Department of Biochemistry, Purdue University , West Lafayette, Indiana, USA.
Department of Biomedical Sciences, Universidad de Extremadura , Badajoz, Spain.
mSphere. 2023 Oct 24;8(5):e0028323. doi: 10.1128/msphere.00283-23. Epub 2023 Aug 18.
A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, and . We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in and that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.
多种诱导型蛋白降解（IPD）系统已被开发为研究蛋白质功能的有力工具。IPD 系统为快速失活几乎任何感兴趣的目标蛋白提供了一种便捷的机制。吲哚乙酸诱导降解（AID）是最常见的 IPD 系统之一，已在多种真核研究模式生物中建立。迄今为止，尚未开发用于致病真菌物种的 IPD 工具。在这里，我们证明了原始的 AID 和第二代 AID2 系统在人致病性酵母 和 中高效且快速地起作用。我们开发了一系列质粒，支持这些病原体的实验室菌株中 AID 系统的使用。这些系统可以在几分钟内诱导目标蛋白的降解超过 95%。在 AID2 的情况下，在低纳摩尔浓度的合成植物生长素类似物 5-金刚烷基吲哚-3-乙酸的情况下达到最大降解。在这两个物种中，生长素诱导的靶标降解成功模拟了基因缺失。该系统应该可以很容易地适应其他真菌物种和临床病原体菌株。我们的研究结果将 AID 系统定义为研究真菌病原体中蛋白质特征的强大而方便的功能基因组学工具。重要性危及生命的真菌感染是一个日益严重的人类健康问题，由于治疗选择有限以及抗药性病原体菌株的进化而变得复杂。确定用于治疗侵袭性真菌感染的新靶点，包括由 引起的感染，是当务之急。在本报告中，我们在 和 中建立并验证了一种诱导型蛋白降解方法，为这些真菌病原体，以及可能的其他真菌病原体物种中的蛋白质功能特征提供了一种新工具。我们预计该工具最终将有助于鉴定和表征有希望的药物靶点以及与毒力和耐药性相关的因素。
