The ability of the pathogenic fungus Candida albicans to switch from a yeast to a hyphal morphology in response to external signals is implicated in its pathogenicity. We used glass DNA microarrays to investigate the transcription profiles of 6333 predicted ORFs in cells undergoing this transition and their responses to changes in temperature and culture medium. We have identified several genes whose transcriptional profiles are similar to those of known virulence factors that are modulated by the switch to hyphal growth caused by addition of serum and a 37 degrees C growth temperature. Time course analysis of this transition identified transcripts that are induced before germ tube initiation and shut off later in the developmental process. A strain deleted for the Efg1p and Cph1p transcription factors is defective in hyphae formation, and its response to serum and increased temperature is almost identical to the response of a wild-type strain grown at 37 degrees C in the absence of serum. Thus Efg1p and Cph1p are needed for the activation of the transcriptional program that is induced by the presence of serum.

Glycolysis is a metabolic pathway that is central to the assimilation of carbon for either respiration or fermentation and therefore is critical for the growth of all organisms. Consequently, glycolytic transcriptional regulation is important for the metabolic flexibility of pathogens in their attempts to colonize diverse niches. We investigated the transcriptional control of carbohydrate metabolism in the human fungal pathogen Candida albicans and identified two factors, Tye7p and Gal4p, as key regulators of glycolysis. When respiration was inhibited or oxygen was limited, a gal4tye7 C. albicans strain showed a severe growth defect when cultured on glucose, fructose or mannose as carbon sources. The gal4tye7 strain displayed attenuated virulence in both Galleria and mouse models as well, supporting the connection between pathogenicity and metabolism. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling revealed that Tye7p bound the promoter sequences of the glycolytic genes and activated their expression during growth on either fermentable or non-fermentable carbon sources. Gal4p also bound the glycolytic promoter sequences and activated the genes although to a lesser extent than Tye7p. Intriguingly, binding and activation by Gal4p was carbon source-dependent and much stronger during growth on media containing fermentable sugars than on glycerol. Furthermore, Tye7p and Gal4p were responsible for the complete induction of the glycolytic genes under hypoxic growth conditions. Tye7p and Gal4p also regulated unique sets of carbohydrate metabolic genes; Tye7p bound and activated genes involved in trehalose, glycogen, and glycerol metabolism, while Gal4p regulated the pyruvate dehydrogenase complex. This suggests that Tye7p represents the key transcriptional regulator of carbohydrate metabolism in C. albicans and Gal4p provides a carbon source-dependent fine-tuning of gene expression while regulating the metabolic flux between respiration and fermentation pathways.

We have determined the transcriptional response of the budding yeast Saccharomyces cerevisiae to cold. Yeast cells were exposed to 10 degrees C for different lengths of time, and DNA microarrays were used to characterize the changes in transcript abundance. Two distinct groups of transcriptionally modulated genes were identified and defined as the early cold response and the late cold response. A detailed comparison of the cold response with various environmental stress responses revealed a substantial overlap between environmental stress response genes and late cold response genes. In addition, the accumulation of the carbohydrate reserves trehalose and glycogen is induced during late cold response. These observations suggest that the environmental stress response (ESR) occurs during the late cold response. The transcriptional activators Msn2p and Msn4p are involved in the induction of genes common to many stress responses, and we show that they mediate the stress response pattern observed during the late cold response. In contrast, classical markers of the ESR were absent during the early cold response, and the transcriptional response of the early cold response genes was Msn2p/Msn4p independent. This implies that the cold-specific early response is mediated by a different and as yet uncharacterized regulatory mechanism.

