The J anus transcription factor H ap X controls fungal adaptation to both iron starvation and iron excess
2014; Springer Nature; Volume: 33; Issue: 19 Linguagem: Inglês
10.15252/embj.201489468
ISSN1460-2075
AutoresFabio Gsaller, Peter Hortschansky, Sarah R. Beattie, Veronika Klammer, Katja Tuppatsch, Beatrix E. Lechner, Nicole Rietzschel, Ernst R. Werner, Aaron A. Vogan, Dawoon Chung, Ulrich Mühlenhoff, Masashi Kato, Robert A. Cramer, Axel A. Brakhage, Hubertus Haas,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle4 August 2014Open Access The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess Fabio Gsaller Fabio Gsaller Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Peter Hortschansky Peter Hortschansky Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Search for more papers by this author Sarah R Beattie Sarah R Beattie Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Veronika Klammer Veronika Klammer Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Katja Tuppatsch Katja Tuppatsch Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Friedrich Schiller University, Jena, Germany Search for more papers by this author Beatrix E Lechner Beatrix E Lechner Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Nicole Rietzschel Nicole Rietzschel Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Ernst R Werner Ernst R Werner Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Aaron A Vogan Aaron A Vogan Department of Biology, McMaster University, Hamilton, ON, Canada Search for more papers by this author Dawoon Chung Dawoon Chung Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Ulrich Mühlenhoff Ulrich Mühlenhoff Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Masashi Kato Masashi Kato Faculty of Agriculture, Meijo University, Nagoya, Japan Search for more papers by this author Robert A Cramer Robert A Cramer Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Axel A Brakhage Corresponding Author Axel A Brakhage Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Friedrich Schiller University, Jena, Germany Search for more papers by this author Hubertus Haas Corresponding Author Hubertus Haas Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Fabio Gsaller Fabio Gsaller Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Peter Hortschansky Peter Hortschansky Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Search for more papers by this author Sarah R Beattie Sarah R Beattie Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Veronika Klammer Veronika Klammer Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Katja Tuppatsch Katja Tuppatsch Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Friedrich Schiller University, Jena, Germany Search for more papers by this author Beatrix E Lechner Beatrix E Lechner Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Nicole Rietzschel Nicole Rietzschel Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Ernst R Werner Ernst R Werner Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Aaron A Vogan Aaron A Vogan Department of Biology, McMaster University, Hamilton, ON, Canada Search for more papers by this author Dawoon Chung Dawoon Chung Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Ulrich Mühlenhoff Ulrich Mühlenhoff Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany Search for more papers by this author Masashi Kato Masashi Kato Faculty of Agriculture, Meijo University, Nagoya, Japan Search for more papers by this author Robert A Cramer Robert A Cramer Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA Search for more papers by this author Axel A Brakhage Corresponding Author Axel A Brakhage Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany Friedrich Schiller University, Jena, Germany Search for more papers by this author Hubertus Haas Corresponding Author Hubertus Haas Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Author Information Fabio Gsaller1,‡, Peter Hortschansky2,‡, Sarah R Beattie3, Veronika Klammer1, Katja Tuppatsch2,4, Beatrix E Lechner1, Nicole Rietzschel5, Ernst R Werner6, Aaron A Vogan7, Dawoon Chung3, Ulrich Mühlenhoff5, Masashi Kato8, Robert A Cramer3, Axel A Brakhage 2,4 and Hubertus Haas 1 1Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria 2Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany 3Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA 4Friedrich Schiller University, Jena, Germany 5Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany 6Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria 7Department of Biology, McMaster