Mevalonate governs interdependency of PNAS PLUS and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus

Sabiha Yasmina,1,2,3, Laura Alcazar-Fuolib,1, Mario Gründlingera,1, Thomas Puempelc, Timothy Cairnsb, Michael Blatzera, Jordi F. Lopezd, Joan O. Grimaltd, Elaine Bignellb, and Hubertus Haasa,3

aDivision of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria; bMicrobiology Section, Imperial College London, London SW7 2AZ, United Kingdom; cDepartment of Microbiology, University of Innsbruck, A-6020 Innsbruck, Austria; and dDepartment of Environmental Chemistry, Institute of Environmental Assessment and Water Studies, Jordi Girona, 08034 Barcelona, Catalonia Spain

Edited by Joan Wennstrom Bennett, Rutgers University, New Brunswick, NJ, and approved October 19, 2011 (received for review April 25, 2011) Aspergillus fumigatus is the most common airborne fungal path- (N5-anhydromevalonyl-N5-hydroxyornithine) was observed first by ogen for humans. In this mold, iron starvation induces production Diekmann and Zähner (7) as a degradation product of fusarinine. of the siderophore triacetylfusarinine C (TAFC). Here we demon- Subsequent studies (8) demonstrated that anhydromevalonic acid strate a link between TAFC and ergosterol biosynthetic pathways, is synthesized from mevalonic acid and that the CoA derivative of which are both critical for virulence and treatment of fungal infec- anhydromevalonic acid, along with N5-hydroxyornithine, forms tions. Consistent with mevalonate being a limiting prerequisite for fusarinine (9). Thus far we have populated the proposed bio- TAFC biosynthesis, we observed increased expression of 3-hy- synthetic scheme (Fig. 1) with five genes encoding respective droxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron A. fumigatus siderophore biosynthetic enzymes (3, 5). However, starvation, reduced TAFC biosynthesis following lovastatin-medi- candidate genes for enzymes converting mevalonate to anhy- ated Hmg1 inhibition, and increased TAFC biosynthesis following dromevalonyl-CoA have not yet been identified in any fungal fi Hmg1 overexpression. We identi ed enzymes, the acyl-CoA ligase species. Moreover, the regulatory link between siderophore and SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of fi ergosterol biosynthesis remains elusive. MICROBIOLOGY mevalonate and TAFC, de ciency of which under iron starvation In A. fumigatus, iron acquisition, including biosynthesis and impaired TAFC biosynthesis, growth, oxidative stress resistance, uptake of siderophores, is repressed by iron via the GATA-type and murine virulence. Moreover, inactivation of these enzymes transcription factor siderophore repressor A (SreA), and ge- alleviated TAFC-derived biosynthetic demand for mevalonate, as fi evidenced by increased resistance to lovastatin. Concordant with nome-wide microarray analysis identi ed 49 SreA-controlled genes (10). Notably, SreA must be distinguished from the basic bilateral demand for mevalonate, iron starvation decreased the – – ergosterol content and composition, a phenotype that is mitigated helix loop helix type transcription factor SrbA, a recently iden- fi in TAFC-lacking mutants. ti ed regulator of ergosterol biosynthesis in A. fumigatus (11). In the current study, we identified the enzymes connecting ergos- siderophore | isoprenoide | statins terol and siderophore biosynthetic pathways within the SreA regulon and elucidated the regulatory link between these path- ost-imposed iron limitation necessitates elaborate iron-ac- ways. This link might be crucial for optimization of treatment of Hquisition mechanisms in pathogenic microbes, whereby fun- infections caused by siderophore-producing fungi. fi gal agents of human disease favor high-af nity reductive iron Results uptake and chaperone-assisted iron assimilation (1). The latter Iron Starvation Up-Regulates Expression of the Gene Encoding 3- mechanism is favored by inhaled spores of the saprobic mold – Aspergillus fumigatus, the most common airborne fungal patho- Hydroxy-3-Methyl-Glutaryl CoA Reductase, hmg1. Mevalonate is a key gen of humans and an agent of life-threatening invasive disease metabolite in the biosynthesis of sterols such as fungal ergosterol in immunocompromised patients (2). In the absence of direct and a variety of nonsterol isoprenoid derivatives (Fig. 1). Mevalo- utilization mechanisms for human iron sources such as heme, nate is synthesized from 3-hydroxy-3-methylglutaryl-CoA (HMG- ferritin, or transferrin, A. fumigatus employs both reductive iron CoA) catalyzed by HMG-CoA reductase, the rate-limiting enzyme assimilation (RIA) and siderophore (low-molecular-mass ferric of sterol biosynthesis. Subsequently, mevalonate is phosphorylated iron chelators)-mediated iron uptake during murine infection (3). by mevalonate kinase. Siderophore biosynthesis is a -specific, virulence-essential biosynthetic process and a significant prospect for rationalized therapeutic intervention. Author contributions: S.Y., L.A.F., M.G., and H.H. designed research; S.Y., L.A.F., M.G., T.P., A. fumigatus produces three major hydroxamate-type side- T.C., M.B., and J.F.L. performed research; T.P., J.O.G., E.B., and H.H. contributed new rophores for iron uptake: extracellular triacetylfusarinine C reagents/analytic tools; S.Y., L.A.F., M.G., T.P., T.C., J.F.L., J.O.G., E.B., and H.H. analyzed (TAFC), which removes iron from transferrin (4), and hyphal data; and S.Y., E.B., and H.H. wrote the paper. ferricrocin (FC) and conidial hydroxyferricrocin (HFC), which The authors declare no conflict of interest. facilitate intracellular sequestration and distribution of iron This article is a PNAS Direct Submission. (5, 6). Genetic abrogation of the entire siderophore system Freely available online through the PNAS open access option. renders A. fumigatus avirulent in a murine model of invasive Data deposition: Gene/protein sequences for SidI (AFUA_1G17190) and sidH (AFUA_ pulmonary aspergillosis; inactivation of either extra- or intra- 3G03410 are found at http://www.cadre-genomes.org.uk/. cellular siderophore biosynthesis results in partial attenuation of 1S.Y., L.A.F., and M.G. contributed equally to this work. virulence (3, 5). 2Present address: Pakistan Council of Scientific and Industrial Research, Lahore, Pakistan. 2 5 The cyclic tripeptide TAFC consists of three N -acetyl-N - 3 5 To whom correspondence may be addressed: E-mail: [email protected] or anhydromevalonyl-N -hydroxyornithines, linked by ester bonds [email protected]. 2 – and is derived from the N -acetyl lacking precursor fusarinine C. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. The presence of the anhydromevalonyl moiety in fusarinine 1073/pnas.1106399108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1106399108 PNAS Early Edition | 1of8 Downloaded by guest on September 24, 2021 Fig. 1. Ergosterol (green) and siderophore (purple) biosynthetic pathways in A. fumigatus. Biosynthesis of both TAFC and FC starts with N5-hydroxylation of ornithine. Subsequently, the hydroxamate group is formed by the transfer of an acyl group from acyl-CoA derivatives to N5-hydroxyornithine. Here the pathways for biosynthesis of TAFC and FC split because of the choice of the acyl group with acetyl for FC and anhydromevalonyl for TAFC. Assembly of the cyclic siderophores fusarinine C and FC is catalyzed by different nonribosomal peptide synthetases (NRPS). TAFC and hydroxyferricrocin are formed by N2- acetylation of fusarinine C and hydroxylation of FC, respectively. Anhydromevalonyl-CoA originates from mevalonate via CoA-esterification and dehydration. Previously identified A. fumigatus genes encoding respective siderophore biosynthetic enzyme activities are indicated in black (3, 5). The functionally characterized sidI and sidH genes (this study) are shown in orange. TAFC, FC, mevalonate, and ergosterol are in red. (Adapted from ref. 5.)