We used transcription profiling in Candida albicans to investigate cellular regulation involving cAMP. We found that many genes require the adenylyl cyclase Cdc35p for proper expression. These include genes encoding ribosomal subunit proteins and RNA polymerase subunit proteins, suggesting that growth could be controlled in part by cAMP-mediated modulation of gene expression. Other genes influenced by loss of adenylyl cyclase are involved in metabolism, the cell wall, and stress response and include a group of genes of unknown function that are unique to C. albicans. The profiles generated by loss of the adenylyl cyclase regulator Ras1p and a downstream effector Efg1p were also examined. The loss of Ras1p function disturbs the expression of a subset of the genes regulated by adenylyl cyclase, suggesting both that the primary role of Ras1p in transcriptional regulation involves its influence on the function of Cdc35p and that there are Ras1p independent roles for Cdc35p. The transcription factor Efg1p is also needed for the expression of many genes; however, these genes are distinct from those modulated by Cdc35p with the exception of a class of hyphal-specific genes. Therefore transcription profiling establishes that cAMP plays a key role in the overall regulation of gene expression in C. albicans, and enhances our detailed understanding of the circuitry controlling this regulation. INTRODUCTIONcAMP is an important regulatory molecule in both prokaryotes and eukaryotes. In fungi this molecule has been implicated in a variety of cellular processes. For example, in Magnaporthe grisea cells with mutations in the gene encoding adenylyl cyclase have a reduced vegetative growth rate, are sterile, and are defective in forming appresoria and thus are unable to infect susceptible rice leaves (Choi and Dean, 1997). In Ustilago maydis, mutants of adenylyl cyclase cause constitutively filamentous growth, (Gold et al., 1994), whereas strains with defects in the gene encoding the regulatory subunit of protein kinase A fail to induce tumors in plants (Gold et al., 1997). In Cryptococcus neoformans, the cAMP signaling pathway regulates several important cellular processes including capsule production, melanin formation, mating, and virulence (Alspaugh et al., 1997). In Neurospora crassa, cAMP regulates morphology, conidiation, mating, and stress responses (Lengeler et al., 2000), whereas in Schizosaccharomyces pombe cAMP signaling plays a role in mating, sporulation, gluconeogenesis, and entry into stationary phase (D'Souza and Heitman, 2001).Perhaps the best studied fungal cAMP signaling network is that of the model eukaryote Saccharomyces cerevisiae (D'Souza and Heitman, 2001), where cAMP is essential for growth and regulates nutrient sensing, stress responses, and pseudohyphal differentiation. Adenylyl cyclase, encoded by the CDC35/CYR1 gene, appears primarily regulated by GTPases. These regulators include a pair of Ras homologues, encoded by RAS1 and RAS2 (Toda et al., 1985), and a G␣ subunit encoded by GPA2 (Kubler et al.,...

Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and, in the S. cerevisiae lineage, this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes aCandida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of theCEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, andSTE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that theC. albicans CST20, HST7, CEK1, andCPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of theHST7 and CPH1 genes, disruption of theCEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

SummaryTreatments that perturb DNA synthesis or mitosis will activate checkpoints that prevent cell cycle progression and cell proliferation. In yeast-form cells of the fungal pathogen Candida albicans , exposure to hydroxyurea (HU) or shutting off expression of the polo-like kinase CaCDC5 blocked nuclear division and spindle elongation, but activated a highly polarized growth mode. We have used transcription profiling both to characterize the initiation and progression of this polar growth pattern and to determine how cell elongation may be linked to the cell cycle in C. albicans . Different gene expression patterns during early stages of cell elongation support the concept that CaCdc5p-depleted and HU-exposed cells were blocked at different stages of the cell cycle, and suggest that different signals may generate the common polarized growth phenotype. Consistent with this, BUB2 expression was modulated in CaCdc5p-depleted cells, and absence of BUB2 prevented the maintenance of cell polarization, resulting in multibudded, pseudohyphal cells with constrictions. In contrast, HU-induced filaments did not modulate or require BUB2 , but were dependent on the GTPase Ras1p. However, at later stages of cell elongation, transcription profiles were more similar, and comparisons with serum-induced hyphae revealed that the cell cycle-arrested filaments expressed several targets of the hyphal signalling pathways. Thus, arresting the yeast cell cycle in S or M phase generates a polarized growth pattern through different mechanisms in C. albicans , and maintenance of the polar growth mode can ultimately lead to the expression of hyphal-associated cell wall and virulence-related factors, in the absence of any external stimuli.

Background: Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common diseasecausing microbial agent.