University, Hamilton, ON, Canada 8Faculty of Agriculture, Meijo University, Nagoya, Japan ‡These authors contributed equally *Corresponding author. Tel: +49 3641 5321001; Fax: +49 3641 5320802; E-mail: [email protected] *Corresponding author. Tel: +43 512 9003 70205; Fax: +43 512 9003 73100; E-mail: [email protected] The EMBO Journal (2014)33:2261-2276https://doi.org/10.15252/embj.201489468 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Balance of physiological levels of iron is essential for every organism. In Aspergillus fumigatus and other fungal pathogens, the transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron consumption and activating iron uptake. Here, we demonstrate that HapX is also essential for iron resistance via activating vacuolar iron storage. We identified HapX protein domains that are essential for HapX functions during either iron starvation or high-iron conditions. The evolutionary conservation of these domains indicates their wide-spread role in iron sensing. We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter. The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes. Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability. Synopsis The fungal transcription factor HapX is involved in adaptation to both iron starvation and excess. HapX and the CCAAT-binding complex cooperatively bind to the promoter of the vacuolar iron transporter gene and regulate its expression to control vacuolar iron storage. HapX controls fungal adaptation to both iron starvation and excess. HapX acts in part through activation or repression of vacuolar iron deposition. Different domains of HapX are required for adaptation to iron starvation or excess. A homodimer of HapX associates with the CCAAT-binding complex at the promoter of the vacuolar iron transporter gene to regulate its expression. Introduction The redox-active metal iron is an indispensable cofactor in a variety of essential cellular processes such as oxidative phosphorylation, biosynthesis of numerous metabolites, and detoxification of oxidative stress. Paradoxically, the same redox property makes this metal potentially toxic by causing oxidative stress (Halliwell & Gutteridge, 1984; Lin et al, 2011). Thus, iron homeostasis requires precise regulation of iron uptake and storage to satisfy the cellular needs but to avoid toxic iron excess. In Aspergillus fumigatus, iron homeostasis is maintained by two central transcription factors, which are interconnected in a negative transcriptional feed-back loop: the GATA-factor SreA and the bZIP-factor HapX (Haas, 2012). During iron sufficiency, SreA represses iron uptake, including reductive iron assimilation and siderophore-mediated iron uptake, to avoid toxic effects (Schrettl et al, 2008). During iron starvation, HapX activates siderophore-mediated iron acquisition and represses iron-consuming pathways, including heme biosynthesis and respiration, to spare iron (Schrettl et al, 2010). As shown in Aspergillus nidulans, HapX functions via physical interaction with the CCAAT-binding complex (CBC) (Hortschansky et al, 2007). The CBC is a heterotrimeric DNA-binding complex, which is conserved in all eukaryotes. In A. nidulans, inactivation of either one of its subunits, phenocopied HapX inactivation with respect to defects in adaptation to iron starvation (Hortschansky et al, 2007). However, the CBC has HapX-independent functions in Aspergillus spp. (Kato, 2005; Thon et al, 2010). Humans lack HapX and genome-wide identification resulted in 5,000–15,000 CBC-binding sites depending on the type of human cell analyzed (Fleming et al, 2013). Deficiency in HapX, but not SreA, attenuates virulence of A. fumigatus in murine models of aspergillosis (Schrettl et al, 2008, 2010), which emphasizes the crucial role of adaptation to iron limitation in pathogenicity. With the exception of Saccharomyces cerevisiae and closely related Saccharomycotina species, most fungal species possess orthologs to SreA and HapX (Haas et al, 2008; Kaplan & Kaplan, 2009). The important role of HapX orthologs in virulence is conserved in Candida albicans, Cryptococcus neoformans, and Fusarium oxysporum (Jung et al, 2010; Chen et al, 2011; Hsu et al, 2011; Lopez-Berges et al, 2012). Both SreA and HapX appear to be regulated post-translationally by iron, blocking HapX function and activating SreA function (Haas et al, 1999; Hortschansky et al, 2007). In Schizosaccharomyces pombe, post-translational iron sensing by the HapX and SreA orthologs involves the monothiol glutaredoxin Grx4 (Labbe et