Recently, genome-wide transcription profiling has indicated that produce non–mevalonate-derived siderophores (e.g., Usti- that expression of the gene encoding HMG-CoA reductase, lago maydis) (12–15). hmg1, is down-regulated in a shift from iron-depleted to iron- replete conditions in A. fumigatus (10), whereas the expression of Lovastatin Inhibits TAFC Production. Inhibition of HMG-CoA re- the gene encoding mevalonate kinase, erg12, is up-regulated. ductase by treatment with 1 μM lovastatin (16) reduced the Northern blot analysis confirmed that iron starvation increases TAFC production to 63% of that in untreated WT A. fumigatus hmg1 transcript levels but decreases erg12 transcript levels (SI without affecting the biomass, suggesting that HMG-CoA re- Appendix, Fig. S1A). Consistent with the down-regulation of ductase plays an essential role in TAFC biosynthesis (Table 1). erg12, the total cellular ergosterol content of A. fumigatus my- Treatment with 8 μM of lovastatin reduced the TAFC pro- celia decreased to 38% during iron-depleted as compared with duction to 14% and the biomass to 16% of the untreated control, iron-replete growth (Fig. 2). Therefore, the observed up-regu- underscoring that HMG-CoA reductase activity is essential for lation of hmg1 expression during iron starvation does not affect both growth and TAFC biosynthesis. ergosterol biosynthesis. We speculated that this expression pat- tern reflects an increased demand for mevalonate as a precursor Overexpression of hmg1 Increases TAFC Production. To assess di- for TAFC biosynthesis, which is induced by iron starvation. rectly the involvement of Hmg1 in TAFC production, we tried to Consistent with this hypothesis, genome-wide transcription pro- delete the hmg1 gene. However, these efforts remained futile, filing data do not indicate that iron availability has any effect on most likely because of the essentiality of this gene. Over- the expression of genes encoding HMG-CoA reductase in fungal expression of hmg1 by placing the gene under the control of the species that do not produce siderophores (e.g., Saccharomyces xylose-inducible xylP promoter (SI Appendix, Figs. S1B and S2C) cerevisiae, Cryptococcus neoformans, and Candida albicans) or (17, 18) resulted in more than twofold increased TAFC pro- duction compared with WT in the absence of lovastatin (Table 1). Consistent with Hmg1 being the target of lovastatin toxicity, hmg1 overexpression increased lovastatin resistance and TAFC

Table 1. Comparison of biomass and TAFC production of WT and hmg1OE in response to lovastatin treatment Lovastatin (μM)

