Eckert S, Wicker M, Muhlschlegel F

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The yeast Candida dubliniensis, an opportunistic human pathogen, has recently been established as a species that is distinct from Candida albicans. While having differing ribosomal DNA sequences and discrete chlamydospore formation conditions, both species show similar patterns of germ tube and hyphae production. We are interested in the pH regulation of yeast-hyphal transition. We recently isolated the genes encoding the enzymes Phr1p and Phr2p, which play an important role in the cell wall biosynthesis, from C. dubliniensis. Their strong similarity in sequence and expression pattern to their C. albicans equivalents suggests that they are subject to pH-dependent regulation by a homologue of CaRim101p. Here we report the cloning of this regulatory gene, CdRim101. An ORF of 2028 nt encodes a deduced protein of 676 amino acids with a molecular weight of 76468 Da. The deduced protein sequence shows 83% identity and 87% similarity to C. albicans Rim101p. Similarities to other related proteins range from 32% for Yarrowia lipolytica Rim101p and Saccharomyces cerevisiae Rim101p and 27% to Kluyveromyces lactis Rim101p to 24% to Aspergillus nidulans PacC. The putative Zink finger DNA binding domains are well conserved. CdRim101 shows a pH-dependent expression pattern similar to CaRim101, with high levels of transcript at pH 7 and absence at pH 4. CdRim101 seems to have stronger expression levels than CaRim101. To investigate the role of CdRim101p for morphology, we transformed the wild-type CdRim101 gene into the C. albicans rim101 deletion mutant CAR26. On solid M199 medium (pH7), CAR26 exclusively grows in yeast form, while C. albicans wild-type strains show strong filamentation. C. dubliniensis wild-type strains show an intermediate phenotype of moderate filamentation. CAR26 was partially complemented by the CdRim101 gene. It filaments almost to the same extent as the C. albicans wild-type, and stronger than the C. dubliniensis wild-type. In an RNA hybridisation experiment, the complemented CAR26 exhibits strong CdRim101 mRNA expression at pH 7 and no detectable transcript at pH 4. To further explore the role of Rim101p in morphogenesis, we plan to delete this gene in C. dubliniensis. We present our CdRim101 deletion strategy, which employs a C. dubliniensis ura3 mutant. We are planning to reverse the above complementation experiment by expressing CaRim101 in a C. dubliniensis rim101 mutant to further examine the effect of this regulator on filamentation.

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The 15 th Congress of the International Society for Human and Animal Mycology
    • ISHAM 15th (2003)