al, 2013). Recently, iron resistance of A. fumigatus was shown to involve SreA-mediated repression of iron uptake and vacuolar iron storage mediated by the vacuolar iron importer CccA (Gsaller et al, 2012). In A. nidulans and A. fumigatus, inactivation of both HapX and SreA is synthetically lethal underlining the critical role of iron homeostasis in cellular survival (Hortschansky et al, 2007; Schrettl et al, 2010). In agreement with their expression pattern and characterized mode of action, the detrimental effects of SreA or HapX inactivation identified so far were confined to growth during iron sufficiency or starvation, respectively, which does not explain the synthetic lethality of their inactivation. Here, we provide an explanation for this synthetic lethality by demonstrating that HapX mediates both repression of vacuolar iron storage during iron starvation and activation of vacuolar iron storage during iron excess, i.e. HapX displays inverse activities depending on the ambient iron availability. In line, we identified protein domains that are essential for mediating adaptation to iron starvation or iron excess, exclusively. Moreover, we demonstrate for the first time that HapX not only acts via protein–protein interaction with the CBC but also directly recognizes an evolutionary conserved motif in the cccA promoter. As the CBC has HapX/iron-independent targets, the latter data reveal the mechanism for discrimination of general CBC and specific HapX/CBC target genes. Results and Discussion HapX mediates iron resistance by activating CccA-mediated vacuolar iron storage HapX functions were analyzed in A. fumigatus ATCC 46645 (Schrettl et al, 2010) and, to facilitate the studies, in its ∆akuA-derivative AfS77, which lacks non-homologous recombination (Hartmann et al, 2010). We did not observe any phenotypic differences between respective ATCC46645- and AfS77-derivative strains (data not shown). For clarity, however, the genetic background used is given for all experiments. Previously, genome-wide transcriptional profiling revealed that during iron starvation HapX activates genes involved in iron acquisition (including siderophore transporter-encoding mirB) and represses the vacuolar iron transporter-encoding cccA as well as numerous genes involved in iron-consuming processes (see below) (Schrettl et al, 2010). CccA-mediated vacuolar iron storage was recently shown to mediate iron resistance (Gsaller et al, 2012). Consistently, the cccA transcript level is upregulated by iron and particularly by SreA-deficiency (Gsaller et al, 2012). The latter is consistent with SreA-deficiency increasing the cellular iron content (Schrettl et al, 2008) but also shows that transcriptional activation of cccA is mediated by an SreA-independent regulatory mechanism. Northern analysis demonstrated that HapX-deficiency (strain ∆hapX) impairs not only repression of cccA during iron starvation but also induction of cccA during a 1-h shift from iron starvation to iron sufficiency as well as during growth in high-iron medium (Fig 1A). As shown previously (Schrettl et al, 2010), HapX-deficiency caused downregulation of mirB during iron starvation, but did not affect repression of mirB by iron (Fig 1A). Figure 1. HapX is important for adaptation to both iron limitation and iron excess HapX represses cccA during iron starvation and activates cccA during iron excess. Northern analysis was performed with liquid cultures under conditions of iron starvation (−Fe), iron sufficiency (+Fe, 0.03 mM FeSO4), and high-iron availability (hFe, 3 mM FeSO4) at 37°C for 24 h or from mycelia shifted for 1 h from −Fe to +Fe (sFe). On agar plates, HapX-deficiency impairs sporulation on BPS-plates, and growth during iron excess. Growth pattern of wild-type (wt), ∆hapX and ∆hapX∆cccA on solid minimal media containing the indicated iron concentration is shown after 48 h at 37°C. The greenish color of the fungal colonies originates from the spore pigment, and its decrease indicates reduced sporulation. The original size of fungal colony photographs is 2.3 × 2.3 cm in all figures. HapX-deficiency impairs submerged growth during both iron starvation and iron excess. Liquid biomass production was monitored after 24 h of growth at 37°C under the indicated iron availability. The data represent the mean ± standard deviation (SD) of biological triplicates. The difference between mutant and wild-type strains was statistically significant during −Fe and hFe but not +Fe (two-tailed, unpaired t-test; P < 0.05). Data information: