Candida albicans Hap43 Is a Repressor Induced under Low-Iron Conditions and Is Essential for Iron-Responsive Transcriptional Regulation and Virulence

ABSTRACT Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies but causes life-threatening infections in immunocompromised patients. In addition to innate and adaptive immunities, hosts also resist microbial infections by developing a mechanism of “natural resistance” that maintains a low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this iron-deprived environment to cause infections. There are three types of iron-responsive transcriptional regulators in fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccharomyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43, which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 under iron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans under low-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for iron acquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demonstrated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in the nucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the global corepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplay among Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other iron-responsive metabolic activities.

[1]  Hubertus Haas,et al.  HapX-Mediated Adaption to Iron Starvation Is Crucial for Virulence of Aspergillus fumigatus , 2010, PLoS pathogens.

[2]  B. Spellberg,et al.  The iron chelator deferasirox enhances liposomal amphotericin B efficacy in treating murine invasive pulmonary aspergillosis. , 2010, The Journal of antimicrobial chemotherapy.

[3]  Alexander D. Johnson,et al.  A Phenotypic Profile of the Candida albicans Regulatory Network , 2009, PLoS genetics.

[4]  P. Blaiseau,et al.  KlAft, the Kluyveromyces lactis Ortholog of Aft1 and Aft2, Mediates Activation of Iron-Responsive Transcription Through the PuCACCC Aft-Type Sequence , 2009, Genetics.

[5]  D. Kornitzer.  Fungal mechanisms for host iron acquisition. , 2009, Current opinion in microbiology.

[6]  A. Sibirny,et al.  Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation , 2009, BioMetals.

[7]  M. Marques,et al.  Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus , 2009, Nucleic acids research.

[8]  S. Labbé,et al.  Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1* , 2009, The Journal of Biological Chemistry.

[9]  A. Sibirny,et al.  Production of flavin mononucleotide by metabolically engineered yeast Candida famata. , 2009, Metabolic engineering.

[10]  B. Spellberg,et al.  Iron acquisition: a novel perspective on mucormycosis pathogenesis and treatment , 2008, Current opinion in infectious diseases.

[11]  B. Séraphin,et al.  Regulation of ARE transcript 3′ end processing by the yeast Cth2 mRNA decay factor , 2008, The EMBO journal.

[12]  Hubertus Haas,et al.  SreA-mediated iron regulation in Aspergillus fumigatus , 2008, Molecular microbiology.

[13]  Hubertus Haas,et al.  Siderophores in fungal physiology and virulence. , 2008, Annual review of phytopathology.

[14]  H. Drakesmith,et al.  Viral infection and iron metabolism , 2008, Nature Reviews Microbiology.

[15]  Ji-Liang Tang,et al.  The Zur of Xanthomonas campestris functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters , 2008, Nucleic acids research.

[16]  J. Rine,et al.  Sre1, an iron-modulated GATA DNA-binding protein of iron-uptake genes in the fungal pathogen Histoplasma capsulatum. , 2008, Biochemistry.

[17]  D. Richardson,et al.  Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence. , 2008, Trends in microbiology.

[18]  Mingchun Li,et al.  Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors , 2008, Eukaryotic Cell.

[19]  Z. Chi,et al.  Isolation and characterization of Candida membranifaciens subsp. flavinogenie W14-3, a novel riboflavin-producing marine yeast. , 2008, Microbiological research.

[20]  D. Tollervey,et al.  The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3′ processing pathway , 2008, Nucleic acids research.

[21]  J. Bähler,et al.  Key Function for the CCAAT-Binding Factor Php4 To Regulate Gene Expression in Response to Iron Deficiency in Fission Yeast , 2008, Eukaryotic Cell.

[22]  J. Lloyd,et al.  Secretion of Flavins by Shewanella Species and Their Role in Extracellular Electron Transfer , 2007, Applied and Environmental Microbiology.

[23]  C. Philpott,et al.  Response to Iron Deprivation in Saccharomyces cerevisiae , 2007, Eukaryotic Cell.

[24]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[25]  B. Spellberg,et al.  The iron chelator deferasirox protects mice from mucormycosis through iron starvation. , 2007, The Journal of clinical investigation.

[26]  D. Barra,et al.  A GATA-type transcription factor regulates expression of the high-affinity iron uptake system in the methylotrophic yeast Pichia pastoris. , 2007, Archives of biochemistry and biophysics.

[27]  Masashi Kato,et al.  Interaction of HapX with the CCAAT‐binding complex—a novel mechanism of gene regulation by iron , 2007, The EMBO journal.

[28]  J. Kronstad,et al.  Iron Regulation of the Major Virulence Factors in the AIDS-Associated Pathogen Cryptococcus neoformans , 2006, PLoS biology.

[29]  Liza Gross.  Iron Regulation and an Opportunistic AIDS-Related Fungal Infection , 2006, PLoS biology.

