Related Experiment Video
Updated: May 26, 2025
Generation of Fluorescent Protein Fusions in Candida Species
Published on: March 4, 2017
PCR-based CRISPR/Cas9 system for fluorescent tagging: A tool for studying Candida parapsilosis virulence
Tibor Nemeth1, Andrea Zarnocki1, Anett Ladanyi1
1Department of Biotechnology and Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
Abstract:
Candida parapsilosis is persistent in a hospital environment hence it is often associated with nosocomial infections especially amongst low-birth weight neonates. Genetic modification is therefore important to characterise the physiological and virulence related properties of this fungus. A PCR-based CRISPR/Cas9 system has been adopted to facilitate the generation of fluorescent tagged prototroph isolates. We examined a total of eight fluorescent protein coding genes, out of which three were found to be applicable for simultaneous utilisation. We investigated three clinical isolates of C. parapsilosis in terms of their adherence to silicone and their uptake by J774.2 murine macrophages in competition assays. Interestingly, we found significant differences between them in both experiments where GA1 isolate was significantly less resistant to macrophage uptake and CDC317 was significantly more adherent to silicone material. In silico analysis of the agglutinin-like sequences (Als) exposed remarkable diversity in this protein family and additionally, the thorough analysis of the ALS genes revealed evidence of formation of a new gene by intrachromosomal recombination in the GA1 isolate. Finally, we provide a step by step protocol for the application of the PCR-based CRISPR/Cas9 system for fluorescently labelling C. parapsilosis isolates.
Insights
Genetic modification of Candida parapsilosis using CRISPR/Cas9 enables fluorescent tagging for studying fungal properties. This research highlights variations in virulence and adherence among clinical isolates, aiding in understanding nosocomial infections.
Area of Science:
- Medical Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Candida parapsilosis is a common cause of hospital-acquired infections, particularly in vulnerable neonates.
- Understanding its physiological and virulence factors is crucial for infection control.
- Genetic tools are needed to study C. parapsilosis isolates effectively.
Purpose of the Study:
- To develop and apply a PCR-based CRISPR/Cas9 system for fluorescently labeling C. parapsilosis.
- To characterize physiological and virulence properties of clinical isolates.
- To investigate variations in adherence and macrophage interaction.
Main Methods:
- Utilized a PCR-based CRISPR/Cas9 system to generate fluorescently tagged C. parapsilosis.
- Screened eight fluorescent protein genes, identifying three for simultaneous use.
- Assessed adherence to silicone and uptake by J774.2 murine macrophages for three clinical isolates.
- Performed in silico analysis of agglutinin-like sequences (Als).
Main Results:
- Successfully generated fluorescently tagged C. parapsilosis isolates.
- Identified significant differences in silicone adherence and macrophage uptake among isolates.
- GA1 isolate showed reduced macrophage resistance, while CDC317 exhibited increased silicone adherence.
- Discovered diversity in Als genes and evidence of a novel gene formation in the GA1 isolate.
Conclusions:
- The PCR-based CRISPR/Cas9 system is effective for fluorescently labeling C. parapsilosis.
- Clinical isolates of C. parapsilosis exhibit distinct adherence and virulence characteristics.
- Genetic variation, including novel gene formation, contributes to the diversity of C. parapsilosis.
More Related Videos
09:56CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans
Published on: November 14, 2018
07:48Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow
Published on: April 1, 2022
Related Concept Videos
02:53CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
01:59CRISPR