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.

An IMP dehydrogenase gene was isolated from Candida albicans on a ϳ2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 g of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 g/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.IMP dehydrogenase (EC 1.1.1.205) is the key enzyme in the de novo biosynthesis of GMP, catalyzing the NAD-dependent oxidation of IMP to XMP. This conversion of IMP to XMP is the rate-limiting step in the biosynthetic pathway of guanine nucleotides, at least in mammals (26). While genes encoding IMP dehydrogenases have been isolated from archaea (11), protozoa (5, 54, 55), bacteria (2,3,16,31,36,47,49), plants (12), invertebrates (46), and mammals (10, 13, 39, 50), the only fungal sequences known are those encoding the putative IMP dehydrogenase genes sequenced in the Saccharomyces cerevisiae genome project (14,27,28). The polymorphic fungus Candida albicans is an opportunistic pathogen in immunocompromised individuals, causing oral and vaginal candidiasis as well as disseminated infections in neutropenic patients. Genetic manipulations of this diploid microorganism have been hampered by the apparent absence of a haploid phase and the dependence of marker systems on the spontaneous occurrence of auxotrophic mutants. Thus, the identification of the IMH3 gene in C. albicans suggested to us that mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, might be used to develop a dominant selectable marker system in Candida. In several other organisms, it has been shown that amplification of an IMP dehydrogenase gene can confer MPA resistance in a gene-dosage-dependent manner by overexpression of the enzyme (9,20,23,25,54). We report the cloning and sequencing of a DNA fragment of C. albicans with a coding region for a protein with high amino acid similarity and identity to known IMP dehydrogenases. Transformation of C.albicans by using a multicopy plasmid vector yielded transformants resistant to the specific IMP dehydrogenase inhibitor MPA. Culturing of transformant cells in the presence of MPA stabilized replicative plasmids, suggesting the feasibility of using IMH3 as a dominant selection marker for genetic studies in C. alb...

Candida albicansis the most importantCandidaspecies causing vulvovaginal candidiasis (VVC). VVC has significant medical and economical impact on women’s health and wellbeing. While current antifungal treatment is reasonably effective, supportive and preventive measures such as application of probiotics are required to reduce the incidence of VVC. We investigated the potential of the probioticsLactobacillus rhamnosusGR-1 andLactobacillus reuteriRC-14 towards control ofC. albicans.In vitroexperiments demonstrated that lactic acid at low pH plays a major role in suppressing fungal growth. Viability staining following cocultures with lactobacilli revealed thatC. albicanscells lost metabolic activity and eventually were killed. Transcriptome analyses showed increased expression of stress-related genes and lower expression of genes involved in fluconazole resistance, which might explain the increased eradication ofCandidain a previous clinical study on conjoint probiotic therapy. Our results provide insights on the impact of probiotics onC. albicanssurvival.

SummaryAn understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.