Strain TAFC/Biomass 00.51248

WT TAFC 1.00 0.97 0.63 0.50 0.25 0.14 Biomass 1.00 1.00 1.00 0.94 0.54 0.16 hmg1OE TAFC 2.06 1.75 1.19 1.15 0.93 0.88 Fig. 2. A. fumigatus total ergosterol content is affected by iron availability, Biomass 1.06 1.16 1.00 1.04 0.88 0.91 TAFC biosynthesis, and hmg1 overexpression. (A) The ergosterol content of the WT strain was measured after growth for 48 h in minimal medium Cultures were grown for 48 h in minimal medium containing xylose as – without iron (Fe ) and with iron (Fe+). (B) The ergosterol contents of ΔsidI, carbon source and lovastatin. The biomass and TAFC production of the WT ΔsidH, and hmg1OE mutants were normalized to that of the WT strain grown without lovastatin were 0.34 g and 511 μmol/g dry weight, respectively. The under the conditions shown in A. Error bars indicate SD for three in- values represent the means of three experiments, normalized to the un- dependent experiments. treated WT. SD in each case was <10%.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1106399108 Yasmin et al. Downloaded by guest on September 24, 2021 biosynthesis under lovastatin treatment. Thus, 8 μM of lovastatin quisition system (3). In agreement, deficiency in sidI or sidH PNAS PLUS decreased the biomass of the WT and hmg1OE strains to 16% impaired production of extracellular TAFC (Fig. 4). Interestingly, and 91% of the untreated WT, respectively. Overexpression of deficiency in sidI, but not sidH, substantially decreased the FC hmg1 also increased the ergosterol content by about 14% in both content (Fig. 4). Complementation of ΔsidI and ΔsidH with the iron-depleted and iron-replete conditions (Fig. 2). Together, respective WT copy of the deleted gene (SI Appendix, Figs. S2B these results indicate that Hmg1 represents a bottleneck in and S6B) restored the WT phenotype (Fig. 3), demonstrating TAFC biosynthesis. that all phenotypic consequences are direct results of the gene deletions. These results demonstrate that SidI and SidH are The Acyl-CoA Ligase SidI and the Enoyl-CoA Hadratase SidH Are essential for TAFC synthesis and that RIA partially compensates Conserved in Siderophore-Producing Fungi. In iron-replete con- for the lack of siderophore-mediated iron uptake. ditions siderophore biosynthesis in A. fumigatus is repressed by In A. fumigatus, iron starvation decreases the cellular iron the GATA transcription factor SreA (10). Mining of the recently content and down-regulates iron-dependent pathways, including identified SreA regulon identified two genes, sidI (AFUA_ heme and iron sulfur cluster biosynthesis (10). Deficiency in sidI 1G17190) and sidH (AFUA_3G03410), encoding candidate or sidH also decreased resistance to hydrogen peroxide-caused enzymes for activation and dehydration, respectively, of meval- oxidative stress (Fig. 3), an effect that might be explained by the onate for TAFC biosynthesis (Fig. 1). sidI and sidH are flanked decreased iron uptake and consequently impaired iron-de- by siderophore biosynthetic genes (5), and Northern analysis pendent antioxidative defense (10). Remarkably, ΔsidI displayed confirmed up-regulation of sidI and sidH during iron starvation significantly higher susceptibility to hydrogen peroxide and iron (10) (SI Appendix, Fig. S3). starvation than did ΔsidH (Fig. 3). Because TAFC biosynthesis is SidI and SidH belong to the acyl-CoA ligase and enoyl-CoA blocked at different steps in ΔsidI and ΔsidH, the phenotypic hydratase protein families, respectively, with members in all differences might be caused by accumulation of different inter- organisms. The best-characterized members are involved in β- mediates with different consequences on fungal metabolism oxidation. Phylogenetic analysis (SI Appendix, Fig. S4) revealed (Discussion). that all closely related SidI homologs are found in Pezizomyco- Comparative analysis of WT, ΔsidH, and ΔsidI virulence was tina species known or believed to produce mevalonate-derived conducted by measuring survival in a neutropenic model of in- siderophores (1). In contrast, non–siderophore-producing Pezi- vasive pulmonary aspergillosis as previously described (5). At an zomycotina such as S. cerevisiae and C. albicans do not possess infectious dose of 5 × 105 spores, the mortality associated with

SidI orthologs. Similarly, Schizosaccharomyces pombe that pro- ΔsidH and ΔsidI infections was lower (71% and 80%, respec- MICROBIOLOGY duces the siderophore ferrichrome (19), which lacks a mevalo- tively) than with WT infections (96%); however, significant nate-derived moiety, does not possess an SidI ortholog. This attenuation was not reproducibly measurable at the level of phylogenetic analysis underscores the possible involvement of murine survival. Further titration of the infectious dose to 9 × SidI in siderophore production and demonstrates its evolution- 104 spores increased the attenuation of virulence in both the ary conservation. As is evident from the phylogenetic analysis ΔsidH (n = 14) and ΔsidI mutants (n = 15) relative to WT (n = (SI Appendix, Fig. S5), only siderophore-producing fungi possess 8) (P = 0.0003 and P = 0.0008 respectively) (Fig. 5). Virulence close SidH homologs. also was attenuated significantly in the ΔsidH and ΔsidI mutants (P = 0.0032 and P = 0.0008, respectively) relative to the cor- Deletion of SidI or SidH Blocks TAFC Biosynthesis, Decreases responding reconstituted strains sidHC (n = 7) and sidIC (n = 8). Resistance to Oxidative Stress, and Attenuates Virulence in a Murine Model of Invasive Pulmonary Aspergillosis. To understand the role Genetic Inactivation of TAFC Biosynthesis Decreases Lovastatin of SidI and SidH, we generated respective gene deletion mutants Susceptibility. Inactivation of TAFC biosynthesis by the deletion in A. fumigatus ATCC 46645 (WT), termed “ΔsidI” and” ΔsidH,” of sidI or sidH increased resistance to lovastatin (8 μM) in iron- respectively (SI Appendix, Figs. S2A and S6A). Consistent with depleted conditions (Fig. 6). Accordingly, the ergosterol content the expression of sidI and sidH being restricted to conditions of of ΔsidI and ΔsidH increased about 13% during iron depletion iron limitation, both ΔsidI and ΔsidH displayed no difference (Fig. 2). These results suggest that inactivation of SidI or SidH from WT with respect to growth rate on iron-replete solid me- increases the mevalonate pool for essential isoprenoid bio- dium (Fig. 3). In contrast, ΔsidI and ΔsidH showed a mild growth synthesis, including ergosterol, by blocking TAFC-mediated reduction during iron depletion and were unable to grow in the mevalonate consumption. In contrast, TAFC biosynthesis is not presence of the ferrous iron-specific chelator bathophenanthro- essential, because A. fumigatus possesses the alternative RIA line disulfonate (BPS), which inhibits RIA and renders side- system. Consistent with TAFC biosynthesis and the expression of rophore-mediated iron uptake the only active high-affinity ac- involved enzymes being repressed by iron, ΔsidI and ΔsidH dis-

