Identification of a Candida albicans Ferrichrome Transporter and Its Characterization by Expression inSaccharomyces cerevisiae

Ardon, Orly; Bussey, Howard; Philpott, Caroline C.; Ward, Diane McVey; Davis‐Kaplan, Sandra; Verroneau, Steeve; Jiang, Bo; Kaplan, Jerry

doi:10.1074/jbc.m108701200

Cited by 60 publications

(65 citation statements)

References 31 publications

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“…The copper dependence of elemental iron uptake contrasts with the copper independence of iron uptake from the siderophore ferrichrome. A single siderophore permease homologue is present in the genome of C. albicans that may mediate ferrichrome uptake (Lesuisse et al, 2001 ;Ardon et al, 2001), C. albicans may even synthesize its own siderophores (Ismail et al, 1985), providing even greater versatility in scavenging iron.…”

Section: Discussionmentioning

confidence: 99%

Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator

et al. 2002

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High-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities : an externally directed surface ferric reductase, a membrane-associated PPD ( pphenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.

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Section: Discussionmentioning

confidence: 99%

Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator

et al. 2002

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“…No vacuolar staining was seen in ⌬cwh36 cells with any of the antibodies. Assembly of the vacuolar H ϩ -ATPase is sequential, with the V 0 subunit being required for the addition of the V 1 subunit to the complex (15). Examination of VMA subunits in ⌬cwh36 cells by Western analysis revealed a marked reduction in the amount of Vph1p, but the V 1 subunits Vma1p and Vma2p were present (Fig.…”

Section: Fig 4 Iron Transport and Fet3p Immunofluorescence Studies mentioning

confidence: 98%

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

Davis‐Kaplan

Ward

Shiflett

et al. 2004

Journal of Biological Chemistry

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We conducted a genome-wide screen in the budding yeast Saccharomyces cerevisiae of 4,792 homozygous diploid deletions to identify genes that function in iron metabolism. Strains unable to grow on iron-restricted medium contained deletions of genes that encode the structural components of the high affinity iron transport system (FET3, FTR1), the iron-sensing transcription factor AFT1 or genes required for the assembly of the transport system. We also identified genes that were not previously known to play a role in iron metabolism. Deletion of the gene CWH36 resulted in a severe growth defect on iron-limited medium, as well as increased sensitivity to Congo red and calcofluor white. The ability of yeast to grow on iron-limited medium requires the activity of the high affinity iron transport system. This system is comprised of two plasma membrane proteins, the multicopper oxidase Fet3p and the transmembrane permease Ftr1p (1, 2). The genes that encode these proteins are under the control of the iron-sensing transcription factor Aft1p (3). In the absence of iron, Aft1p translocates from the cytosol to the nucleus and induces the transcription of at least 15 different genes that comprise the iron-regulon. Fet3p and Ftr1p must be coordinately synthesized for both proteins to be properly targeted through the secretory apparatus to the cell surface (4, 5). The activity of the multicopper oxidase Fet3p requires copper and copper loading of apoFet3p is dependent upon the expression of genes involved in copper transport. Copper enters the cell via the cell surface copper transporters Ctr1p, Ctr3p, and can be transported into the post-Golgi compartment in which apoFet3p is loaded via the copper transporter Ccc2p (1, 2, 6). Proper targeting of the Fet3p/Ftr1p complex to the cell surface also requires genes involved in vesicular traffic. In the absence of proper copper homeostasis or appropriate vesicular trafficking, an inactive apoFet3/Ftr1p complex is translocated to the cell surface (7,8), and cells are unable to grow on low iron medium.To identify genes required for proper targeting/expression of Fet3p, we took advantage of the low iron growth phenotype of cells lacking a functional Fet3p. Using a genomic screen, which employed an arrayed collection of homozygous diploid deletion strains, we identified genes required for growth on iron-limited medium. In particular, we characterize a novel gene that when deleted leads to defective vacuolar acidification, failure to copper load apoFet3, and consequently defective iron transport. his3⌬1/his3⌬1, ura3⌬0/ura3⌬0, leu2⌬0/leu2⌬0, lys2⌬0/ϩ, met15⌬0/ϩ) from Research Genetics was employed for the genetic screen. A 96-pin replicator was used to transfer 1 l of a cell suspension from wells in a master plate to wells in a 96-well plate containing 100 l of YPD (1% yeast extract, 2% peptone (Difco) 1.0% agarose, and 2.0% dextrose)/well. Cells were grown to saturation in a 30°C incubator (usually 48 h). The volume in the well was then brought to ϳ250 l (outside wells lost more liquid ...

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“…Genes encoding ferric reductases have been cloned from C. albicans (22), and there have been several reports on possible siderophore production by C. albicans (23)(24)(25). Either the siderophore-bound iron may be released by the ferric reductases and then picked up by the high-affinity uptake system (18, 26 -28), or the siderophoreiron complex is transported into the cell via receptor-mediated mechanisms (28,29). Thus, it is likely that the siderophoremediated iron binding may function upstream of and provide free iron to the oxidase-permease iron uptake system.…”

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confidence: 99%

“…These proteins belong to the major facilitator superfamily, whose members have been found in many organisms. Currently, one siderophore transporter gene (CaARN1) has been found in C. albicans, and its function has been investigated by heterologous expression in S. cerevisiae (28,29). Ardon et al (29) reported that CaArn1p expressed in S. cerevisiae specifically mediates the uptake of FC 1 -iron, but does not transport iron bound to FOB.…”

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confidence: 99%

Characterization and Functional Analysis of the Siderophore-Iron Transporter CaArn1p in Candida albicans

Bai

Zheng

et al. 2002

Journal of Biological Chemistry

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Siderophores are small organic compounds with high affinity for ferric iron. Microorganisms commonly acquire iron via siderophore secretion and uptake. Here we report the characterization of the siderophore transporter CaArn1p in the fungal pathogen Candida albicans. Deletion of CaARN1 reduced the ability of C. albicans to use iron bound to the hydroxamate-type siderophore ferrichrome and abolished it when two high-affinity iron permease genes (CaFTR1 and CaFTR2) were also deleted, indicating a role of CaArn1p as well as the permeases in ferrichrome-iron uptake. Caarn1⌬ (but not Caftr1⌬Caftr2⌬) assimilated iron from another hydroxamate-type siderophore, ferrioxamine B, suggesting that iron uptake from this compound depends on the permeases, but not on CaArn1p. Northern blot analysis revealed that the transcription repressor CaTup1p repressed CaARN1 expression under iron-replete conditions via the DNA-binding protein Rfg1p. Green fluorescent protein-tagged CaArn1p was observed predominantly in the plasma membrane, with some in the cytoplasm as distinct spots. The number of these spots increased with the increase in ferrichrome concentration, suggesting that CaArn1p internalization might be a mechanism for ferrichrome-iron uptake or for recycling the transporter. Caarn1⌬ did not show reduced virulence when injected into the blood stream of mice, implying that CaArn1p is not required for iron uptake along this route of infection.

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Identification of a Candida albicans Ferrichrome Transporter and Its Characterization by Expression inSaccharomyces cerevisiae

Cited by 60 publications

References 31 publications

Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator

Reductive iron uptake by Candida albicans: role of copper, iron and the TUP1 regulator

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

Characterization and Functional Analysis of the Siderophore-Iron Transporter CaArn1p in Candida albicans

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