Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1.

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.

NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42 degrees C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

Functional isolation of the Candida albicans FCR3 gene encoding a bZip transcription factor homologous to Saccharomyces cerevisiae Yap3p.

We have isolated a C. albicans gene, named FCR3 (for fluconazole resistance 3), based upon its ability to suppress the FCZ hypersusceptibility of a Saccharomyces cerevisiae mutant strain (JY312) lacking the transcription factors Pdr1p and Pdr3p. The FCR3 ORF (1200 bp) encodes a 399 amino acid protein containing a basic leucine zipper (bZip) domain. Fcr3p displays the highest level of sequence homology with the S. cerevisiae Yap3p protein (34% identity, 45% similarity). We had previously shown that deletion of the PDR5 gene encoding a multidrug transporter completely abolished the ability of FCR3 to suppress the FCZ hypersusceptibility of JY312, suggesting that FCR3 confers FCZ resistance by activating PDR5 expression. We show here that the beta-galactosidase activity of a PDR5 promoter-lacZ construct in JY312 is increased two-fold upon FCR3 overexpression, demonstrating that FCR3 regulates PDR5 at the transcriptional level. We also show that FCR3 overexpression not only suppresses the hypersusceptibility of JY312 to 4-nitroquinoline-N-oxide (4-NQO) but also confers higher levels of resistance to this compound as compared to the wild-type KY320 strain. Since PDR5 is not involved in 4-NQO resistance, this result indicates that FCR3 can also activate the transcription of other genes that can confer 4-NQO resistance. Finally, Northern blot analysis indicates that FCR3 encodes a single 2.4 kb RNA transcript in C. albicans, suggesting that the FCR3 mRNA contains long 5' and/or 3' untranslated regions. The nucleotide sequence of the FCR3 gene has been deposited at GenBank under Accession No. AF342983.

Mitochondrial control of iron homeostasis. A genome wide analysis of gene expression in a yeast frataxin-deficient strain.

Deletion of YFH1, the yeast frataxin homologue gene, elicits mitochondrial iron accumulation and alters cellular iron homeostasis. Here, we report a genome wide analysis of gene expression in a yfh1(DeltaYFH1) deleted strain. Frataxin deficiency results in enhanced expression of some 70 genes including a set of genes, called the iron regulon, that are under the control of the iron-sensing transcription factor AFT1. Five new AFT1-dependent genes, YOR382w, YOR383c, YDR534c, YLR136c, and YLR205c were found. The first three genes presumably encode cell-wall glycosylphosphatidylinositol anchor proteins and exhibit a 30-100-fold increased expression. The triple deletion of these genes decreases efficiency in utilization of the iron of ferrioxamine B by the yeast cell. YLR136c bears homology to tristetraproline proteins, which are post-transcriptional regulators in mammalian cells. Deletion of YLR136c increases the mRNA levels of iron regulon members. YLR205c bears homology to heme oxygenases. Our data show that frataxin deficiency elicits iron mobilization from all iron sources in an AFT1-dependent manner. Wild-type and DeltaYFH1 glycerol-grown cells exhibit similar high respiration rates, no mitochondrial iron accumulation, and high expression of the iron regulon, suggesting that under these conditions little iron is extruded from mitochondria. These data suggest that the activity of Yfh1p is not essential in cells grown on glycerol. This study has also revealed unexpected links between mitochondria and remote metabolic pathways since frataxin deficiency also enhances the expression of genes such as HSP30, that escape to AFT1 control. Finally, no oxidative stress gene is induced.

Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae.

Three Candida albicans genes, designated FCR (for fluconazole resistance), have been isolated by their ability to complement the fluconazole (FCZ) hypersensitivity of a Saccharomyces cerevisiae mutant lacking the transcription factors Pdr1p and Pdr3p. Overexpression of any of the three FCR genes in the pdr1 pdr3 mutant resulted in increased resistance of the cells to FCZ and cycloheximide and in increased expression of PDR5, a gene coding for a drug efflux transporter of the ATP-binding cassette superfamily and whose transcription is under the control of Pdr1p and Pdr3p. Deletion of PDR5 in the pdr1 pdr3 strain completely abrogated the ability of the three FCR genes to confer FCZ resistance, demonstrating that PDR5 is required for FCR-mediated FCZ resistance in S. cerevisiae. The FCR1 gene encodes a putative 517-amino-acid protein with an N-terminal Zn2C6-type zinc finger motif homologous to that found in fungal zinc cluster proteins, including S. cerevisiae Pdr1p and Pdr3p. We have constructed a C. albicans CAI4-derived mutant strain carrying a homozygous deletion of the FCR1 gene and analyzed its ability to grow in the presence of FCZ. We found that the fcr1Delta/fcr1Delta mutant displays hyperresistance to FCZ and other antifungal drugs compared to the parental CAI4 strain. This hyperresistance could be reversed to wild-type levels by reintroduction of a plasmid-borne copy of FCR1 into the fcr1Delta/fcr1Delta mutant. Taken together, our results indicate that the FCR1 gene behaves as a negative regulator of drug resistance in C. albicans and constitute the first evidence that FCZ resistance can result from the inactivation of a regulatory factor such as Fcr1p.

AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily.

We have isolated a Candida albicans gene that confers resistance to the azole derivative fluconazole (FCZ) when overexpressed in Saccharomyces cerevisiae. This gene encodes a protein highly homologous to S. cerevisiae yAP-1, a bZip transcription factor known to mediate cellular resistance to toxicants such as cycloheximide (CYH), 4-nitroquinoline N-oxide (4-NQO), cadmium, and hydrogen peroxide. The gene was named CAP1, for C. albicans AP-1. Cap1 and yAP-1 are functional homologues, since CAP1 expression in a yap1 mutant strain partially restores the ability of the cells to grow on toxic concentrations of cadmium or hydrogen peroxide. We have found that the expression of YBR008c, an open reading frame identified in the yeast genome sequencing project and predicted to code for a multidrug transporter of the major facilitator superfamily, is dramatically induced in S. cerevisiae cells overexpressing CAP1. Overexpression of either CAP1 or YAP1 in a wild-type strain results in resistance to FCZ, CYH, and 4-NQO, whereas such resistance is completely abrogated (FCZ and CYH) or strongly reduced (4-NQO) in a ybr008c deletion mutant, demonstrating that YBR008c is involved in YAP1- and CAP1-mediated multidrug resistance. YBR008c has been renamed FLR1, for fluconazole resistance 1. The expression of an FLR1-lacZ reporter construct is strongly induced by the overexpression of either CAP1 or YAP1, indicating that the FLR1 gene is transcriptionally regulated by the Cap1 and yAP-1 proteins. Taken collectively, our results demonstrate that FLR1 represents a new YAP1-controlled multidrug resistance molecular determinant in S. cerevisiae. A similar detoxification pathway is also likely to operate in C. albicans.

Cis- and trans-acting elements determining induction of the genes of the gamma-aminobutyrate (GABA) utilization pathway in Saccharomyces cerevisiae.

In S. cerevisiae, gamma-aminobutyrate (GABA) induces transcription of the UGA genes required for its utilization as a nitrogen source. Analysis of the 5' region of the UGA1 and UGA4 genes led to the identification of a conserved GC-rich sequence (UASGABA) essential to induction by gamma-aminobutyrate. Alone, this UASGABA element also supported some levels of reporter gene transcription in the presence of gamma-aminobutyrate. To be effective, UASGABA requires two positive-acting proteins that both contain a Cys6-Zn2 type zinc-finger motif, namely pathway-specific Uga3p and pleiotropic Uga35p(Dal81p/DurLp). Further analysis of the UGA4 gene revealed that Gln3p, a global nitrogen regulatory protein containing a GATA zinc-finger domain, is required in order to reach high levels of gamma-aminobutyrate-induced transcription. The Gln3p factor exerts its function mainly through a cluster of 5'-GAT(A/T)A-3'(UASGATA) situated just upstream from UASGABA. The role of Gln3p is less predominant in UGA1 than in UGA4 gene expression. We propose that tight coupling between the UASGABA and UASGATA elements enables the cell to integrate, according to its nitrogen status, the induced expression levels of UGA4.

Two mutually exclusive regulatory systems inhibit UASGATA, a cluster of 5'-GAT(A/T)A-3' upstream from the UGA4 gene of Saccharomyces cerevisiae.

The S. cerevisiae Uga43(Dal80) protein down-regulates the expression of multiple nitrogen pathway genes. It contains a zinc-finger motif similar to the DNA-binding domain of the vertebrate GATA family of transcription factors; this domain is known to direct binding to 5'-GATA-3' core sequences. The inducible UGA4 gene, which encodes the specific gamma-aminobutyrate permease, undergoes strong repression by Uga43p. This study shows that the 5' region of UGA4 contains a UAS element made of four directly repeated 5'-CGAT(A/T) AG-3' sequences. This element, called UASGATA, can potentially confer to the UGA4 gene high-level expression in the absence of inducer, but this potential activity is inhibited by two distinct repression systems. One system is Uga43p-dependent; it operates in cells grown on a poor nitrogen source. The other is the nitrogen repression system, which relies on Ure2p and glutamine and operates when a good nitrogen source is present. Nitrogen repression also blocks the synthesis of Uga43p, making the two repression systems mutually exclusive. Previous studies have shown that expression supported by 5'-GATA-3'-containing UAS elements requires Gln3p, another global nitrogen regulatory factor containing a GATA zinc-finger domain. Although Gln3p contributes to UASGATA activity, evidence suggests that a second factor can potentially direct expression through UASGATA. Expression conferred by this putative factor is subject to both Uga43p- and Ure2p-mediated repression. The role of UASGATA in the expression of the UGA4 gene is discussed in relation to its sensitivity to the two distinct repression systems.