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.

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.

Superoxide dismutases (SOD) convert superoxide radicals into less damaging hydrogen peroxide. The opportunistic human pathogen Candida albicans is known to express CuZnSOD (SOD1) and MnSOD (SOD3) in the cytosol and MnSOD (SOD2) in the mitochondria. We identified three additional CuZn-containing superoxide dismutases, SOD4, SOD5, and SOD6, within the sequence of the C. albicans genome. The transcription of SOD5 was up-regulated during the yeast to hyphal transition of C. albicans, and SOD5 was induced when C. albicans cells were challenged with osmotic or with oxidative stresses. SOD5 transcription was also increased when cells were grown on nonfermentable substrates as the only carbon source. The Rim101p transcription factor was required for all inductions observed, whereas the Efg1p transcription factor was specifically needed for serum-modulated expression. Deletion of SOD5 produced a viable mutant strain that showed sensitivity to hydrogen peroxide when cells were grown in nutrient-limited conditions. Sod5p was found to be necessary for the virulence of C. albicans in a mouse model of infection. However, the sod5 mutant strain showed the same resistance to macrophage attack as its parental strain, suggesting that the loss of virulence in not due to an increased sensitivity to macrophage attack.

ABSTRACTPhagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals againstCandida albicansinfection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains ofC. albicans. We used an end point dilution assay to monitor the killing ofC. albicansat low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candidainteraction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutantCandidacells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candidaactivity. Continued coculture of theCandidaand macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In generalCandidacells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defectivecla4mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant ofC. albicansvirulence.

ABSTRACT Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42pG12V or CaCdc42pD118A ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42pG12V was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.

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.

The pathogenic fungus Candida albicans is capable of responding to a wide variety of environmental cues with a morphological transition from a budding yeast to a polarized filamentous form. We demonstrate that the Ras homologue of C. albicans, CaRas1p, is required for this morphological transition and thereby contributes to the development of pathogenicity. However, CaRas1p is not required for cellular viability. Deletion of both alleles of the CaRAS1 gene caused in vitro defects in morphological transition that were reversed by either supplementing the growth media with cAMP or overexpressing components of the filament-inducing mitogen-activated protein (MAP) kinase cascade. The induction of filament-specific secreted aspartyl proteinases encoded by the SAP4-6 genes was blocked in the mutant cells. The defects in filament formation were also observed in situ after phagocytosis of C. albicans cells in a macrophage cell culture assay and, in vivo, after infection of kidneys in a mouse model for systemic candidiasis. In the macrophage assay, the mutant cells were less resistant to phagocytosis. Moreover, the defects in filament formation were associated with reduced virulence in the mouse model. These results indicate that, in response to environmental cues, CaRas1p is required for the regulation of both a MAP kinase signalling pathway and a cAMP signalling pathway. CaRas1p-dependent activation of these pathways contributes to the pathogenicity of C. albicans cells through the induction of polarized morphogenesis. These findings elucidate a new medically relevant role for Ras in cellular morphogenesis and virulence in an important human infectious disease.

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.

ABSTRACT 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 a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 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 , and STE12 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 the C. albicans CST20 , HST7 , CEK1 , and CPH1 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 the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1 delta/cek1 delta null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 microliter suspension containing 1 x 10(5), 1 x 10(6) and 1 x 10(7) blastopores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1 delta/cek1 delta null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

BACKGROUND:The pathogenic fungus Candida albicans is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of mycelia displaying hyphae with branches and lateral buds. Hyphae have been observed to adhere to and invade host tissues more readily than the yeast form, suggesting that filamentous growth may contribute to the virulence of this major human pathogen. A molecular and genetic understanding of the potential role of morphological switching in the pathogenicity of C. albicans would be of significant benefit in view of the increasing incidence of candidiasis. RESULTS:The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of cells of the budding yeast Saccharomyces cerevisiae deleted for the STE20 gene and the CLA4 gene. CaCLA4 encodes a member of the Ste20p family of serine/threonine protein kinases and is characterized by a pleckstrin homology domain and a Cdc42p-binding domain in its amino-terminal non-catalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vitro, in both synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The gene deletions reduced colonization of the kidneys in infected mice and suppressed C. albicans virulence in the mouse model. CONCLUSIONS:Our results demonstrate that the function of the CaCla4p protein kinase is essential for virulence and morphological switching of C. albicans in a mouse model. Thus, hyphal formation of C. albicans mediated by CaCla4p may contribute to the pathogenicity of this dimorphic fungus, suggesting that regulators of morphological switching may be useful targets for antifungal drugs.

The pheromone signal in the yeast Saccharomyces cerevisiae is transmitted by the beta and gamma subunits of the mating response G-protein. The STE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G beta mutation. The same genetic screen identified BEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designated MDG1 (multicopy suppressor of defective G-protein). The MDG1 gene was independently isolated in a search for multicopy suppressors of a bem1 mutation. The MDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein from Aequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion of MDG1 causes sterility in cells in which the wild-type G beta has been replaced by partly defective G beta derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted for STE20 is partially suppressed by multiple copies of BEM1 and CDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels of STE20 and BEM1 are capable of suppressing a temperature-sensitive mutation in CDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae . CST20 encodes a homolog of the Ste20p/p65 PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7 , encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20 . Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans , one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans .

Two recessive mutations in the Saccharomyces cerevisiae SIG1 (suppressor of inhibitory G-protein) gene have been identified by their ability to suppress the signalling defect of dominant-negative variants of the mating response G-protein beta-subunit. The mutations and deletion of SIG1 enhance the sensitivity of the cells to pheromone and stimulate the basal transcription of a mating specific gene, FUS1, suggesting that Sig1p plays a negatively regulatory role in G(beta gamma)-mediated signal transduction. An additional function of Sig1p in vegetatively growing cells is suggested by the finding that the mutations and deletion of SIG1 cause temperature-sensitive growth defects. The SIG1 gene encodes a protein with a molecular weight of 65 kDa that contains at the amino-terminus two zinc finger-like sequence motifs. Epistasis experiments localize the action of Sig1p within the pheromone signalling pathway at a position at or shortly after the G-protein. We propose that Sig1p represents a novel negative regulator of G(beta gamma)-mediated signal transduction.

In the yeast Saccharomyces cerevisiae the G‐protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant‐negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G‐protein mediated signal transduction, the activation of a protein kinase through G beta gamma.