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

项目摘要 免疫受损个体的真菌感染是一个不断升级的世界卫生问题。 最近医院中多药耐药耳念珠菌致死性暴发和 通常是良性的真菌，如C. glabrata强调了问题的严重性。当前治疗 真菌感染的选择仅限于几种抗真菌药物， 无效。目前迫切需要新的抗真菌分子靶点，以应对药物- 耐药病原体。本项目将描述一个新发现的C。白念珠菌毒力和耐药性 Cdc 14蛋白磷酸酶。我们最近的工作揭示了C.白念珠菌Cdc 14 调节细胞壁完整性、分隔、棘白菌素敏感性和菌丝发育，所有过程都与 毒性重要的是，即使是Cdc 14活性水平的适度降低也会严重损害病毒的毒力。 侵袭性念珠菌病小鼠模型。相反，Cdc 14对于正常的发育、生长和发育是有害的。 动物的细胞分裂Cdc 14在真菌中高度保守，其独特而严格的活性位点特异性 这意味着开发有效和高选择性的抑制剂应该是可以实现的， 对其他蛋白磷酸酶的挑战。我们的总体目标是， Cdc 14在C.白色念珠菌在目标1中， cdc 14对细胞壁完整性和分隔的调控。在目标2中，我们将描述Cdc 14对菌丝生长的调控作用。 启动和维护。在目标3中，我们将描述Cdc 14本身受 细胞壁应力和菌丝诱导信号。在目标1和2中，我们将采用无偏见的组学方法， 确定Cdc 14的相关底物和Cdc 14控制下的转录回路。在目标1中， 直接表征由Cdc 14缺陷引起的细胞壁缺陷。我们还将测试特定的模型， Cdc 14分别在目的1和2中具有促进细胞壁完整性和菌丝起始的功能。在目标3中， 将重点关注无序Cdc 14 C末端尾部的磷酸化调节，这是整合 模式真菌中的调节信号。我们将使用定量磷酸化蛋白质组学来了解 磷酸化C.白念珠菌Cdc 14在细胞壁应激、胞质分裂/分隔和菌丝起始过程中的作用 分化所有这三个目标将结束与结构功能分析使用生化，细胞生物学， 以及蜡虫和小鼠感染测定来表征Cdc 14功能的生理学意义， 磷酸调节，包括发病机制的重要性。总的来说，结果将定义分子 Cdc 14促进几种与生物学过程相关的生物学过程的机制，这些机制将有助于 评估其作为抗真菌靶点的未来潜力。Cdc 14底物和效应物的鉴定可能 提供额外的候选抗真菌靶点。Cdc 14的结构、活性和生物学特性高度保守， 真菌界的特异性意味着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.