Development and application of auxin-inducible degradation in Candida pathogens

项目总结 免疫受损个体的机会性真菌感染是一个不断升级的世界卫生问题， 世界卫生组织最近的一份报告强调了这一点。多药耐药致死性暴发 医院中的金黄色念珠菌和正常良性共生真菌种类中耐药性的上升 光滑假丝酵母突出了这个问题的严重性。即使是严重的新冠肺炎病例也容易发生继发性 被曲霉和念珠菌等真菌病原体感染，这可能是致命的。目前的治疗方案 真菌感染仅限于少数几类正在变得越来越无效的抗真菌药物。那里 迫切需要新的分子靶点来开发抗真菌药物来处理耐药病原体。 我们的中心目标是在念珠菌中建立生长素诱导降解(AID)技术，以 能够对毒力和耐药因子进行功能研究，并作为促进抗真菌靶点的工具 抗真菌药物发现早期阶段的验证。援助提供快速和特定的目标耗尽 与其他常用的蛋白质功能研究方法相比，该方法具有重要的优势 人物刻画。在目标1中，我们将设计分子生物学试剂和菌株，并建立方案，以 在白色念珠菌、光滑念珠菌和金黄色念珠菌中验证和实施改良的AID系统。我们的新系统 应适用于任何菌株，包括临床分离的念珠菌病原物种。验证 实验将使用我们实验室确定的新的毒力和抗药性因子。在《目标2》中，我们将结合 两种常见动物感染模型对假丝酵母菌的辅助技术 幼虫和免疫抑制小鼠，为药物的早期靶点验证和体内模拟创建系统 对发病机制的影响。这些系统也将应用于我们的新型候选抗真菌靶标。援助是一种 强大的功能基因组学工具，将使真菌病原体的新研究机会成为可能。试剂 和在项目期间建立的方案将提供给研究界，工作 将建立一个蓝图，将AID的使用扩大到其他不同的真菌病原体。总体而言，这项技术 平台将解决确定和验证可行的抗真菌新靶点的迫切需要 治疗方面的发展。

PROJECT SUMMARY Opportunistic fungal infection of immune-compromised individuals is an escalating world health problem, recently highlighted in a report from the World Health Organization. Lethal outbreaks of multi drug-resistant Candida auris in hospitals and the rise of drug resistance in normally benign commensal fungal species like Candida glabrata highlight the severity of the problem. Even severe COVID-19 cases facilitate secondary infection by fungal pathogens like Aspergillus and Candida that can be lethal. Current treatment options for fungal infections are limited to a few antifungal drug classes that are becoming increasingly ineffective. There is a pressing need for new molecular targets for antifungal development to deal with drug-resistant pathogens. Our central objective is to establish auxin-inducible degradation (AID) technology in Candida pathogens to enable functional studies of virulence and drug resistance factors and as a tool to facilitate antifungal target validation in the early stages of antifungal drug discovery. AID provides rapid and specific depletion of target proteins of interest and has key advantages over other common methods for protein functional characterization. In Aim 1 we will engineer molecular biology reagents and strains, and establish protocols, to validate and implement a modified AID system in C. albicans, C. glabrata, and C. auris. Our novel system should be applicable in any strain, including clinical isolates, of Candida pathogen species. Validation experiments will use novel virulence and drug resistance factors identified in our labs. In Aim 2 we will combine AID technology in Candida species with two common animal infection models, Galleria mellonella (waxworm) larvae and immunosuppressed mice, to create systems for early target validation and in vivo simulation of drug effects on pathogenesis. These systems will also be applied to our novel candidate antifungal targets. AID is a powerful functional genomics tool that will enable new research opportunities in fungal pathogens. Reagents and protocols established during the project will be made available to the research community, and the work will establish a blueprint for expanding AID use to other diverse fungal pathogens. Overall, this technological platform will address the pressing need for identification and validation of viable new targets for antifungal therapeutic development.