[30]  B. Spellberg,et al.  Deferiprone iron chelation as a novel therapy for experimental mucormycosis. , 2006, The Journal of antimicrobial chemotherapy.

[31]  Nuria Pujol‐Carrion,et al.  Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae , 2006, Journal of Cell Science.

[32]  R. Lill,et al.  Iron-sulfur protein biogenesis in eukaryotes: components and mechanisms. , 2006, Annual review of cell and developmental biology.

[33]  S. Labbé,et al.  A Transcription Factor Cascade Involving Fep1 and the CCAAT-Binding Factor Php4 Regulates Gene Expression in Response to Iron Deficiency in the Fission Yeast Schizosaccharomyces pombe , 2006, Eukaryotic Cell.

[34]  A. Dancis,et al.  Reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) is dependent on CaFRE10 ferric reductase for Candida albicans grown in unbuffered media. , 2006, Microbiology.

[35]  C. Philpott,et al.  Iron-dependent metabolic remodeling in S. cerevisiae. , 2006, Biochimica et biophysica acta.

[36]  D. Winge,et al.  Role of Glutaredoxin-3 and Glutaredoxin-4 in the Iron Regulation of the Aft1 Transcriptional Activator in Saccharomyces cerevisiae* , 2006, Journal of Biological Chemistry.

[37]  J. Tamarit,et al.  Colorimetric assay for the quantitation of iron in yeast. , 2006, Analytical biochemistry.

[38]  H. Rogers,et al.  Natural resistance, iron and infection: a challenge for clinical medicine. , 2006, Journal of medical microbiology.

[39]  J. Morschhäuser,et al.  Candida albicans MTLα tup1Δ mutants can reversibly switch to mating‐competent, filamentous growth forms , 2005 .

[40]  David S. McNabb,et al.  Novel Regulatory Function for the CCAAT-Binding Factor in Candida albicans , 2005, Eukaryotic Cell.

[41]  M. Moore,et al.  The Aspergillus fumigatus Siderophore Biosynthetic Gene sidA, Encoding l-Ornithine N5-Oxygenase, Is Required for Virulence , 2005, Infection and Immunity.

[42]  Jean-Michel Camadro,et al.  Direct Activation of Genes Involved in Intracellular Iron Use by the Yeast Iron-Responsive Transcription Factor Aft2 without Its Paralog Aft1 , 2005, Molecular and Cellular Biology.

[43]  C. Russell,et al.  Expression of one-hybrid fusions with Staphylococcus aureus lexA in Candida albicans confirms that Nrg1 is a transcriptional repressor and that Gcn4 is a transcriptional activator. , 2005, Fungal genetics and biology : FG & B.

[44]  Yang-Nim Park,et al.  Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans , 2005, Eukaryotic Cell.

[45]  A. Sibirny,et al.  Positive selection of mutants defective in transcriptional repression of riboflavin synthesis by iron in the flavinogenic yeast Pichia guilliermondii. , 2005, FEMS yeast research.

[46]  T. Rouault,et al.  Iron–sulphur cluster biogenesis and mitochondrial iron homeostasis , 2005, Nature Reviews Molecular Cell Biology.

[47]  R. Lill,et al.  Iron-sulfur-protein biogenesis in eukaryotes. , 2005, Trends in biochemical sciences.

[48]  D. Thiele,et al.  Coordinated Remodeling of Cellular Metabolism during Iron Deficiency through Targeted mRNA Degradation , 2005, Cell.

[49]  Ken Haynes,et al.  Siderophore Biosynthesis But Not Reductive Iron Assimilation Is Essential for Aspergillus fumigatus Virulence , 2004, The Journal of experimental medicine.

[50]  R. Kolter,et al.  The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. , 2004, Gene.

[51]  George Newport,et al.  Regulatory networks affected by iron availability in Candida albicans , 2004, Molecular microbiology.

[52]  M. Hentze,et al.  Balancing Acts Molecular Control of Mammalian Iron Metabolism , 2004, Cell.

[53]  D. Howard.  Iron gathering by zoopathogenic fungi. , 2004, FEMS immunology and medical microbiology.

[54]  Ledion Bitincka,et al.  MatGAT: An application that generates similarity/identity matrices using protein or DNA sequences , 2003, BMC Bioinformatics.

[55]  S. Macaskill,et al.  Gcn4 co‐ordinates morphogenetic and metabolic responses to amino acid starvation in Candida albicans , 2002, The EMBO journal.

[56]  E. Craig,et al.  A specialized mitochondrial molecular chaperone system:¶A role in formation of Fe/S centers , 2002, Cellular and Molecular Life Sciences CMLS.

[57]  M. Schaller,et al.  The Siderophore Iron Transporter of Candida albicans (Sit1p/Arn1p) Mediates Uptake of Ferrichrome-Type Siderophores and Is Required for Epithelial Invasion , 2002, Infection and Immunity.

