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.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11⌬/mre11⌬, rad50⌬/rad50⌬, and rad52⌬/rad52⌬ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2⌬/msh2⌬, pms1⌬/ pms1⌬, and yku80⌬/yku80⌬ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11⌬/mre11⌬ and rad50⌬/rad50⌬ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11⌬/mre11⌬, rad50⌬/rad50⌬, or rad52⌬/rad52⌬ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2⌬/msh2⌬ and pms1⌬/pms1⌬ (MMR mutants) and rad50⌬/rad50⌬ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

SummaryCandida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type ( MAT ) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae . We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTL a and MTL a a a a strains, whereas disruptions in STE20 had no effect. A disruption in KEX2 , a gene involved in processing the S. cerevisiae pheromone Mf a a a a , prevented mating in MTL a a a a but not MTL a cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTL a but not in MTL a a a a cells. Disruption of two cell wall genes, ALS1 and INT1 , had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1 . The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.

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 ␣ mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the ␣ pheromone gene. Here we report the isolation and characterization of the gene MF␣1, which codes for the precursor of the ␣ mating pheromone in C. albicans. Two active ␣-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 mf␣1 null mutant of C. albicans. The mf␣1 null mutant fails to mate as MTL␣, while MTLa mf␣1 cells are still mating competent. Experiments performed with the synthetic ␣-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTL␣ kex2 mutant strain when added exogenously, thereby confirming their roles as ␣ mating pheromones.Candida albicans is the most common opportunistic fungal pathogen of humans and mostly infects immunosuppressed patients (3). It inhabits diverse niches, which include the gastrointestinal tract and the vagina, and causes infection of skin, mucous membranes, and the bloodstream. Since each of these sites of infection presumably requires differences in gene expression, a great deal of effort has been spent in looking at how such adaptation occurs in Candida. Much of this effort was predicated on the characterization of C. albicans as an asexual obligate diploid.That this fungus is asexual was disproved after the C. albicans genome sequencing project revealed the presence of sequences homologous to the Saccharomyces cerevisiae MAT (mating-type) loci. The C. albicans homologues of the MAT loci, the MTL (mating-type-like) loci, were found to be heterozygous in common laboratory strains tested (17). Strains homozygous for the MTL locus were generated either by a deletion strategy (18) or by loss, induced by growth on sorbose as the sole carbon source (19), of one homologue of chromosome 5, the site of the MTL loci (29). Strains thus constructed were able to mate both under laboratory conditions (29) and in the animal host (18). These studies set the stage for dissecting the mating pathway in C. albicans.Mating in fungi has been examined in both yeasts and molds of various sorts. Certain general parts of the process seem to hold across genera and indeed across the kingdom. These include dissimilar regulatory genes (mating-type loci), soluble phe...