Cdc14 phosphatase - novel roles in drug resistance, virulence, and the response to cell wall stress in fungal pathogens

项目总结 免疫受损个体的机会性真菌感染是一个日益严重的世界卫生问题。 近期医院多重耐药金黄色念珠菌的致死性暴发和#年耐药性的上升 正常情况下，像光滑假单胞菌这样的良性共生真菌突出了问题的严重性。目前的治疗方法 治疗真菌感染的选择仅限于少数几类正在变得越来越多的抗真菌药物 效果不佳。迫切需要新的抗真菌分子靶点来处理药物- 抗药性病原体。该项目将表征一种新发现的白色念珠菌的毒力和耐药性。 该因子为CDC14蛋白磷酸酶。我们最近的工作发现了白色念珠菌cdc14在 调节细胞壁完整性、隔膜、棘球菌素敏感性和菌丝发育，所有这些过程都与 致命性。重要的是，即使是cdc14活性水平的适度下降也会严重损害一种 侵袭性念珠菌病小鼠模型。相反，cdc14对于正常发育、生长和 动物的细胞分裂。Cdc14在真菌中高度保守，具有独特而严格的活性部位特异性。 意味着开发有效和高度选择性的抑制剂应该是可以实现的，这一点已经 一直在挑战其他蛋白磷酸酶。我们的总体目标是通过以下方式来描述这些机制 其中CDC14调控白念珠菌毒力相关的生物过程。在目标1中，我们将描述 CDC14对细胞壁完整性和隔膜的调节。在目标2中，我们将描述菌丝的cdc14调节。 启动和维护。在目标3中，我们将描述cdc14本身受 细胞壁应力和菌丝诱导信号。在目标1和目标2中，我们将使用公正的组学方法来 确定CDC14的相关底物和在CDC14控制下的转录电路。在目标1中，我们将 直接表征由镉14缺乏引起的细胞壁缺陷。我们还将测试特定型号的 在AIMS 1和AIMS 2中，CDC14分别具有促进细胞壁完整性和菌丝起始的功能。在《目标3》中我们 将重点放在无序的CDC14 C-末端尾巴的光调节上，这是整合 模型真菌中的调控信号。我们将使用定量磷酸蛋白质组学来了解动态 白念珠菌CDC14在细胞壁胁迫、胞质分裂/隔膜和菌丝起始过程中的磷酸化 差异化。这三个目标都将以结构-功能分析结束，使用生化，细胞生物学， 以及蜡虫和小鼠感染实验，以表征CDC14功能和生理意义 磷调节，包括在发病机制中的重要性。总而言之，结果将定义分子 CDC14促进几个与毒力相关的生物过程的机制将在 评估其作为抗真菌靶标的未来潜力。Cdc14底物和效应器的鉴定可以 提供其他候选抗真菌靶点。CDC14的结构、活性和结构高度保守 整个真菌王国的特异性意味着这一结果将与许多其他真菌病原体相关。

PROJECT SUMMARY Opportunistic fungal infection of immune-compromised individuals is an escalating world health problem. Recent lethal outbreaks of multi drug-resistant Candida auris in hospitals and the rise of drug resistance in normally benign commensal fungi like C. glabrata highlight the severity of the problem. 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. This project will characterize a newly identified C. albicans virulence and drug resistance factor, the Cdc14 protein phosphatase. Our recent work has uncovered novel roles for C. albicans Cdc14 in regulating cell wall integrity, septation, echinocandin sensitivity, and hyphal development, all processes tied to virulence. Importantly, even modest reduction in Cdc14 activity level severely compromises virulence in a mouse model of invasive candidiasis. In contrast, Cdc14 is dispensable for normal development, growth, and cell division in animals. Cdc14 is highly conserved in fungi and its unique and strict active site specificity implies that development of potent and highly selective inhibitors should be achievable, something that has been challenging with other protein phosphatases. Our overall objective is to characterize the mechanisms by which Cdc14 regulates virulence-associated biological processes in C. albicans. In Aim 1 we will characterize Cdc14 regulation of cell wall integrity and septation. In Aim 2 we will characterize Cdc14 regulation of hyphal initiation and maintenance. In Aim 3 we will characterize the mechanisms by which Cdc14 itself is regulated by cell wall stress and hypha-inducing signals. In Aims 1 and 2 we will employ unbiased omics approaches to identify the relevant substrates of Cdc14 and the transcriptional circuits under Cdc14 control. In Aim 1 we will directly characterize the cell wall defects arising from Cdc14-deficiency. We will also test specific models for Cdc14 function in promoting cell wall integrity and hyphal initiation in Aims 1 and 2, respectively. In Aim 3 we will focus on phosphoregulation of the disordered Cdc14 C-terminal tail, which is a hub for integration of regulatory signals in model fungi. We will use quantitative phosphoproteomics to understand the dynamic phosphorylation of C. albicans Cdc14 during cell wall stress, cytokinesis/septation, and initiation of hyphal differentiation. All three aims will conclude with structure-function analyses using biochemical, cell biological, and waxworm and mouse infection assays to characterize the physiological significance of Cdc14 function and phosphoregulation, including the importance for pathogenesis. Collectively, the results will define the molecular mechanisms by which Cdc14 promotes several virulence-related biological processes that will be useful in assessing its future potential as an antifungal target. The identification of Cdc14 substrates and effectors may provide additional candidate antifungal targets. The high conservation of Cdc14 structure, activity, and specificity across the fungal kingdom implies the results will be relevant to many other fungal pathogens.