Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens

Abstract

ABSTRACT The auxin-inducible degron (AID) technology is a convenient and powerful tool for protein functional characterization in a broad array of eukaryotic species. We recently demonstrated that the original AID and improved AID2 systems are very effective at rapid protein depletion in Candida albicans, and described a limited set of reagents for their use in certain auxotrophic lab strains. With an eye toward broader applicability with improved flexibility, we report here a new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, such as clinical isolates and the reference strain SC5314. We adapted a common recyclable antibiotic marker system for the required genome editing steps, and developed a strategy for simultaneous CRISPR/Cas9-mediated tagging of both target alleles. We also developed a composite all-in-one tagging cassette that combines the degron tag and the OsTIR1F74A gene for single-step strain engineering. We added a fluorescent protein tag option and designed and validated an approach for N-terminal tagging that retains natural promoter control. We also compared the effectiveness of the two commonly used synthetic auxins, 5-phenyl-indole-3-acetic acid and 5-adamantyl-indole-3-acetic acid, and the two common OsTIR1 variants, F74A and F74G, and provide guidelines for using the new AID2 system. Finally, using the novel all-in-one cassette, we demonstrate that the AID2 system also works in Candida auris, albeit less effectively under some conditions. The new reagents should enhance the convenience and accessibility of the AID2 system for the Candida research community. IMPORTANCE Invasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate the discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high‑value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species. Invasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate the discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high‑value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species.