Nina Agabian

A Human-Curated Annotation of the Candida albicans Genome

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.

Metabolic specialization associated with phenotypic switching in Candida albicans

Phase and antigenic variation are mechanisms used by microbial pathogens to stochastically change their cell surface composition. A related property, referred to as phenotypic switching, has been described for some pathogenic fungi. This phenomenon is best studied in Candida albicans, where switch phenotypes vary in morphology, physiology, and pathogenicity in experimental models. In this study, we report an application of a custom Affymetrix GeneChip representative of the entire C. albicans genome and assay the global expression profiles of white and opaque switch phenotypes of the WO-1 strain. Of 13,025 probe sets examined, 373 ORFs demonstrated a greater than twofold difference in expression level between switch phenotypes. Among these, 221 were expressed at a level higher in opaque cells than in white cells; conversely, 152 were more highly expressed in white cells. Affected genes represent functions as diverse as metabolism, adhesion, cell surface composition, stress response, signaling, mating type, and virulence. Approximately one-third of the differences between cell types are related to metabolic pathways, opaque cells expressing a transcriptional profile consistent with oxidative metabolism and white cells expressing a fermentative one. This bias was obtained regardless of carbon source, suggesting a connection between phenotypic switching and metabolic flexibility, where metabolic specialization of switch phenotypes enhances selection in relation to the nutrients available at different anatomical sites. These results extend our understanding of strategies used in microbial phase variation and pathogenesis and further characterize the unanticipated diversity of genes expressed in phenotypic switching.

Regulatory networks affected by iron availability in Candida albicans

Iron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip© representing the entire Candida albicans genome, we examined the changes in genome‐wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high‐iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell‐surface properties to others which affect mitochondrial function, iron transport and virulence‐related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell‐surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/biosynthesis. Comparisons between wild‐type and SFU1‐null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron‐responsive. Together, these results not only expand our understanding of global iron regulation in C. albicans, but also provide insights into the potential role of iron availability in C. albicans virulence.

Genomic evidence for a complete sexual cycle in Candida albicans

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

Differential Expression of Candida albicans Secreted Aspartyl Proteinase and Phospholipase B Genes in Humans Correlates with Active Oral and Vaginal Infections

The in vivo expression of Candida albicans secreted aspartyl proteinase (SAP1-SAP8) and phospholipase B (PLB1 and PLB2) genes was analyzed in 137 human subjects with oral and vaginal candidiasis or carriage. Total RNA was isolated from whole unstimulated saliva or vaginal swabs, and the expression of SAP1-8 and PLB1-2 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. A spectrum of SAP gene expression profiles was obtained from different C. albicans strains during symptomatic disease and asymptomatic carriage. SAP2 and SAP5 were the most common genes expressed during both infection and carriage. SAP1, SAP3, SAP4, SAP7, SAP8, and PLB1 expression was correlated with oral disease, whereas SAP1, SAP3, and SAP6-SAP8 expression was correlated with vaginal disease. Furthermore, SAP1, SAP3, and SAP8 were preferentially expressed in vaginal, rather than oral, infections. This study demonstrates the differential expression of the hydrolytic enzyme genes in humans and correlates the expression of specific Candida species virulence genes with active disease and anatomical location.

Genome-Wide Transcription Profiling of the Early Phase of Biofilm Formation by Candida albicans

ABSTRACT The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by most microorganisms to survive in changing environments. Biofilm formation proceeds through distinct developmental phases and impacts not only medicine but also industry and evolution. In organisms such as the opportunistic pathogen Candida albicans, the ability to grow as biofilms is also an important mechanism for persistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medical devices. The early stage of C. albicans biofilm is characterized by the adhesion of single cells to the substratum, followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changes in the transcriptome begin within 30 min of contact with the substrate and include expression of genes related to sulfur metabolism, in particular MET3, and the equivalent gene homologues of the Ribi regulon in Saccharomyces cerevisiae. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathway for cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production in C. albicans.

KEX2 INFLUENCES CANDIDA ALBICANS PROTEINASE SECRETION AND HYPHAL FORMATION

Candida albicans possesses at least seven differentially expressed genes that encode virulence-related secretory aspartyl proteinases (Saps). Sap DNA sequences predict post-translational processing at lysine-arginine residues in the preproteins, reminiscent of the maturation of Saccharomyces cerevisiae α-factor, where a prepropolypeptide is converted into a biologically active pheromone by Kex2, a subtilisin-like proprotein convertase. To investigate involvement of a C. albicans KEX2 homologue in Sap activation, a genetic selection was performed based on KEX2 function. A kex2strain of S. cerevisiae was transformed with a C. albicans genomic DNA library and screened for the production of active α-factor. Positive clones were assayed for killer toxin activity, another Kex2-dependent phenotype. Plasmids that rescued both defects contained a sequence encoding a protein homologous to S. cerevisiae Kex2. Both alleles of the C. albicans KEX2 were inactivated by successive mutations. Null mutants continued to secrete active Sap2; however, the enzyme was abnormally processed and secreted at reduced levels. Unexpectedly, null mutants were incapable of forming hyphae, instead differentiating into aberrantly shaped cells. The ability to normally process Sap2 and form hyphae was restored upon transformation of null mutants with aKEX2-containing plasmid.

A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.

Candida albicans Modulates Host Defense by Biosynthesizing the Pro-Resolving Mediator Resolvin E1

Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.

Role of Calcineurin in Stress Resistance, Morphogenesis, and Virulence of a Candida albicans Wild-Type Strain

ABSTRACT By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.