Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans

ABSTRACT Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 μM, a plateau between 1 and 25 μM, and a second steep rise above 25 μM, consistent with high- and low-affinity uptake systems. AKm for high-affinity uptake was estimated to be 0.6 μM Fe(II); 1 μM was used for standardized uptake assays. At this concentration, the uptake rate was 110 ± 3 pmol/106 cells/h. Iron repletion (15 μM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0°C or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.

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Identification and Characterization of TUP1-Regulated Genes in Candida albicans

Genetics ◽

10.1093/genetics/156.1.31 ◽

2000 ◽

Vol 156 (1) ◽

pp. 31-44 ◽

Cited By ~ 7

Author(s):

Burkhard R Braun ◽

W Steven Head ◽

Ming X Wang ◽

Alexander D Johnson

Keyword(s):

Candida Albicans ◽

Systemic Infection ◽

Subtractive Hybridization ◽

Filamentous Growth ◽

Surface Proteins ◽

Infection Model ◽

Ferric Reductase ◽

Pathogenesis Related Protein ◽

Plant Pathogenesis ◽

Rnase T2

Abstract TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans. Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1. One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5. These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins. The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2. The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions. We produced knockout strains in C. albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media. However, two animal models for C. albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Δ and rbt4Δ strains had significantly reduced virulence. TUP1 appears, therefore, to regulate many genes in C. albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.

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Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors

Eukaryotic Cell ◽

10.1128/ec.00108-08 ◽

2008 ◽

Vol 7 (7) ◽

pp. 1168-1179 ◽

Cited By ~ 71

Author(s):

Yong-Un Baek ◽

Mingchun Li ◽

Dana A. Davis

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Candida Albicans ◽

Transcription Factors ◽

Iron Acquisition ◽

Mammalian Host ◽

Regulatory Sequence ◽

Ferric Reductase ◽

Reductase Gene ◽

Ferric Reductases

ABSTRACT Iron is an essential nutrient that is severely limited in the mammalian host. Candida albicans encodes a family of 15 putative ferric reductases, which are required for iron acquisition and utilization. Despite the central role of ferric reductases in iron acquisition and mobilization, relatively little is known about the regulatory networks that govern ferric reductase gene expression in C. albicans. Here we have demonstrated the differential regulation of two ferric reductases, FRE2 and FRP1, in response to distinct iron-limited environments. FRE2 and FRP1 are both induced in alkaline-pH environments directly by the Rim101 transcription factor. However, FRP1 but not FRE2 is also induced by iron chelation. We have identified a CCAAT motif as the critical regulatory sequence for chelator-mediated induction and have found that the CCAAT binding factor (CBF) is essential for FRP1 expression in iron-limited environments. We found that a hap5Δ/hap5Δ mutant, which disrupts the core DNA binding activity of CBF, is unable to grow under iron-limited conditions. C. albicans encodes three CBF-dependent transcription factors, and we identified the Hap43 protein as the CBF-dependent transcription factor required for iron-limited responses. These studies provide key insights into the regulation of ferric reductase gene expression in the fungal pathogen C. albicans.

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The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31752-0 ◽

1994 ◽

Vol 269 (35) ◽

pp. 22039-22045 ◽

Cited By ~ 6

Author(s):

E. Nègre ◽

T. Vogel ◽

A. Levanon ◽

R. Guy ◽

T.J. Walsh ◽

...

Keyword(s):

Candida Albicans ◽

Binding Site ◽

Binding Domain ◽

Affinity Binding ◽

Collagen Binding ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site

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Increased high-affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1-dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans

Molecular Microbiology ◽

10.1046/j.1365-2958.2003.03692.x ◽

2003 ◽

Vol 50 (2) ◽

pp. 391-409 ◽

Cited By ~ 62

Author(s):

Yong-Sun Bahn ◽

Janet Staab ◽

Paula Sundstrom

Keyword(s):

Gene Expression ◽

Candida Albicans ◽

Cyclic Amp ◽

High Affinity ◽

Germ Tubes

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The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4342-4350.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4342-4350

Author(s):

D G Roman ◽

A Dancis ◽

G J Anderson ◽

R D Klausner

Keyword(s):

Fission Yeast ◽

Iron Uptake ◽

Ferric Iron ◽

Reductase Activity ◽

Iron Acquisition ◽

Protein A ◽

Mutant Gene ◽

Predicted Amino Acid Sequence ◽

Mrna Levels ◽

Ferric Reductase

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

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Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae

Journal of Fungi ◽

10.3390/jof7090756 ◽

2021 ◽

Vol 7 (9) ◽

pp. 756

Author(s):

Suzie Kang ◽

Hyewon Seo ◽

Min-Gyu Lee ◽

Cheol-Won Yun

Keyword(s):

Saccharomyces Cerevisiae ◽

Iron Uptake ◽

Nitrogen Starvation ◽

Copper Metabolism ◽

Nitrogen Utilization ◽

Deletion Mutants ◽

Uptake System ◽

Wild Type ◽

High Affinity ◽

Uptake Activity

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

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