, 8:89-193. 34. Schrettl M, Kim HS, Eisendle M, Kragl C, Nierman WC, Heinekamp T, Werner ER, Jacobsen I, Illmer P, Yi H et al.: SreA-mediated iron regulation in Aspergillus fumigatus. Mol Microbiol 2008, 70:27-43. 35. Schrettl M, Beckmann N, Varga J, Heinekamp T, Jacobsen ID, Jochl C, Moussa TA, Wang S, Gsaller F, Blatzer M et al.: HapXmediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus. PLoS Pathog 2010, 6:e1001124. 36. Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S, Thon M, Kniemeyer O, Abt B, Seeber B, Werner ER et al.: Interaction of HapX with the CCAAT-binding complex – a novel mechanism of gene regulation by iron. EMBO J 2007, 26:3157-3168. 37. Petrik M, Haas H, Schrettl M, Helbok A, Blatzer M, Decristoforo C: In vitro and in vivo evaluation of selected 68Ga-siderophores for infection imaging. Nucl Med Biol 2012, 39:361-369. 38. Yasmin S, Alcazar-Fuoli L, Grundlinger M, Puempel T, Cairns T,  Blatzer M, Lopez JF, Grimalt JO, Bignell E, Haas H: Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus. Proc Natl Acad Sci U S A 2012, 109:E497-E504. The authors found that the key intermediate in the siderophore and ergosterol biosynthetic pathways is mevalonate, and identified the two genes encoding mevalonate synthesis in A. fumigatus. This paper elucidates the critical biochemical link between these two pathways that are essential for virulence and indicate that statin drugs not only inhibit sterol production but also the synthesis of siderophores derived from mevalonate. 39. Barker BM, Kroll K, Vodisch M, Mazurie A, Kniemeyer O, Cramer RA: Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygencontrolled fermenter. BMC Genomics 2012, 13 62-2164-13-62. Current Opinion in Microbiology 2013, 16:692–699

40. Willger SD, Puttikamonkul S, Kim KH, Burritt JB, Grahl N, Metzler LJ, Barbuch R, Bard M, Lawrence CB, Cramer RA Jr: A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus. PLoS Pathog 2008, 4:e1000200. 41. Blatzer M, Barker BM, Willger SD, Beckmann N, Blosser SJ,  Cornish EJ, Mazurie A, Grahl N, Haas H, Cramer RA: SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus. PLoS Genet 2011, 7:e1002374. This is the first paper to reveal a role for the sterol regulatory element binding protein, SrbA in regulating iron acquisition in low iron and hypoxic conditions, and links SrbA activity to HapX and SreA. 42. Linde J, Hortschansky P, Fazius E, Brakhage AA, Guthke R, Haas H: Regulatory interactions for iron homeostasis in Aspergillus fumigatus inferred by a Systems Biology approach. BMC Syst Biol 2012, 6 6-0509-6-6. 43. Vodisch M, Scherlach K, Winkler R, Hertweck C, Braun HP, Roth M, Haas H, Werner ER, Brakhage AA, Kniemeyer O: Analysis of the Aspergillus fumigatus proteome reveals metabolic changes and the activation of the pseurotin A biosynthesis gene cluster in response to hypoxia. J Proteome Res 2011, 10:2508-2524. 44. Zhou S, Fushinobu S, Kim SW, Nakanishi Y, Wakagi T, Shoun H: Aspergillus oryzae flavohemoglobins promote oxidative damage by hydrogen peroxide. Biochem Biophys Res Commun 2010, 394:558-561. 45. Schinko T, Berger H, Lee W, Gallmetzer A, Pirker K, Pachlinger R, Buchner I, Reichenauer T, Guldener U, Strauss J: Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism. Mol Microbiol 2010, 78:720-738. 46. Liu H, Gravelat FN, Chiang LY, Chen D, Vanier G, Ejzykowicz DE, Ibrahim AS, Nierman WC, Sheppard DC, Filler SG: Aspergillus fumigatus AcuM regulates both iron acquisition and gluconeogenesis. Mol Microbiol 2010, 78:1038-1054. 47. Hagag S, Kubitschek-Barreira P, Neves GW, Amar D, Nierman W, Shalit I, Shamir R, Lopes-Bezerra L, Osherov N: Transcriptional and proteomic analysis of the Aspergillus fumigatus DeltaprtT protease-deficient mutant. PLoS ONE 2012, 7:e33604. 48. Feng X, Krishnan K, Richie DL, Aimanianda V, Hartl L, Grahl N, Powers-Fletcher MV, Zhang M, Fuller KK, Nierman WC et al.: HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus. PLoS Pathog 2011, 7:e1002330. 49. Willger SD, Cornish EJ, Chung D, Fleming BA, Lehmann MM, Puttikamonkul S, Cramer RA: Dsc orthologs are required for hypoxia adaptation, triazole drug responses, and fungal virulence in Aspergillus fumigatus. Eukaryot Cell 2012, 11:1557-1567. 50. Jain R, Valiante V, Remme N, Docimo T, Heinekamp T,  Hertweck C, Gershenzon J, Haas H, Brakhage AA: The MAP kinase MpkA controls cell wall integrity, oxidative stress response, gliotoxin production and iron adaptation in Aspergillus fumigatus. Mol Microbiol 2011, 82:39-53. The authors provide the first evidence that iron acquisition pathways are downregulated by MAP kinase even under iron limitation suggesting that this pathway acts as a counterbalance to the HapX/SreA system. 51. Muller S, Baldin C, Groth M, Guthke R, Kniemeyer O, Brakhage AA, Valiante V: Comparison of transcriptome technologies in the pathogenic fungus Aspergillus fumigatus reveals novel insights into the genome and MpkA dependent gene expression. BMC Genomics 2012, 13 519-2164-13-519. 52. Petrik M, Franssen GM, Haas H, Laverman P, Hortnagl C,  Schrettl M, Helbok A, Lass-Florl C, Decristoforo C: Preclinical evaluation of two 68Ga-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging. Eur J Nucl Med Mol Imaging 2012, 39:1175-1183. The radiolabelled siderophores were taken up in vivo and the method was able distinguish between mild and severe infections in rats. This work shows the clinical applicability of this new method for diagnosing invasive aspergillosis. www.sciencedirect.com

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53. Ibrahim AS, Gebremariam T, French SW, Edwards JE Jr, Spellberg B: The iron chelator deferasirox enhances liposomal amphotericin B efficacy in treating murine invasive pulmonary aspergillosis. J Antimicrob Chemother 2010, 65:289-292. 54. Ibrahim AS, Gebremariam T, Luo G, Fu Y, French SW, Edwards JE Jr, Spellberg B: Combination therapy of murine mucormycosis or aspergillosis with iron chelation, polyenes, and echinocandins. Antimicrob Agents Chemother 2011, 55:1768-1770.

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55. Moree WJ, Phelan VV, Wu C, Bandeira N, Cornett DS, Duggan BM,  Dorrestein PC: Interkingdom metabolic transformations captured by microbial imaging mass spectrometry. Proc Natl Acad Sci U S A 2012, 109:13811-13816. The powerful MALDI-TOF and MALDI-FT-IR methodology combined with MS/MS analysis allowed the authors to visualize and identify siderophores and other metabolites secreted at the interface between A. fumigatus and the bacterium, Pseudomonas aeruginosa. This work highlights the complexity of microbial metabolism, and competition for iron, in mixed cultures.

Current Opinion in Microbiology 2013, 16:692–699