Fig. 3. Both ΔsidI and ΔsidH are highly susceptible to iron starvation and oxidative stress. The fungal WT strain, mutant strains, and complemented strains – + were grown for 48 h on minimal medium agar under iron-depleted (Fe ) or iron-replete (Fe ) conditions without or with H2O2 (2 mM) or BPS (250 μM). (Left) Radial growth of the WT strain. (Right) Growth of the mutant and complemented strains normalized to WT.

Yasmin et al. PNAS Early Edition | 3of8 Downloaded by guest on September 24, 2021 Fig. 4. SidI and SidH are required for the synthesis of TAFC. HPLC analysis of supernatant and cell extract from cultures of WT, ΔsidH, and ΔsidI strains grown – in iron-depleted conditions (Fe ) for 24 h. Units are given in milli absorption units (mAU).

played WT-like susceptibility to lovastatin and ergosterol content 7). Comparative GC-MS analysis of the neutral lipid fraction in iron-replete conditions. obtained from a WT strain, the siderophore-deficient L-orni- thine-N5-monooxygenase mutant, ΔsidA (3), the TAFC-non- Iron Depletion Reduces Cellular Ergosterol Content in WT but Less in synthesizing mutants, ΔsidF, ΔsidH, ΔsidI, and ΔsidD (5), and TAFC Nonsynthesizing Strains. Collectively, the data shown above a ferricrocin-nonsynthesizing mutant ΔsidC (5) revealed iron- suggest a model in which bilateral demand for mevalonate occurs dependent alterations in sterol composition (Fig. 7). In all strains under iron-depleted conditions, imposed by the requirement for the major sterol was ergosterol; 15 additional sterol constituents siderophore biosynthesis. Therefore, we examined in more detail were identified by comparison with known sterol spectra (SI cellular sterol content in WT and siderophore biosynthetic Appendix, Table S1). mutants under iron-replete and iron-starvation conditions (Fig. In A. fumigatus ergosterol biosynthesis involves several iron- dependent enzymes, which operate at different levels of the pathway. To permit an appraisal of sterol intermediates within the context of potential iron-dependent rate limitations, we partitioned the analyzed sterols into two groups, according to the positioning of heme-dependent enzymes [lanosterol 14-α-deme- thylase (Erg11) and sterol C-22 desaturase (Erg5)] or oxo-diiron enzymes [methyl sterol oxidase (Erg25) and sterol C-5 desaturase (Erg3)] in the sterol biosynthetic pathway (20). In our classifica- tion the category of C-4 methyl sterols, derived from lanosterol via eburicol and sequentially demethylated at C-14 and C-4 by Erg11, Erg24, Erg25, Erg26, and Erg27, includes five di-trite- tramethylcholesta-8,24-dien-3β-ol compounds: 4,4,14-trimethyl- cholesta-8,24-dien-3β-ol, 4α,24-dimethylcholesta-8,24(28)-dien-

Fig. 5. In neutropenic mice virulence in ΔsidH and ΔsidI siderophore mutants is attenuated relative to isogenic WT (ATCC46645) and recon- stituted strains. Inocula of 9 × 104 conidiospores were administered in- tranasally under anesthesia in 40 μL of saline. Mice were weighed twice daily Fig. 6. Deficiency in SidI or SidH increases resistance to lovastatin. Ap- after infection, and the experimental end point was determined by a weight proximately 50 conidia were spotted on minimal medium agar without or loss of 20% relative to the day of infection. sidHC and sidIC indicate recon- with iron containing 0–8 μM lovastatin. Radial growth was measured after stituted strains. Murine survival was compared between groups using 48 h of incubation. The values represent the means of three experiments Kaplan–Meier plots and log rank analysis. normalized to the untreated WT. The SD was <5%.

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Fig. 7. Ergosterol biosynthesis in A. fumigatus. Accumulated intermediates are represented in bar plots as the percentage of total sterols. (A) Ergosterol. Asterisks indicate significantly different sterol content (P < 0.05 by nonparametric Mann–Whitney test). (B) Accumulation of (i) C-4 methyl sterols and (ii) Δ7 sterols according to current understanding of the ergosterol biosynthetic pathway. Fe represents iron-replete (+) or iron-depleted (−) conditions.

