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Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
Published on: March 19, 2015
Involvement of the Noncanonical Polyadenylation Polymerase Cid14 in Fungal Azole Resistance in the Pathogen
Chenxi Li1, Sihui Zhen2, Xiaoyu Ma1
1Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, Institute of Biochemistry and Molecular Biology, College of Life Sciences, Beijing Normal University (CLS-BNU), Beijing 100875, China.
Abstract:
The yeast noncanonical polyadenylation polymerase Cid14 was originally identified from fission yeast and plays a critical role in the TRAMP complex. This protein is a cytoplasmic cofactor and regulator of RNA-degrading exosomes. Cid14 is highly conserved from yeast to animals and has been demonstrated to play key roles in the regulation of RNA surveillance, nutrition metabolism, and growth in model organisms, but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Cid14 protein, named CID14, in the fungal pathogen C. neoformans. To obtain insights into the function of Cid14, we created a mutant strain, cid14Δ, with the CRISPR-Cas9 editing tool. Disruption of CID14 impaired cell membrane stability. Further investigations revealed the defects of the cid14Δ mutant in resistance to low carbohydrate levels. Meanwhile, significantly, the ability to grow under flucytosine stress was decreased in the cid14Δ mutant. More importantly, our results showed that the cid14Δ mutant does not affect yeast virulence but exhibits multidrug resistance to azole. Our work is the first to suggest that Cid14 plays critical roles in azole resistance by affecting Afr1, which is chiefly responsible for azole excretion in the ABC (ATP-binding cassette) transporter.
Insights
The fungal protein Cid14, crucial for RNA processing, impacts cell membrane stability and azole antifungal drug resistance in Cryptococcus neoformans. This study reveals Cid14
Area of Science:
- Molecular Biology
- Mycology
- Antifungal Drug Resistance
Background:
- Cid14 is a conserved protein regulating RNA processing and metabolism in model organisms.
- Its role in the fungal pathogen Cryptococcus neoformans (C. neoformans) was previously uncharacterized.
- C. neoformans poses a significant threat, necessitating research into its fundamental biology and drug resistance mechanisms.
Purpose of the Study:
- To identify and characterize the Cid14 homolog in C. neoformans.
- To investigate the function of C. neoformans Cid14 (CID14) in fungal physiology and stress response.
- To explore the potential role of CID14 in antifungal drug resistance.
Main Methods:
- Gene identification and cloning of CID14 in C. neoformans.
- CRISPR-Cas9 gene editing to create a cid14 deletion mutant (cid14Δ).
- Phenotypic analysis of the cid14Δ mutant under various stress conditions, including flucytosine and azole exposure, and assessment of virulence and membrane stability.
Main Results:
- The CID14 gene was identified and characterized in C. neoformans.
- Disruption of CID14 led to impaired cell membrane stability and defects in growth under low carbohydrate conditions.
- The cid14Δ mutant showed reduced growth under flucytosine stress but exhibited multidrug resistance to azole antifungals, linked to the ABC transporter Afr1.
Conclusions:
- Cid14 plays a critical role in maintaining cell membrane stability and nutrient metabolism in C. neoformans.
- CID14 is essential for resistance to flucytosine but contributes to azole resistance, likely through regulation of the efflux pump Afr1.
- This study provides novel insights into the function of Cid14 in fungal pathogens and its implications for antifungal drug resistance strategies.
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