Németh, Tibor Mihály and Zarnocki, Andrea and Ladanyi, Anett and Papp, Csaba Gergő and Ayaydin, Ferhan and Szebeni, Gábor and Gácser, Attila (2025) PCR-based CRISPR/Cas9 system for fluorescent tagging: A tool for studying Candida parapsilosis virulence. PLOS ONE, 20 (2). No. e0312948. ISSN 1932-6203
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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.
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| Additional Information: | Funding Agency and Grant Number: EU's Horizon 2020 research and innovation program [739593, TKP2021-EGA-28]; Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund [143373, 142877]; Louis-Jeantet Foundation; Hungarian Scientific Research Fund [NKFIH K 147510]; Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences [BO/00561/21, BO/00582/22/8]; New National Excellence Program of the Ministry of Human Capacities [UNKP-23-5]; NKFIH [PIACI_FOKUSZ-2024-00036]; [HUN- REN 2001007] Funding text: We listed the funders of our work and those who were funded. FA and AG: EU's Horizon 2020 research and innovation program under grant agreement No. 739593 (https://research-and-innovation.ec.europa.eu/funding/funding-opportunit ies/funding-programmes- and-open-calls/horizon-2020_en) AG: Project no TKP2021-EGA-28 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021- EGA funding scheme. (https://nkfih.gov.hu/english/nrdi-fund/thematic-excellence-pro- gramme-tkp2021/call-for-applications) AG: This research was supported by the grant HUN- REN 2001007 (https://hun-ren.hu/en) AG: This research was supported by the Louis-Jeantet Foundation (https://www.jeantet.ch/en/local-re- search/) AG: Hungarian Scientific Research Fund (NKFIH K 147510) (https://nkfih.gov. hu/for-the-applicants) TN: Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the FK 22 funding scheme, project no. 143373 (https://nkfih.gov.hu/for-the-applicants) TN: Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/00561/21 (https://mta.hu/english) TN: UNKP-23-5 New National Excellence Program of the Ministry of Human Capacities. (https://www.unkp.gov.hu/palyazatok/bolyai-fel- sooktatasi-fiatal-oktatoi-kutatoi-osztondij) GJS: Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the FK 22 funding scheme, project no. 142877 (https://nkfih.gov.hu/for-the-applicants) GJS: Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/00582/22/8 (https://mta.hu/english) GJS: UNKP-23-5 New National Excellence Program of the Ministry of Human Capacities. (https://www.unkp.gov. hu/palyazatok/bolyai-felsooktatasi-fiatal-ok- tatoi- kutatoi-osztondij) GJS: 2023-1.1.1- PIACI_FOKUSZ-2024-00036 (NKFIH) (https://nkfih.gov.hu/palyazoknak/nkfi-alap) the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. |
| Subjects: | Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia R Medicine / orvostudomány > R1 Medicine (General) / orvostudomány általában |
| SWORD Depositor: | MTMT SWORD |
| Depositing User: | MTMT SWORD |
| Date Deposited: | 02 Sep 2025 14:32 |
| Last Modified: | 02 Sep 2025 14:32 |
| URI: | https://real.mtak.hu/id/eprint/223208 |
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