Search

Although the fungus Candida albicans is a commensal colonizer of humans, the organism is also an important opportunistic pathogen. Most infections caused by C. albicans arise from organisms that were previously colonizing the host as commensals, and therefore successful establishment of colonization is a prerequisite for pathogenicity. To elucidate fungal activities that promote colonization, an analysis of the transcription profile of C. albicans cells recovered from the intestinal tracts of mice was performed. The results showed that within the C. albicans colonizing population, cells expressed genes characteristic of the laboratory-grown exponential phase and genes characteristic of post-exponential-phase cells. Thus, gene expression both promoted the ability to grow rapidly (a characteristic of exponential-phase cells) and enhanced the ability to resist stresses (a characteristic of post-exponential-phase cells). Similarities in gene expression in commensal colonizing cells and cells invading host tissue during disease were found, showing that C. albicans cells adopt a particular cell surface when growing within a host in both situations. In addition, transcription factors Cph2p and Tec1p were shown to regulate C. albicans gene expression during intestinal colonization.

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Gα subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Gα subunit in Saccharomyces cerevisiae , and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gβ subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Gα and Gβ subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.

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.

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., Synopsis 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.

Candida albicans , the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus ( MTL ). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene ( KEX2 ) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1 , which codes for the precursor of the α mating pheromone in C. albicans . Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans . To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans . The mfα1 null mutant fails to mate as MTLα , while MTL a mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTL a cells of C. albicans . These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.

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 CaCdc42p G12V or CaCdc42p D118A ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p G12V 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 .

In a screen for Candida albicans genes capable of supressing a ste20Δ mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855. Copyright © 2000 John Wiley & Sons, Ltd.

In Saccharomyces cerevisiae MATa cells, export of the a-factor mating pheromone is mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of transporters and a close homologue of mammalian multidrug transporter P-glycoproteins (Pgps). We have used functional complementation of a ste6delta mutation to isolate a gene encoding an ABC transporter capable of a-factor export from the pathogenic yeast, Candida albicans. This gene codes for a 1323-amino acid protein with an intramolecular duplicated structure, each repeated half containing six potential hydrophobic transmembrane segments and a hydrophilic domain with consensus sequences for an ATP-binding fold. The predicted protein displays significant sequence similarity to S. cerevisiae Ste6p and mammalian Pgps. The gene has been named HST6, for homologue of STE6. A high degree of structural conservation between the STE6 and the HST6 loci with respect to DNA sequence, physical linkage and transcriptional arrangement indicates that HST6 is the C. albicans orthologue of the S. cerevisiae STE6 gene. We show that the HST6 gene is transcribed in a haploid-specific manner in S. cerevisiae, consistent with the presence in its promoter of a consensus sequence for Mata1p-Matalpha2p binding known to mediate the repression of haploid-specific genes in S. cerevisiae diploid cells. In C. albicans, HST6 is expressed constitutively at high levels in the different cell types analysed (yeast, hyphae, white and opaque), demonstrating that HST6 transcription is not repressed in this diploid yeast, unlike in diploid S. cerevisiae, and suggesting a basic biological function for the Hst6p transporter in C. albicans. The strong similarity between Hst6p and the multidrug transporter Pgps also raises the possibility that Hst6p could be involved in resistance to antifungal drugs in C. albicans.

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source. © 2013 Boyles et al.

Although gastrointestinal colonization by the opportunistic fungal pathogen Candida albicans is generally benign, severe systemic infections are thought to arise due to escape of commensal C. albicans from the gastrointestinal (GI) tract. The C. albicans transcription factor Efg1p is a major regulator of GI colonization, hyphal morphogenesis, and virulence. The goals of this study were to identify the Efg1p regulon during GI tract colonization and to compare C. albicans gene expression during colonization of different organs of the GI tract. Our results identified significant differences in gene expression between cells colonizing the cecum and ileum. During colonization, efg1 − null mutant cells expressed higher levels of genes involved in lipid catabolism, carnitine biosynthesis, and carnitine utilization than did colonizing wild-type (WT) cells. In addition, during laboratory growth, efg1 − null mutant cells grew to a higher density than WT cells. The efg1 − null mutant grew in depleted medium, while WT cells could grow only if the depleted medium was supplemented with carnitine, a compound that promotes the metabolism of fatty acids. Altered gene expression and altered growth capability support the ability of efg1 − cells to hypercolonize naïve mice. Also, Efg1p was shown to be important for transcriptional responses to the stresses present in the cecum environment. For example, during colonization, SOD5 , encoding a superoxide dismutase, was highly upregulated in an Efg1p-dependent manner. Ectopic expression of SOD5 in an efg1 − null mutant increased the fitness of the efg1 − null mutant cells during colonization. These data show that EFG1 is an important regulator of GI colonization.

Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating., IMPORTANCE The mitogen-activated protein (MAP) kinase cascade is an extensively used signaling module in eukaryotic cells, and the ability to regulate these modules is critical for ensuring proper responses to a wide variety of stimuli. One way that cells regulate this signaling module is through scaffold proteins that insulate related pathways against cross talk, improve signaling efficiency, and ensure that signals are connected to the correct response. The Ste5 scaffold of the S. cerevisiae mating response is a well-studied representative of this class of proteins. Using bioinformatics, structural modeling, and molecular genetic approaches, we have investigated the equivalent scaffold in the pathogenic yeast Candida albicans. We show that the C. albicans protein is structurally distinct from that of Saccharomyces cerevisiae but still provides similar functions. Increases in pathway complexity have been associated with changes in scaffold connectivity, and overall, the tethering capacity of the scaffolds has been more conserved than their structural organization.

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.

In the opaque state, MTL a and MTL α strains of Candida albicans are able to mate, and this mating is directed by a pheromone-mediated signaling process. We have used comparisons of genome sequences to identify a C. albicans gene encoding a candidate a -specific mating factor. This gene is conserved in Candida dubliniensis and is similar to a three-gene family in the related fungus Candida parapsilosis but has extremely limited similarity to the Saccharomyces cerevisiae MFA1 (Sc MFA1 ) and Sc MFA2 genes. All these genes encode C-terminal CAAX box motifs characteristic of prenylated proteins. The C. albicans gene, designated Ca MFA1 , is found on chromosome 2 between ORF19.2165 and ORF19.2219. MFA1 encodes an open reading frame of 42 amino acids that is predicted to be processed to a 14-amino-acid prenylated mature pheromone. Microarray analysis shows that MFA1 is poorly expressed in opaque MTL a cells but is induced when the cells are treated with α-factor. Disruption of this C. albicans gene blocks the mating of MTL a cells but not MTL α cells, while the reintegration of the gene suppresses this cell-type-specific mating defect.

Background: The 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined. Results: Supercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome. Conclusion: Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution., National Institute of Allergy and Infectious Diseases (U.S.) (contract N01 AI05406), National Institute of Allergy and Infectious Diseases (U.S.) (grant R01 AI 16567), National Research Council (U.S.). Genome Health Initiative, Canadian Institutes for Health Research (grant HOP 67260), Canadian Institutes for Health Research (grant MOP 42516), Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research)

Candida albicans contains a functional mating response pathway that is similar to the well-studied system of Saccharomyces cerevisiae. We have characterized a regulator of G protein signaling (RGS) homolog in C. albicans with sequence similarity to the SST2 gene of Saccharomyces cerevisiae. Disruption of this gene, which had been designated SST2, causes an opaque MTLa/MTLa derivative of strain SC5314 to show hypersensitivity to the C. albicans alpha-factor. This hypersensitivity generates an enhanced cell cycle arrest detected in halo assays but reduces the overall mating efficiency of the cells. Transcriptional profiling of the pheromone-regulated gene expression in the sst2 mutant shows a pattern of gene induction similar to that observed in wild-type cells, but the responsiveness is heightened. This involvement of an RGS in the sensitivity to pheromone is consistent with the prediction that the mating response pathway in C. albicans requires the activation of a heterotrimeric G protein., English16400182

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., 200247

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 .

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.

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 theCaCDC35 gene encoding C. albicansadenylyl 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 ofCaCDC35 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. Homozygouscacdc35Δ 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.

We have used a genomic library of Candida albicans to transform Saccharomyces cerevisiae and screened for genes that act similarly to dominant negative mutations by interfering with pheromone-mediated cell cycle arrest. Six different plasmids were identified from 2000 transformants; four have been sequenced. One gene (CZF1) encodes a protein with structural motifs characteristic of a transcription factor. A second gene (CCN1) encodes a cyclin homologue, a third (CRL1) encodes a protein with sequence similarity to GTP-binding proteins of the RHO family, and a fourth (CEK1) encodes a putative kinase of the ERK family. Since CEK1 confers a phenotype similar to that of the structurally related S. cerevisiae gene KSS1 but cannot complement a KSS1 defect, it is evident that dominant negative selection can identify proteins that complementation screens would miss. Because dominant negative mutations exert their influence even in wild-type strain backgrounds, this approach should be a general method for the analysis of complex cellular processes in organisms not amenable to direct genetic analysis.

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.