Genetic manipulation of the pathogenic yeast Candida parapsilosis.
Candida parapsilosis is an important human pathogen, responsible for severe cases of systemic candidiasis and one of the leading causes of mortality in neonates. In this report, we describe the first system for genetic manipulation of C. parapsilosis. We isolated and subsequently determined DNA sequences of genes encoding galactokinase ( CpGAL1) and orotidine-5'-phosphate decarboxylase ( CpURA3) from a genomic DNA library of C. parapsilosis by functional complementation of corresponding mutations in Saccharomyces cerevisiae. The predicted protein products, Gal1p and Ura3p, displayed a high degree of homology with corresponding sequences of C. albicans and S. cerevisiae, respectively. A collection of galactokinase-deficient ( gal1) strains of C. parapsilosis was prepared using direct selection of mutagenized cells on media containing 2-deoxy-galactose. Additionally, we constructed a plasmid vector carrying CpGAL1 as a selection marker and a genomic DNA fragment with an autonomously replicating sequence activity that transforms the C. parapsilosis gal1 mutant strain with high efficiency. This system for genetic transformation of C. parapsilosis may significantly advance the study of this human pathogen, greatly improving our understanding of its biology and virulence, with implications for drug development.
Nosek
Jozef
J
Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 84215 Bratislava, Slovakia. nosek@fns.uniba.sk
Adamíková
Lubica
L
Zemanová
Júlia
J
Tomáska
Lubomír
L
Zufferey
Rachel
R
Mamoun
Choukri Ben
CB
D002175:Candida / Q000235:genetics*; D005819:Genetic Markers; D005822:Genetic Vectors; D015698:Genomic Library; D030781:Organisms, Genetically Modified; D010957:Plasmids
D005819:Genetic Markers