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Mevalonate Governs Interdependency of Ergosterol and Siderophore

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Mevalonate Governs Interdependency of Ergosterol and Siderophore Mevalonate governs interdependency of ergosterol 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 fungus-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,
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