3β-ol, 4,4,14,24-tetramethylcholesta-8,24(28)-dien-3β-ol, 4α,24- to antifungal drugs that target ergosterol biosynthesis (Table 2). dimethylcholesta-7,24(28)-dien-3β-ol, and 4,4,24-trimethylcho- BPS-mediated iron chelation heightened the susceptibility of the lesta-8,24(28)-dien-3β-ol. The category of Δ7 sterols, which are WT strain and the FC-lacking ΔsidC mutant to both voriconazole desaturated at carbon 5 by the Erg3 enzyme to produce and amphotericin B. The susceptibility of ΔsidI and ΔsidH cannot 24-methylcholesta-5,7,24(28)-trien-3β-ol sterol, includes three be tested under this condition, because their growth is inhibited 24-methylcholesta-Δ7–3β-ol isomers: 24-methylcholesta-7,22- dien-3β-ol, 24-methylcholesta-7,24(28)-dien-3β-ol, and 4α,24- dimethylcholesta-7,24(28)-dien-3β-ol. Table 2. MIC of voriconazole and amphotericin B for WT and Iron limitation negatively impacted the ergosterol content of siderophore-deficient mutants independence of iron availability the cell in all strains tested (Fig. 7A). The observed reduction in MIC amphotericin B (μg/ ergosterol content was greater for the WT and ΔsidC mutants MIC voriconazole (μg/mL) mL) than for TAFC-nonsynthesizing mutants ΔsidA, ΔsidD, ΔsidF, – – – – ΔsidH, and ΔsidI. C-4 methyl and Δ7 sterols accumulated (Fig. Strain Fe BPS* Fe Fe+ Fe BPS Fe Fe+ Δ 7B) under iron starvation in the WT and sidC strains but Δ Δ sidA No growth 0.064 0.125 No growth 0.19 0.50 drastically less in the TAFC-nonsynthesizing mutants sidA, Δ Δ Δ Δ Δ sidI No growth 0.125 0.125 No growth 1.00 1.00 sidD, sidF, sidH, and sidI. ΔsidH No growth 0.125 0.125 No growth 1.00 1.00 ΔsidC 0.032 0.125 0.125 0.38 1.00 1.00 Iron Starvation Increases A. fumigatus Susceptibility to Voriconazole WT 0.064 0.125 0.125 0.50 1.00 1.00 and Amphotericin B. We next tested the effect of iron availability or the inactivation of siderophore biosynthesis on susceptibility *Fe–,Fe+, and Fe–BPS are as described in Fig. 3.

