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Generation of Fluorescent Protein Fusions in Candida Species
Published on: March 4, 2017
Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker
Attila Gácser1, Siegfried Salomon, Wilhelm Schäfer
1Center of Applied Molecular Biology, University of Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany.
Abstract:
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.
Insights
Researchers developed an efficient transformation method for Candida parapsilosis, a key fungal pathogen. This new technique utilizes a dominant selectable marker for improved molecular genetic studies of clinical isolates.
Area of Science:
- Medical Mycology
- Molecular Biology
- Yeast Genetics
Background:
- Candida parapsilosis is an increasingly significant human pathogenic fungus, frequently implicated in hospital-acquired infections.
- Understanding the molecular genetics of prototrophic clinical isolates is crucial for combating C. parapsilosis infections.
Purpose of the Study:
- To develop an efficient transformation system for prototrophic clinical isolates of Candida parapsilosis.
- To establish a method utilizing a dominant selectable marker for genetic manipulation.
Main Methods:
- Transformation of C. parapsilosis using a plasmid vector encoding the Candida albicans inosine monophosphate dehydrogenase (IMH3) gene for mycophenolic acid (MPA) resistance.
- Comparison of transformation efficiencies between electroporation and lithium acetate (LiAc) methods.
- Analysis of transformants using polymerase chain reaction (PCR) and Southern hybridization to confirm gene presence and integration.
Main Results:
- A modified electroporation method yielded a high number of mitotically stable transformants with clear MPA resistance.
- The LiAc method demonstrated very poor transformation efficiency.
- PCR and Southern hybridization confirmed the presence and genomic integration of the Candida albicans IMH3(r) gene in C. parapsilosis transformants.
Conclusions:
- This study presents the first reported method for transforming prototrophic clinical isolates of Candida parapsilosis using a dominant selectable marker.
- The developed electroporation-based transformation system is efficient and provides stable transformants.
- This technique offers a valuable tool for future research into the biology and virulence of this important pathogenic yeast.