The iron-sensitive phenotype of ∆cccA was previously analyzed in Gsaller et al (2012) and was further characterized in Fig 2. Moreover, the response of hapX transcript levels to a 1-h shift from iron starvation to sufficiency (sFe) was analyzed in Fig 4. Strains are derivatives of A. fumigatus AfS77. Download figure Download PowerPoint The role of HapX in transcriptional control of cccA during iron excess implicated a role of HapX in iron detoxification. In agreement, HapX-deficiency not only decreased sporulation on agar plates in the presence of the iron starvation-inducing, iron-specific chelator bathophenanthroline disulfonate (BPS) and decreased biomass production in liquid cultures during iron starvation, as shown previously (Schrettl et al, 2010), but also dramatically decreased growth on solid and in liquid high-iron media (Fig 1B and C). As reported previously (Schrettl et al, 2010), HapX-deficiency did affect neither growth rate nor sporulation under iron-replete conditions. A mutant strain lacking both HapX and CccA, ∆hapX∆cccA, displayed the same growth pattern as ∆hapX on solid and in liquid media (Fig 1B and C). The epistasis of HapX- to CccA-deficiency strongly suggests that lack of cccA expression is responsible for the ∆hapX growth defect during iron excess. Taken together, HapX acts as a Janus-type transcription factor mediating both repression and activation of cccA and consequently vacuolar iron storage depending on the ambient iron availability. HapX additionally controls CccA-independent mechanisms involved in iron detoxification Notably, HapX-deficiency rendered A. fumigatus more susceptible to iron toxicity than CccA-deficiency on solid (Fig 2A) and in liquid (Supplementary Table S1) high-iron media. These data indicate that HapX is also required for the activity of iron detoxification mechanisms other than CccA-mediated iron storage. In support, conditional expression of cccA using the xylose-inducible xylP promoter (Zadra et al, 2000; Gsaller et al, 2012) increased iron resistance of ∆hapX under inducing but not repressing conditions (Fig 2A; compare strains ∆hapX and ∆hapXcccAOE). However, the radial growth of this strain did not reach that of the wild-type or the ∆cccAcccAOE strain (a cccA deletion mutant expressing cccA under control of the inducible xylP promoter). Compared to ∆hapX, ∆hapXcccAOE also displayed a significant decrease in growth and sporulation on BPS- and low-iron agar plates, demonstrating that activation of vacuolar iron storage is particularly detrimental in a HapX-deficient background. Figure 2. HapX-deficiency renders A. fumigatus more susceptible to iron toxicity than CccA-deficiency and impairs induction of genes involved in iron-consuming processes Strains were grown on solid minimal medium with the given iron availability under xylP-driven cccAOE non-inducible (0% xylose) and inducible (0.5% xylose) conditions for 48 h at 37°C. Northern blot analysis was performed after liquid growth for 24 h at 37°C under iron limitation and a subsequent 1-h shift to iron sufficiency (sFe). rRNA is shown as a control for RNA quantity and quality. Data information: Strains are derivatives of A. fumigatus ATCC 46645. Download figure Download PowerPoint As previously indicated by genome-wide transcriptional profiling (Schrettl et al, 2010), apart from cccA numerous other genes involved in iron-consuming processes are repressed by HapX during iron starvation. Northern analysis revealed that in a 1-h shift from iron-limited to iron-replete conditions, which reflects short-term iron excess, HapX-deficiency impairs the transcriptional activation not only of cccA but also of genes encoding iron-consuming functions (Fig 2B). These proteins include the iron–sulfur cluster-containing LeuA (α-isopropylmalate isomerase) and AcoA (aconitase), involved in leucine biosynthesis and the TCA cycle, respectively, the heme-containing CycA (cytochrome c) involved in respiration, and the heme biosynthesis protein HemA (δ-aminolevulinic acid synthase). These data indicate that HapX might help to detoxify iron excess via general upregulation of iron-dependent proteins and processes. In agreement with the iron-detoxifying activity of iron-dependent proteins, overexpression of iron–sulfur cluster enzymes has been shown to attenuate iron toxicity in S. cerevisiae (Li et al, 2011). HapX levels are significantly higher during iron starvation compared to sufficiency or excess of iron In contrast to iron sufficiency, A. fumigatus hapX and its orthologs in A. nidulans, F. oxysporum, C. albicans, S. pombe, and C. neoformans, are transcriptionally upregulated by iron starvation (Mercier et al, 2006; Hortschansky et al, 2007; Jung et al, 2010; Schrettl et al, 2010; Hsu et al, 2011; Lopez-Berges et al, 2012). Remarkably, Northern analysis did not detect hapX transcripts during iron excess despite the HapX requirement under this condition (Fig 1A). To increase the sensitivity of transcript detection, hapX transcript levels were quantified by qRT-PCR and compared to that of sreA (Fig 3A). This analysis confirmed highest hapX expression during iron starvation, i.e. 33-fold higher compared to iron sufficiency, and 17-fold downregulation after a 1-h shift from iron starvation to iron sufficiency. As reported previously (Schrettl et al, 2008), sreA expression was increased (about threefold) during iron sufficiency compared to iron starvation and highly upregulated (about 29-fold) during a 1-h shift from iron starvation to iron sufficiency. During iron excess, a condition in which SreA was previously found to be important for iron resistance (Schrettl et al, 2008; Gsaller et al, 2012), the sreA transcript level was about threefold increased compared to that of hapX (data not shown), i.e. hapX was clearly expressed, although below the Northern sensitivity level. Figure 3. HapX production decreases during ambient and high-iron availability qRT-PCR revealing iron-dependent sreA and hapX transcript abundance. Transcript levels of hapX and sreA were determined during iron starvation (−Fe), iron sufficiency (+Fe, 0.03 mM), iron excess (hFe, 3 mM) and after a 1-h shift from iron starvation to iron sufficiency (sFe) and normalized to that of γ-actin (AFUA_6G04740) using the method. Data represent the mean ± SD of two biological and three PCR technical replicates and are presented relative to the transcript levels during iron sufficiency. All differences found are statistically significant with exception of the hapX transcript level during iron sufficiency compared to high-iron conditions (two-tailed, unpaired t-test; P < 0.05). In epifluorescence microscopy, HapXVENUS is detectable in the nuclei only during iron starvation. 104 spores of the respective strain were grown in 24-well plates in liquid media at 37°C for 18 h. DAPI was used for staining of nuclei. Western blot analysis after GFP-trap enrichment revealing significantly increased HapXVENUS production during iron starvation. The molecular mass of HapXVENUS is 80.2 kDa (27.2 kDa Venus + 53 kDa HapX). Data information: Strains are derivatives of A. fumigatus AfS77. Download figure Download PowerPoint To analyze the expression and localization of A. fumigatus HapX at the protein level, we generated an A. fumigatus strain expressing HapX N-terminally tagged with the Venus fluorescent protein (a derivative of yellow fluorescent protein) (Nagai et al, 2002), under the control of the endogenous hapX promoter in single copy at the hapX locus in ∆hapX (strain hapXVENUS). This cured all mutant phenotypes on solid and in liquid media, indicating that the HapXVENUS protein is fully functional (Supplementary Table S1). In agreement with the transcriptional data, in epifluorescence microscopy HapXVENUS was detectable during iron starvation but not during iron sufficiency, iron excess or a 1-h shift from iron starvation to iron sufficiency (Fig 3B). As previously observed in A. nidulans and S. pombe (Hortschansky et al, 2007; Mercier & Labbe, 2009), A. fumigatus HapXVENUS localized to the nucleus during iron starvation. These data indicate that lower protein levels of HapX are required for its functions during iron excess compared to iron starvation. Consistently, S-tagged HapX (strain hapXR) was detectable only during iron starvation (Fig 4E) but not during iron excess (data not shown) in Western blot analyses. These data also demonstrate that expression pattern-based prediction of gene functions can be misleading. In order to increase the sensitivity of HapX protein detection, we enriched HapXVENUS by GFP-trap, a commercially available GFP pull-down (Rothbauer et al, 2008), before Western blot analysis with a GFP-directed antiserum was applied. This way, the HapXVENUS protein was detected in mycelia grown under iron starvation, iron-replete as well as high-iron conditions with the lowest amount present under high-iron conditions (Fig 3C). Under iron starvation, significant HapX proteolyses was found. Most likely, the Venus-HapX degradation was caused during the non-denaturating GFP enrichment procedure. The