Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid

Abstract

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...

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