Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications. Candida albicans is a commonly encountered fungal pathogen usually responsible for superficial infections (thrush and vaginitis). However, an estimated 30% of severe fungal infections, most due to Candida, result in death. Those who are most at risk include individuals taking immune-suppressive drugs following organ transplantation, people with HIV infection, premature infants, and cancer patients undergoing chemotherapy. Current therapies for this pathogen are made more difficult by the significant secondary effects of anti-fungal drugs that target proteins that are also found in the human host. Recent sequencing and assembly of the genome for the fungal pathogen C. albicans used simple automated procedures for the identification of putative genes. Here, we report a detailed annotation of the 6,354 genes that are present in the genome sequence of this organism, essentially writing the dictionary of the C. albicans genome. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that are absent from the human genome and whose products might be targeted for antifungal therapy. The results of these efforts will thus ensure that the Candida research community has uniform and comprehensive genomic information for medical research, for the development of functional genomic tools as well as for future diagnostic and therapeutic applications.

Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans . The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1 -mediated drug resistance is achieved in this fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2 but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction of MDR1 expression in wild-type C. albicans , but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans . Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans , we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA ₃ ) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA ₃ ) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA ₃ ). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA ₃ or Cap1p-CSE-HA ₃ (the binding ratio was at least twofold; P ≤ 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3′ ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets ( P ≤ 0.02) included 11 genes involved in the OSR ( CAP1 , GLR1 , TRX1 , SOD1 , CAT1 , and others), 13 genes involved in response to drugs ( PDR16 , MDR1 , FLU1 , YCF1 , FCR1 , and others), 4 genes involved in phospholipid transport ( PDR16 , GIT1 , RTA2 , and orf19.932), and 3 genes involved in the regulation of nitrogen utilization ( GST3 , orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5′-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans . To better understand Upc2p function in C. albicans , we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters ( P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis ( NCP1 , ERG11 , ERG2 , and others), 18 genes encoding ribosomal subunits ( RPS30 , RPL32 , RPL12 , and others), 3 genes encoding drug transporters ( CDR1 , MDR1 , and YOR1 ), 4 genes encoding transcription factors ( INO2 , ACE2 , SUT1 , and UPC2 ), and 6 genes involved in sulfur amino acid metabolism ( MET6 , SAM2 , SAH1 , and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5′-VNCGBDTR that includes the previously characterized Upc2p binding site 5′-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets ( ERG11 , MDR1 , CDR1 , YOR1 , SUT1 , SMF12 , and CBP1 ). The analysis of ERG11 , MDR1 , and CDR1 transcripts in wild-type and upc2 Δ/ upc2 Δ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.

A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1 - and CDR2 -mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2 , including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2 . Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN ₄ CGG), including TAC1 . In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1 -dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans .