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Published on: March 4, 2017
Enhancing the chemical transformation of Candida parapsilosis
Tibor Németh1, Joshua D Nosanchuk2,3, Csaba Vagvolgyi1
1Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
Abstract:
Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20-55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at -80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.
Insights
Researchers optimized chemical transformation for Candida parapsilosis, a key cause of neonatal infections. The new method significantly increases transformation efficiency and allows for long-term storage of competent cells, aiding virulence studies.
Area of Science:
- Medical Mycology
- Molecular Biology
- Yeast Genetics
Background:
- Candida parapsilosis is a significant cause of invasive fungal infections, particularly in neonates.
- Understudied molecular and physiological characteristics hinder understanding of C. parapsilosis pathobiology.
- Chemical transformation is crucial for genetic manipulation but efficiency decreases with DNA size.
Purpose of the Study:
- To develop and validate an improved chemical transformation protocol for Candida parapsilosis.
- To optimize conditions for maximizing transformation efficiency, regardless of DNA fragment size.
- To establish a method for long-term storage of transformation-competent C. parapsilosis cells.
Main Methods:
- Systematic optimization of cell density, incubation times, and chemical enhancers (e.g., dimethyl sulfoxide).
- Evaluation of dithiothreitol pretreatment effects on colony recovery.
- Testing the modified protocol on various C. parapsilosis isolates, including prototrophic strains.
- Development of a cryopreservation medium for competent yeast cells.
Main Results:
- A 20-55-fold increase in transformation efficiency was achieved, dependent on DNA fragment size.
- Dimethyl sulfoxide enhanced efficiency, while dithiothreitol pretreatment negatively impacted recovery.
- The optimized protocol demonstrated significantly improved transformant recovery in 5 out of 6 tested isolates.
- A cryopreservation medium enabling cell maintenance at -80°C for up to 6 weeks was identified.
Conclusions:
- The modified chemical transformation protocol substantially enhances efficiency and reliability for C. parapsilosis genetic studies.
- The ability to store competent cells reduces workload and streamlines experimental procedures.
- These advancements will facilitate deeper investigation into the genetic underpinnings of C. parapsilosis virulence.