highly increased degradation during iron starvation conditions can be explained by the strong induction of protease activity during iron starvation conditions (data not shown and Supplementary Table S2). However, it cannot be ruled out that this proteolysis reflects a higher HapX turnover during iron starvation, which might be related to the increased transcript level under this condition. The reduced HapX protein content during iron excess compared to iron starvation might be explained by the reduced number of target genes expressed under this condition. Figure 4. CRR-B and, to a lesser degree, CRR-A are crucial for iron detoxification but not adaptation to iron starvation Schematic view of the HapX Cys and domain organization including comparisons of HapX orthologs from A. fumigatus, A. nidulans, C. albicans, S. pombe, Ustilago maydis Yap1, and S. cerevisiae Yap5. Strains were grown for 48 h at 37°C on agar plates with the given iron concentration. Production of biomass during iron starvation (−Fe), iron sufficiency (0.03 mM, +Fe), and iron excess (3 mM, hFe), as well as production of siderophores under iron starvation was monitored after liquid growth for 24 h at 37°C. The data represent the mean ± SD of biological triplicates; the values were normalized to that of strain hapXR carrying a non-mutated S-tagged hapX. Statistically significant differences compared to hapXR are shown in red (two-tailed, unpaired t-test; P < 0.05). Original data with standard deviations are given in Supplementary Table S1. Northern blot analyses were performed after liquid growth for 24 h at 37°C under iron limitation (−Fe) or after a subsequent 1-h shift into iron sufficiency (sFe). rRNA is shown as a control for RNA quantity and quality. Western blot analyses were performed after liquid growth for 24 h at 37°C under iron limitation using antisera recognizing the S-tag for detection of HapX, or porin as control for loading. We were unable to detect S-tagged HapX during iron sufficiency or high-iron conditions with this method (data not shown). Data information: For simplicity, only one mutant per CRR is shown, the respective, phenotypically identical second mutant is shown in Supplementary Fig S3. Strains are derivatives of A. fumigatus AfS77. Download figure Download PowerPoint Two cysteine-rich regions (CRR), CRR-A and CRR-B, are crucial for HapX-mediated iron resistance Aspergillus fumigatus HapX, 491 amino acid residues in length, contains the following domains: a "b(ZIP)" basic and a "coiled-coil" domain, which together mediate DNA-binding in bZIP-type transcription factors, and an N-terminal CBC-binding domain that is essential for HapX function due to its requirement for interaction with the CBC subunit HapE (Hortschansky et al, 2007). Moreover, HapX harbors the enormous number of 19 cysteine residues (Cys), whereby 16 are organized in 4 clusters, termed CRR-A, CRR-B, CRR-C, and CRR-D containing four Cys each (Fig 4). Two single Cys (Cys115 and Cys126) are localized in the coiled-coil region, and another one (Cys422) is localized in the C-terminus. The importance of these Cys is supported by their evolutionary conservation, for example all Cys are conserved in seven Aspergillus species (Supplementary Fig S1); CRR-A, CRR-B, CRR-C as well as the C-terminal Cys are conserved even in distantly related fungal species such as C. albicans (Fig 4A and Supplementary Fig S2). Due to the potential role of Cys in iron sensing (Lill et al, 2012), we studied the impact of 11 of these Cys on HapX functions by site-directed mutagenesis replacing Cys by alanine residues (Fig 4 and Supplementary Fig S3). This analysis included all three single as well as two Cys from each CRR. For simplicity, only one mutant per CRR is shown in Fig 4, the respective, phenotypically identical second mutant is shown in Supplementary Fig S3. All analyzed hapX versions, including the non-mutated (strain hapXR), were expressed under the control of the endogenous promoter, contained a C-terminal S-tag (Terpe, 2003) and were integrated at the hapX locus in the ∆hapX strain. Mutations in CRR-B (strains hapXB1C277A and hapXB3C286A) dramatically decreased adaptation to iron excess, similar to HapX-deficiency (∆hapX), reflected by decreased radial growth under high-iron conditions, decreased biomass production in high-iron media as well as impaired transcriptional induction of cccA and leuA during a shift from iron starvation to iron sufficiency (Fig 4 and Supplementary F
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