Development of <i>ptxD</i> /Phi as a new dominant selection system for genetic manipulation in <i>Cryptococcus neoformans</i>

<i>Cryptococcus neoformans</i> is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic 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 (<i>ptxD</i>)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in <i>C. neoformans</i>. A codon-optimized <i>ptxD</i> gene from <i>Pseudomonas stutzeri</i> was cloned under the <i>TEF</i> promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the <i>ptxD</i> gene into the <i>C. neoformans</i> genome and assessed the impact of <i>ptxD</i> integration on cell growth and virulence factors. The <i>ptxD</i>/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that <i>ptxD</i> integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the <i>ADE2</i> gene using this system, confirming its applicability for gene deletion. Taken together, the <i>ptxD</i>/Phi system provides a robust and versatile tool for genetic manipulation in <i>C. neoformans</i>, facilitating further research into its biology and pathogenicity.IMPORTANCE<i>Cryptococcus neoformans</i> is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of <i>C. neoformans</i>, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.