[58]  Y. Mukai,et al.  Fep1, an Iron Sensor Regulating Iron Transporter Gene Expression in Schizosaccharomyces pombe * , 2002, The Journal of Biological Chemistry.

[59]  Tetsuo Kobayashi,et al.  Isolation of genes encoding novel transcription factors which interact with the Hap complex from Aspergillus species. , 2002, Biochimica et biophysica acta.

[60]  D. Soll,et al.  Roles of TUP1 in Switching, Phase Maintenance, and Phase-Specific Gene Expression in Candida albicans , 2002, Eukaryotic Cell.

[61]  P. Brown,et al.  A second iron-regulatory system in yeast independent of Aft1p , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[62]  F. Tekaia,et al.  Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1 , 2001, Molecular microbiology.

[63]  P. Blaiseau,et al.  Aft2p, a Novel Iron-regulated Transcription Activator That Modulates, with Aft1p, Intracellular Iron Use and Resistance to Oxidative Stress in Yeast* , 2001, The Journal of Biological Chemistry.

[64]  H. Haas,et al.  Identification of members of the Aspergillus nidulans SREA regulon: genes involved in siderophore biosynthesis and utilization. , 2001, Biochemical Society transactions.

[65]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[66]  J. Posey,et al.  Lack of a role for iron in the Lyme disease pathogen. , 2000, Science.

[67]  N. Ramanan,et al.  A high-affinity iron permease essential for Candida albicans virulence. , 2000, Science.

[68]  A. Johnson,et al.  TUP1, CPH1 and EFG1 make independent contributions to filamentation in candida albicans. , 2000, Genetics.

[69]  E. Lesuisse,et al.  Iron uptake by the yeast Pichia guilliermondii. Flavinogenesis and reductive iron assimilation are co-regulated processes , 1999, Biometals.

[70]  D. Howard.  Acquisition, Transport, and Storage of Iron by Pathogenic Fungi , 1999, Clinical Microbiology Reviews.

[71]  H. Haas,et al.  The Aspergillus nidulans GATA Factor SREA Is Involved in Regulation of Siderophore Biosynthesis and Control of Iron Uptake* , 1999, The Journal of Biological Chemistry.

[72]  M. Bolotin-Fukuhara,et al.  HAP4, the glucose‐repressed regulated subunit of the HAP transcriptional complex involved in the fermentation–respiration shift, has a functional homologue in the respiratory yeast Kluyveromyces lactis , 1999, Molecular microbiology.

[73]  G. Marzluf,et al.  Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa , 1998, Molecular and General Genetics MGG.

[74]  M. Schaller,et al.  Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity , 1998, Molecular microbiology.

[75]  P. Robbins,et al.  Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene , 1998, Infection and Immunity.

[76]  D. McNabb,et al.  The Saccharomyces cerevisiae Hap5p homolog from fission yeast reveals two conserved domains that are essential for assembly of heterotetrameric CCAAT-binding factor , 1997, Molecular and cellular biology.

[77]  A. Johnson,et al.  Control of filament formation in Candida albicans by the transcriptional repressor TUP1. , 1997, Science.

[78]  H. Haas,et al.  Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene. , 1997, Gene.

[79]  R. Klausner,et al.  AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. , 1995, The EMBO journal.

[80]  A. Brown,et al.  Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans , 1994, Molecular microbiology.

[81]  P. Xu,et al.  urbs1, a gene regulating siderophore biosynthesis in Ustilago maydis, encodes a protein similar to the erythroid transcription factor GATA-1. , 1993, Molecular and cellular biology.

[82]  D. Irwin,et al.  Isogenic strain construction and gene mapping in Candida albicans. , 1993, Genetics.

[83]  J. Covès,et al.  Reduction and mobilization of iron by a NAD(P)H:flavin oxidoreductase from Escherichia coli. , 1993, European journal of biochemistry.

[84]  P. Williamson,et al.  Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization , 1992, Journal of bacteriology.

[85]  B. Halliwell,et al.  Biologically relevant metal ion‐dependent hydroxyl radical generation An update , 1992, FEBS letters.

[86]  L. Guarente,et al.  Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. , 1989, Genes & development.

[87]  B. Halliwell,et al.  Oxygen toxicity, oxygen radicals, transition metals and disease. , 1984, The Biochemical journal.

[88]  F. Archibald.  Lactobacillus plantarum, an organism not requiring iron , 1983 .

[89]  J. Morschhäuser,et al.  Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms. , 2005, Molecular microbiology.

[90]  A. Gillum,et al.  Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations , 2004, Molecular and General Genetics MGG.

[91]  S. Rupp.  LacZ assays in yeast. , 2002, Methods in enzymology.

[92]  Richard D Klausner,et al.  Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator. , 2002, Microbiology.

[93]  P. Macheroux,et al.  UV-visible spectroscopy as a tool to study flavoproteins. , 1999, Methods in molecular biology.

[94]  A. Demain,et al.  Riboflavin oversynthesis. , 1972, Annual review of microbiology.