PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis.     

The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins.

A hyphally regulated gene (HYR1) from the dimorphic human pathogenic fungus Candida albicans was isolated and characterized. Northern (RNA) analyses showed that the HYR1 mRNA was induced specifically in response to hyphal development when morphogenesis was stimulated by serum addition and temperature elevation, increases in both culture pH and temperature, or N-acetylglucosamine addition. The HYR1 gene sequence revealed a 937-codon open reading frame capable of encoding a protein with an N-terminal signal sequence, a C-terminal glycosylphosphatidylinositol-anchoring domain, 17 potential N glycosylation sites, and a large domain rich in serine and threonine (51% of 230 residues). These features are observed in many yeast cell wall proteins, but no homologs are present in the databases. In addition, Hyr1p contained a second domain rich in glycine, serine, and asparagine (79% of 239 residues). The HYR1 locus in C. albicans CAI4 was disrupted by "Ura-blasting," but the resulting homozygous delta hyr1/delta hyr1 null mutant displayed no obvious morphological phenotype. The growth rates for yeast cells and hyphae and the kinetics of germ tube formation in the null mutant were unaffected. Aberrant expression of HYR1 in yeast cells, when an ADH1-HYR1 fusion was used, did not stimulate hyphal formation in C. albicans or pseudohyphal growth in Saccharomyces cerevisiae. HYR1 appears to encode a nonessential component of the hyphal cell wall.     

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.     

Snf7p, a component of the ESCRT-III protein complex, is an upstream member of the RIM101 pathway in Candida albicans

The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pathway in C. albicans. We identified C. albicans homologs to S. cerevisiae Snf7p, Vps4p, and Bro1p and generated mutants in the cognate gene. We found that snf7Delta/Delta mutants, but not vps4Delta/Delta nor bro1Delta/Delta mutants, had phenotypes similar to, but more severe than, those of RIM101 pathway mutants. We found that the constitutively active RIM101-405 allele partially rescued snf7Delta/Delta mutant phenotypes. The vps4Delta/Delta mutant had subtle phenotypes, but these were not rescued by the RIM101-405 allele. Further, we found that the snf7Delta/Delta, vps4Delta/Delta, and bro1Delta/Delta mutants did not efficiently localize the vital dye FM4-64 to the vacuole and that it was often accumulated in an MVB-like compartment. This phenotype was not rescued by RIM101-405 or observed in RIM101 pathway mutants. These results suggest that Snf7p may serve two functions in the cell: one as a RIM101 pathway member and one for MVB transport to the vacuole.     

Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 gene, which is involved in cell wall beta-glucan synthesis.

The KRE1 gene of Saccharomyces cerevisiae, sacKRE1, appears to be involved in the synthesis of cell wall beta-glucan. S. cerevisiae strains with mutations in the KRE1 gene produce a structurally altered cell wall (1----6)-beta-glucan, which results in resistance to K1 killer toxin. We isolated the canKRE1 gene from Candida albicans by its ability to complement a kre1 mutation in S. cerevisiae and confer sensitivity to killer toxin. Sequence analysis revealed that the predicted protein encoded by canKRE1 shares an overall structural similarity with that encoded by sacKRE1. The canKRE1 protein is composed of an N-terminal signal sequence, a central domain of 46% identity with the sacKRE1 protein, and a C-terminal hydrophobic tract. These structural and functional similarities imply that the canKRE1 gene carries out a function in C. albicans cell wall assembly similar to that observed for sacKRE1 in S. cerevisiae.     

Phenotypic switching in Candida albicans is controlled by a SIR2 gene

We report the cloning of a gene from the human fungal pathogen Candida albicans with sequence and functional similarity to the Saccharomyces cerevisiae SIR2 gene. Deletion of the gene in C. albicans produces a dramatic phenotype: variant colony morphologies arise at frequencies as high as 1 in 10. The morphologies resemble those described previously as part of a phenotypic switching system proposed to contribute to pathogenesis. Deletion of SIR2 also produces a high frequency of karyotypic changes. These and other results are consistent with a model whereby Sir2 controls phenotypic switching and chromosome stability in C.albicans by organizing chromatin structure.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.