Yasmin et al. PNAS Early Edition | 5of8 Downloaded by guest on September 24, 2021 by BPS (Fig. 3). The WT strain displayed the same susceptibility The Ustilago maydis siderophore ferrichrome A requires an- to voriconazole and amphotericin B in media containing 10 μM other isoprenoid pathway intermediate (30): HMG-CoA is iron and in low-iron conditions. Inactivation of the entire side- converted to methylglutaconyl-CoA by the enoyl-CoA hydratase rophore system (ΔsidA), however, increased the susceptibility to Fer4 and then is used by the acylase Fer5 to acylate N5- both drugs, in particular during low-iron conditions. hydroxyornithine that is generated from ornithine by the mon- Sid1. Phylogenetic analysis indicates that A. fumigatus Discussion SidH and U. maydis Fer4 are not orthologs (SI Appendix, Fig. Differences in iron handling between mammals and siderophore- S5), suggesting recruitment of different enoyl-CoA hydratases in producing fungi such as Aspergilli might serve to improve the the evolution of siderophore biosynthesis in these two fungi. efficacy of antifungal therapies. Several lines of evidence indicate Mevalonate is an important intermediate of isoprenoid bio- that A. fumigatus faces iron limitation during mammalian in- synthetic pathways such as that of fungal ergosterol and essen- fection: (i) Defects impairing adaptation to iron limitation, such tial nonsterol isopreonoids (31). Production of mevalonate is as inactivation of the siderophore system or the iron regulator tightly controlled to satisfy cellular requirements for all these HapX, attenuate virulence in murine models of pulmonary as- products but to avoid accumulation of these products to toxic pergillosis (3, 5, 21); (ii) RIA and the siderophore system are levels (32, 33). Like S. cerevisiae, A. fumigatus possesses two transcriptionally up-regulated during initiation of murine in- putative isoforms of HMG-CoA reductase encoded by hmg1 fection (22); and (iii) TAFC-chelated 68Ga, the uptake of which (AFUA_2G03700) and hmg2 (AFUA_1G11230) (34). In S. is induced by iron starvation, allows in vivo PET imaging of in- cerevisiae, Hmg1 and Hmg2 contribute 83% and 17% of the total vasive pulmonary aspergillosis in rats (23). Consistent with these activity, respectively (35). In A. fumigatus, the individual contri- findings, human protection against A. fumigatus includes growth bution of the two putative isoforms is unknown. In Northern blot inhibition by polymorphonuclear leukocytes via lactoferrin-me- analyses, we detected only hmg1, but not hmg2, expression (SI diated iron depletion and possibly siderocalin-mediated scav- Appendix, Fig. S1A). For isoprenoid biosynthesis, mevalonate is enging of siderophores (24, 25). Moreover, increased bone phosphorylated by the mevalonate-kinase Erg12 (Fig. 1). marrow iron stores represent an independent risk factor for in- Several lines of evidence indicate a tight interconnection of A. vasive aspergillosis (26). Inversely, iron chelators were shown to fumigatus TAFC and isoprenoid metabolism by the common improve treatment with azoles and amphotericin B in vitro and intermediate mevalonate: (i) Iron starvation down-regulates the in vivo (27, 28). cellular ergosterol content but up-regulates TAFC production; Siderophore production in A. fumigatus follows an array of (ii) in accordance with the ergosterol content, iron starvation different enzymatic steps (Fig. 1), five of which have been down-regulates the erg12 transcript level; (iii) in accordance with identified recently at the gene level (5). In this study, we iden- TAFC production, iron starvation up-regulates the hmg1 tran- tified two additional TAFC biosynthetic genes, sidI and sidH, script level; (iv) inhibition of HMG-CoA reductase by lovastatin encoding proteins with similarity to acyl-CoA ligases and enoyl- treatment decreases TAFC production; (v) overexpression of hmg1 increases TAFC production, lovastatin resistance, and er- CoA hydratases, respectively. Involvement of such enzymes in gosterol content; (vi) deletion of sidI or sidH (blocking TAFC the biosynthesis of fusarinine-type siderophores was proposed biosynthesis-mediated mevalonate consumption) increases lova- initially by Anke and Diekmann (9) working with Fusarium statin resistance and ergosterol content; and (vii) iron starvation cubense. Previously, mevalonate was thought to be converted to and in particular genetic inactivation of siderophore biosynthesis anhydromevalonate before ligation of CoA (9). However, later increases the susceptibility to the ergosterol-targeting drugs studies on the substrate stereochemistry of enoyl-CoA hydra- voriconazole and amphotericin B. tases concluded that the CoA derivative (having acidic hydrogens Down-regulation of the cellular ergosterol content by iron at C-2 favorable for syn elimination), and not mevalonate itself, starvation, as shown here in A. fumigatus (Fig. 7A) and previously should be the actual substrate (29). Therefore, it is most likely for the siderophore-lacking yeasts C. albicans and S. cerevisiae that the acyl-CoA ligase SidI converts mevalonate to mevalonyl- (20, 36), is likely a consequence of the iron dependence of sev- CoA and that the enoyl-CoA hydratase SidH converts meva- 5 eral enzymes of the late ergosterol biosynthetic pathway, e.g., lonyl-CoA to anhydromevalonyl-CoA, which is transferred to N - Erg11/Cyp51, Erg3, and Erg5 (20, 37). In A. fumigatus, iron hydroxyornithine by the transacylase SidF (Fig. 1). All known starvation results additionally in accumulation of C-4 methyl and fungal siderophores are derived from ornithine as a precursor Δ7 sterols, especially in TAFC-nonsynthesizing strains (Fig. 7B). (1). For fusarinine-type and coprogen-type siderophores, which Accumulation of Δ7 sterols also is caused by inactivation of the are produced by a wide array of fungal species, mevalonate is iron-dependent Erg3 in A. fumigatus (38), indicating that iron a second precursor; here we characterize the genes encoding the starvation indeed limits Erg3 activity. The increased levels of C-4 enzymatic steps for formation of the mevalonate-derived anhy- sterol intermediates during iron starvation might be attributable dromevalonyl-CoA. to decreased carbon-4 demethylation, most likely because of the Elimination of sidI or sidH results in the loss of TAFC pro- reduction of Erg25 activity (Fig. 7B). Inactivation of TAFC bio- duction, leaving the fungus relying solely on RIA. Apart from synthesis increased the ergosterol content and reduced levels of impairing TAFC biosynthesis, deficiency in sidH and in particular C-4 methyl and Δ7 sterol intermediates during iron starvation, in sidI caused increased susceptibility to iron starvation and to indicating that the sterol composition reflects the impact of oxidative stress, likely reflecting iron starvation because iron is, as iron and mevalonate availability, both of which are affected by heme, a cofactor of oxidative stress-detoxifying enzymes such as TAFC production. Consistent with an increased requirement catalases or peroxidases. The phenotypic differences between for mevalonate during iron-depleted conditions for TAFC syn- mutant strains deficient in sidH and sidI might be caused by the thesis, lovastatin susceptibility was higher in iron-depleted than accumulation of different pathway intermediates (mevalonate in iron-replete conditions [minimal inhibitory concentration (MIC) and mevalonyl-CoA, respectively) with different consequences. of 8 μMvs.10μM)] and increased the resistance of mutants de- In further agreement, deficiency in sidI, but not in sidH, sidD,or ficientinSidIorSidH(Fig.6). sidF (5), decreased the intracellular FC content, possibly by dis- The polyene amphotericin B, azoles, and echinocandins are turbing the acetyl-CoA homeostasis required for FC biosynthesis. the drugs of choice for the treatment of aspergillosis. However, The hypersusceptibility to hydrogen peroxide of ΔsidI compared associated toxicities and development of resistances demand new with ΔsidH might be related to the concomitant decrease in FC, antimycotic agents (39, 40). Statins are fungicidal for A. fumi- which plays a role in resistance to oxidative stress (5). gatus (41) but, although they act synergistically with azoles against

