Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker.
Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.
Gácser
Attila
A
Center of Applied Molecular Biology, University of Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany.
Salomon
Siegfried
S
Schäfer
Wilhelm
W
D000935:Antifungal Agents / Q000494:pharmacology; D002175:Candida / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D002177:Candidiasis / Q000382:microbiology; D025141:Drug Resistance, Fungal / Q000235:genetics*; D018274:Electroporation; D005819:Genetic Markers; D006801:Humans; D007168:IMP Dehydrogenase / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009173:Mycophenolic Acid / Q000494:pharmacology; D012641:Selection, Genetic; D014170:Transformation, Genetic
D000935:Antifungal Agents; D005819:Genetic Markers; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid