Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans
Issued Date
2025-01-01
Resource Type
eISSN
21650497
Scopus ID
2-s2.0-85214494868
Pubmed ID
39565132
Journal Title
Microbiology Spectrum
Volume
13
Issue
1
Rights Holder(s)
SCOPUS
Bibliographic Citation
Microbiology Spectrum Vol.13 No.1 (2025)
Suggested Citation
Khongthongdam M., Phetruen T., Chanarat S. Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans. Microbiology Spectrum Vol.13 No.1 (2025). doi:10.1128/spectrum.01618-24 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/103070
Title
Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans
Author(s)
Author's Affiliation
Corresponding Author(s)
Other Contributor(s)
Abstract
Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significantthreat to immunocompromised individuals, particularly those with HIV/AIDS. Effectivegenetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectivelyselected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affectcell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirmingits applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.