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1106399108 Yasmin et al. Downloaded by guest on September 24, 2021 S. cerevisiae, C. albicans, C. neoformans, and clinically important fragment was inserted at the respective site in the multiple cloning site of PNAS PLUS Zygomycetes spp (42–44), they seem not to do so for A. fumigatus pUC18, giving rise to pUC-CoA. A 2.7-kb SspI-HindIII fragment of the pyr- (45, 46). So far, the inhibitory effect of statins has been attrib- ithiamine resistance gene (ptrA) from plasmid pSK275 was inserted into SspI/ uted solely to the inhibition of isoprenoid biosynthesis, which HindIII-digested pUC-CoA to create pSY06. This construct was introduced into the ΔsidI strain by protoplast transformation. Transformants were se- mainly affects sterol biosynthesis but also affects the synthe- lected using 0.1 mg/l pyrithiamine. To confirm homologous integration, sis of dolichol, heme-A, isopentenyl tRNA, carotenoids, and a Southern blot of XbaI-digested genomic DNA from the complemented ubiquinone as well as protein prenylation (31). We show here strain was probed with exon 2–3(SI Appendix, Fig. S2B). that statins also inhibit biosynthesis of mevalonate-derived To reconstitute the ΔsidH strain with a functional sidH copy, a 7.9-kb siderophores, which are essential virulence determinants of an- EcoRI-HindIII fragment released from the cosmid pANsCosSidD was subcl- imal- and plant-pathogenic fungi (5, 47). Our data indicate that oned into plasmid pPhleo carrying the phleomycin resistance marker gene antifungal drug design and testing should consider the metabolic (ble) (52). The resulting 14.2-kb plasmid psidHc was linearized with HindIII and used to transform A. fumigatus ΔsidH. Phleomycin-resistant trans- changes caused by iron starvation. Given the importance of the c siderophore and ergosterol biosynthetic pathways for fungal formants with single homologous reconstitution of the sidH gene (sidH virulence and antifungal treatment, the molecular insights pro- strain) were selected by Southern blot analysis (SI Appendix, Fig. S6B). vided by our analyses may open new possibilities in the fight Overexpression of hmg1. A 1.6-kb fragment encoding the N-terminal region against fungal infections. of the hmg1 gene (the entire gene is 3.6 kb) was amplified from A. fumi- gatus genomic DNA using oligonucleotides hmg-3 and hmg-4 to create an Material and Methods NcoI site at the hmg1 start codon and a SphI site 1.6 kb downstream from Fungal Strains and Growth Conditions. A. fumigatus strains used in this study the start codon. The NcoI/SphI-digested amplified fragment was inserted in are listed in SI Appendix, Table S2. The strains were cultured routinely at 37 °C the respective sites of pGEM(5zf+), yielding plasmid pHMG-GEM. Next, the on minimal medium described previously (48) containing 1% glucose as the 1.7-kb fragment of the xylP promoter was amplified using plasmid pXyluidA- carbon source and 20 mM glutamine as the nitrogen source. Iron-replete P (17) as template and oligonucleotides xylP-R and xylP-F, introducing an μ medium contained 10 M FeSO4. For high-iron conditions, FeSO4 was added NcoI site in the xylP start codon and an NotI site at bp −1674. After digestion fi to a nal concentration of 1.5 mM FeSO4; addition of iron was omitted for with NcoI/NotI, the fragment was inserted into the equally cleaved pHMG- iron-depleted medium. Xylose instead of glucose was used as the carbon GEM plasmid, resulting in pHMG-xyl. Then, the ptrA-containing the 1.9-kb source for induction of expression of hmg1 under the control of the xylP PstI-NdeI fragment from pSK275 was inserted into the respective site of promoter (17, 18). For radial growth assays, 50 conidia of respective strains pHMG-xyl. The resulting plasmid pHMG was used to transform A. fumigatus were spotted onto described solid minimal medium. Lovastatin (Alexis) was WT as described in SI Appendix, Fig. S2C.

