Identifying infection-associated genes of Candida albicans in the postgenomic era.

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

the hyphal-associated adhesin and invasin Als3 of Candida albicans mediates iron acquisition from host ferritin.

Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the baker's yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Deltaals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Deltaals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes.

Glycosylphosphatidylinositol-anchored proteases of Candida albicans target proteins necessary for both cellular processes and host-pathogen interactions.

Intracellular and secreted proteases fulfill multiple functions in microorganisms. In pathogenic microorganisms extracellular proteases may be adapted to interactions with host cells. Here we describe two cell surface-associated aspartic proteases, Sap9 and Sap10, which have structural similarities to yapsins of Saccharomyces cerevisiae and are produced by the human pathogenic yeast Candida albicans. Sap9 and Sap10 are glycosylphosphatidylinositol-anchored and located in the cell membrane or the cell wall. Both proteases are glycosylated, cleave at dibasic or basic processing sites similar to yapsins and Kex2-like proteases, and have functions in cell surface integrity and cell separation during budding. Overexpression of SAP9 in mutants lacking KEX2 or SAP10, or of SAP10 in mutants lacking KEX2 or SAP9, only partially restored these phenotypes, suggesting distinct target proteins of fungal origin for each of the three proteases. In addition, deletion of SAP9 and SAP10 modified the adhesion properties of C. albicans to epithelial cells and caused attenuated epithelial cell damage during experimental oral infection suggesting a unique role for these proteases in both cellular processes and host-pathogen interactions.

Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis.

The inositol/choline responsive element (ICRE) functions as a UAS element mediating coordinate expression of structural genes required for yeast phospholipid biosynthesis. However, ICRE motifs could be detected upstream of various genes apparently not involved in lipid metabolism. In this work we investigated the expression pattern of selected genes containing ICRE promoter motifs, as identified by in silico analysis (ARG4, ERG20, FAR8, GPD2, RSF1, URA8, VHT1 and YEL073C). It turned out that the presence of an ICRE upstream of a gene of unknown function indeed allows to conclude for regulation by phospholipid precursors, which is mediated by activators Ino2/Ino4 and the repressor Opi1. We also demonstrated in vitro binding of Ino2/Ino4 heterodimers to promoter regions. Thus, our analysis supports the view that identification of regulatory elements by a database search provides evidence for a specific pattern of gene expression. Activation by pathway-specific regulators may suggest a physiological function for as yet uncharacterized genes.

CandidaDB: a genome database for Candida albicans pathogenomics.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

Candida albicans proteinases and host/pathogen interactions.

Candida infections are common, debilitating and often recurring fungal diseases and a problem of significant clinical importance. Candida albicans, the most virulent of the Candida spp., can cause severe mucosal and life-threatening systemic infections in immunocompromised hosts. Attributes that contribute to C. albicans virulence include adhesion, hyphal formation, phenotypic switching and extracellular hydrolytic enzyme production. The extracellular hydrolytic enzymes, especially the secreted aspartyl proteinases (Saps), are one of few gene products that have been shown to directly contribute to C. albicans pathogenicity. Because C. albicans is able to colonize and infect almost every tissue in the human host, it may be crucial for the fungus to possess a number of similar but independently regulated and functionally distinct secreted proteinases to provide sufficient flexibility in order to survive and promote infection at different niche sites. The aim of this review is to explore the functional roles of the C. albicans proteinases and how they may contribute to the host/pathogen interaction in vivo.

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1.

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.

Reduced expression of the hyphal-independent Candida albicans proteinase genes SAP1 and SAP3 in the efg1 mutant is associated with attenuated virulence during infection of oral epithelium.

The transition of Candida albicans from a yeast to a hyphal form is controlled by several transcriptional factors, including the key regulators Cph1 and Efg1, and is considered an important virulence attribute. These factors, especially Efg1, regulate the expression of hyphal-associated genes e.g. SAP4-SAP6. In order to investigate the relevance of these transcriptional regulators for hyphal-independent SAP genes, recently constructed cph1 and efg1 single mutants and a cph1/efg1 double mutant lacking these factors were tested during interaction with oral epithelium and polymorphonuclear neutrophils. In contrast to the parental wild-type strain and the cph1 mutant, the efg1 and the cph1/efg1 mutants did not produce hyphal forms in all experiments and were less capable of damaging epithelial cells and neutrophil granulocytes. The attenuated epithelial lesions of these mutants were correlated not only with reduced expression of the hyphal-associated gene SAP4, but also with the lack of SAP1 and SAP3 expression previously shown to be important for oral infections. An efg1 mutant strain carrying a plasmid-borne copy of the EFG1 gene regained hyphal growth, damage of keratinocytes, granulocytes and the expression of SAP1 and SAP3. Although efg1 and cph1/efg1 mutants did not produce germ tubes during infection, expression of the hyphal-associated genes SAP5 and SAP6 was not completely abolished. A reduced capacity to stimulate an epithelial immune response manifested by a delayed onset of IL-1beta, IL-8 and TNF expression was only observed in the cph1/efg1-infected tissue. These results provide further evidence for a combined regulation of different virulence factors, such as dimorphism and expression of SAP genes. Furthermore, it could be demonstrated that the lack of Efg1 also caused reduced expression of hyphal-independent SAP genes. Both the EFG1 and the CPH1 gene products are necessary for adequate induction of an immune response.

Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs.

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.