converted to its active form by hydrolyzing in ethanolic NaOH as described MICROBIOLOGY previously (49). The plates were incubated at 37 °C, and radial growth was Analysis of Siderophores and Ergosterol. TAFC and FC were characterized by measured after 48 h. For propagation of plasmids, Escherichia coli strain reversed-phase HPLC chromatography as described previously (53). α μ DH5 was grown in LB medium supplemented with ampicillin (100 g/mL). Sterols were extracted following 24-h growth in liquid minimal medium under iron-depleted or iron-replete conditions as previously described (54). Phylogenetic Analysis of SidI and SidH. The amino acid sequences of SidI and To 100–200 mg of freeze-dried mycelia, 3 mL of 25% alcoholic (3:2 metha- SidH homologs were retrieved by BLAST search. The alignment was created nol:ethanol) potassium hydroxide solution was added. After vortexing for with the multiple alignment algorithm of CLUSTAL W version 1.83. For 1 min, the mixture was incubated at 85 °C for 1 h. Sterols were extracted phylogenetic analysis Phylip Drawgram version 3.6a3 (http://bioweb2.pas- twice with a mixture of 1 mL and 1.5 mL hexane and, after vigorous vortex teur.fr/phylogeny) was used. mixing, the upper hexane layer was transferred to a clean glass tube and evaporated. Samples were spiked with 0.1 mg of androstanol (Sigma) as an Deletion of A. fumigatus SidI and SidH. The sidI and sidH genes were deleted internal standard for recovery and quantitation. Trimethylsilyl ether deriv- using the bipartite marker technique as described in ref. 50 (SI Appendix, atives of fungal sterols dissolved in toluene/N,O-bis(trimethylsilyl)trifluo- Figs. S2A and S6A). Flanking regions of sidI were amplified by PCR. A 1.8-kb roacetamide (BSTFA; 1:1; Merck) were analyzed on a Trace GC-DSQ-II single- fragment at the 5′ region was amplified with primers sidI-1 and sidI-2 (SI quadrupole mass spectrometer (Thermo Scientific) on a 60 m × 0.25 mm i.d.× Appendix, Table 3) containing add-on restriction sites for ClaI and NotI, re- 0.25 μm film thickness 5% diphenyl- and 95% dimethyl-polysiloxane (HP- spectively, and then was cleaved with NotI. The 1.4-kb flanking region at the 5MS; Agilent) capillary column using He as carrier gas at 1.2 mL/min. Injector, 3′ end was amplified with primers sidI-3 and sidI-4 containing add-on BamHI transfer line, and ion source were at 300, 200, and 250 °C, respectively. and StuI restriction sites, respectively, and then was cleaved with BamHI. Sterols were identified by comparison of mass spectra and retention time Subsequently, the NotI-BamHI 3kb-fragment from pHYTK containing the data (54). Relative sterol composition was expressed as the ratio of raw peak hygromycin resistance-thymidine kinase gene (hph-Tk) cassette was ligated areas with respect to the total sterol content. Each experiment was repeated with the sidI 5′ and 3′ flanking regions, respectively. Each ligation product three times. The significance of content (P < 0.05) was determined by non- then was used as template for the amplification of the bipartite hph-Tk parametric Mann–Whitney test. marker fragment and flanking region using sidI-5 and hph-14 for the 5′ fl ′ fl anking region plus partial hph-Tk and sidI-6 and hph-15 for 3 anking Northern Blot Analyses. Total RNA was isolated from mycelia by using RNA- region plus partial hph-Tk. Solv reagent as described by the manufacturer (Omega Bio-Tek). For the To generate ΔsidH,the5′ flanking region of sidH (1.7 kb) was amplified Northern analysis 10 μg of total RNA was used. RNA was fractionated in using sidH-1 and sidH-4 containing add-on restriction sites for NotI, and the formaldehyde-agarose gels, blotted onto Hybond-N membranes (Amersham 3′ flanking region (1.3 kb) was amplified using sidH-2 and sidH-3 containing Biosciences), and hybridized with digoxigenin-labeled probes (Boehringer add-on restriction sites for BamHI. Subsequently, the NotI–BamHI fragment Mannheim). Hybridization probes were generated by PCR amplification of hph-Tk released from pHYTK was ligated with the sidH 5′ and 3′ flanking using oligonucleotides listed in SI Appendix, Table S3. regions, respectively. Each ligation product then was used as template for fi fl the ampli cation of the bipartite hph-Tk marker fragment and anking Analysis of Fungal Burden in Murine Lungs. Virulence of A. fumigatus strains ′ fl region (as described for sidI) using sidH-5 and hph-14 for the 5 anking was assayed in a murine model for invasive pulmonary aspergillosis described ′ fl region plus partial hph-Tk and sidH-6 and hph-15 for the 3 anking region previously (55). Briefly, outbred male mice (strain CD1, 20–28 g; Charles River plus partial hph-Tk. Laboratories) were used for animal experiments. Immunosuppression was Bipartite marker constructs for sidI and sidH were used to transform WT A. carried out by s.c. injection of 112 mg/kg hydrocortisone acetate and i.p. in- fumigatus. Transformation was done essentially as described by Punt et al. jection of 150 mg/kg cyclophosphamide as described previously (55). Bacterial (51). The transformants were selected on medium containing 200 μg/mL of infections were prevented by adding 1 g/L tetracycline and 64 mg/L ciproxicin hygromycin B. Homologous recombination at the desired locus was con- to the drinking water. Inocula of 5 × 105 or 9 × 104 conidiospores in 40 μLof firmed by Southern blot analysis (SI Appendix, Figs. S2A and S6A). saline were prepared by harvesting spores from 5-d-old slices of solid medium followed by filtration through Miracloth (Calbiochem) and washing with Complementation of the SidI and SidH Deletions. A 3.3-kb fragment con- saline. Mice were anesthetized by inhalation of isofluorane and infected by taining the sidI WT copy was amplified from genomic DNA using primers intranasal instillation. Infected mice were weighed twice daily after infection. sidI-5 and sidI-6 (SI Appendix, Table 3). After cleavage with XbaI-Acc65I, this The end point for survival analyses was determined by a weight loss of 20%

Yasmin et al. PNAS Early Edition | 7of8 Downloaded by guest on September 24, 2021 relative to the day of infection. Murine survival was compared between agar plate with different iron availability containing a lawn of conidia. groups using Kaplan–Meier plots and log rank analysis. Growth inhibition was measured after 48 h by direct observation of the All work using animals in this study was carried out pursuant to Project plates at 37 °C. License PPL/70/6487 authorized by the Home Office pursuant to the Animals (Scientific Procedures) Act 1986 (widely acknowledged to be the most rig- Accession Numbers. The genes and gene products (including accession orous legislation of its type in the world). Details of the relevant legislation numbers at http://www.cadre-genomes.org.uk/) studied in this work are and its application can be found at: http://www.homeoffice.gov.uk/science- hmg1 (AFUA_2G03700), hmg2 (AFUA_1G11230), erg12 (AFUA_4G07780), research/animal-research/. All infections were performed under isofluorane sidI (AFUA_1G17190), sidH (AFUA_3G03410), sidF (AFUA_3G03400), sidD anesthesia, and all efforts were made to minimize suffering. (AFUA_3G034290), sidA (AFUA_2G07680), and sidC AFUA_1G17200).

Drug Susceptibility Testing. E-test strips (Biomerieux) impregnated with ACKNOWLEDGMENTS. This work was supported by Grants FWF P-18606-B11 a gradient of voriconazole or amphotericin B were used according to the and FWF P21643-B11 from the Austrian Science Foundation and by Grant manufacturer’s instructions. Each strip was placed onto a minimal medium UNI-0404/593 from the Tyrolean Science Foundation.

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