Candida albicans responses to antifungals and cell wall stress

描述(由申请人提供)： 念珠菌病是美国第四大最常见的血液感染，与其他形式的系统性念珠菌病相关，尽管接受了抗真菌药物的治疗，但死亡率仍高达40%或更高。白色念珠菌的细胞壁是白念珠菌病发病机制的核心，但将细胞壁调节与毒力联系起来的机制才刚刚开始被了解。最近，我们证明了白色念珠菌对胞壁活性抗真菌卡泊芬净的自然反应是对磷脂酰肌醇-(4，5)-二磷酸(PI(4，5)P2)和葡聚糖素的快速离域。此外，我们还发现了一个新的白色念珠菌PI(4，5)P2-Septin途径，该途径可以调节白念珠菌感染小鼠的细胞壁完整性和毒力。我们假设，在药物暴露或侵袭性念珠菌病期间，激活或下调PI(4，5)P2-Septin途径的能力(即平衡调节)对于白念珠菌对细胞壁压力的最佳反应是必要的。本项目的目标是证明我们的平衡调控假说，验证所提出的PI(4，5)P2-Septin途径，并确定其输出。我们将追求三个具体目标。第一个目的是证明平衡的PI(4，5)P2调节与卡泊芬净的保护反应相关。PI(4，5)P2的动态反应将与PI(4，5)的细胞活力相关 P2调节突变体和卡泊芬净敏感和耐药白念珠菌菌株。第二个目的是在卡泊芬净暴露和侵袭性念珠菌病期间建立PI(4，5)P2和其他PI(4，5)P2-Septin途径成分之间的相互作用。相互作用将通过在延时活细胞成像过程中跟踪组件、演示物理相互作用以及通过荧光共振能量转移(FRET)可视化细胞内的相互作用来评估。突变体中PI(4，5)P2的水平与PKC-MAPK细胞壁完整性通路的激活直接相关。第三个目标是将转录因子连接到PI(4，5)2-Septin途径，并确定参与卡泊芬净反应和致病的转录产物和途径靶点。在小鼠的腹内念珠菌病期间，将使用RNA-Seq来定义转录输出，这是一种全面量化基因表达的基本上没有偏见的方法。该项目采用了一系列创新技术来研究与抗真菌耐药性和念珠菌病发病机制相关的新途径。因此，它很可能会产生通过其他研究无法获得的临床有用的见解。我们的发现将具有重要意义，因为它们将解释PI(4，5)P2-Septin途径如何调控细胞壁完整性、棘球绦虫易感性和耐药性以及发病机制，并将该途径置于这些过程的其他调节因素的背景下。该项目将开辟新的研究途径，详细定义PI(4，5)P2-Septin途径在不同类型念珠菌病中的分子和细胞机制，以及细胞壁调节对与宿主相互作用的影响。

DESCRIPTION (provided by applicant): Candidemia, the fourth most common bloodstream infection in the U.S., and other forms of systemic candidiasis are associated with mortality rates of 40% or more despite treatment with antifungal agents. The Candida albicans cell wall is central to the pathogenesis of candidiasis, but mechanisms that link cell wall regulation and virulence are only beginning to be understood. Recently, we demonstrated that C. albicans rapidly delocalizes phosphatidylinositol-(4, 5)-bisphosphate (PI (4, 5) P2) and septins as part of the natural response to the cell wall-active antifungal caspofungin. Furthermore, we identified a novel C. albicans PI (4, 5) P2-septin pathway that regulates cell wall integrity and virulence among mice with candidiasis. We hypothesize that the ability to activate or down-regulate the PI (4, 5) P2-septin pathway as dictated by the environment (i.e., balanced regulation) is necessary for optimal C. albicans responses to cell wall stress during drug exposure or invasive candidiasis. The objectives of this project are to prove our balanced regulation hypothesis, validate the proposed PI (4, 5) P2-septin pathway, and identify its outputs. We will pursue three specific aims. The first aim is to demonstrate that balanced PI (4, 5) P2 regulation correlates with protective responses to caspofungin. Dynamic PI (4, 5) P2 responses will be correlated with cellular viability in PI (4, 5) P2- regulatory mutants and caspofungin-susceptible and -resistant C. albicans strains. The second aim is to establish interactions between PI (4, 5) P2 and other PI (4, 5) P2-septin pathway components during caspofungin exposure and invasive candidiasis. Interactions will be assessed by tracking components during time-lapse live cell imaging, demonstrating physical interactions, and visualizing interactions within cells by fluorescence resonance energy transfer (FRET). PI (4, 5) P2 levels in pathway mutants will be directly correlated with PKC- MAPK cell wall integrity pathway activation. The third aim is to link transcription factors to the PI (4, 5) 2-septin pathway, and identify transcriptional outputs and pathway targets that contribute to caspofungin responses and pathogenesis. Transcriptional outputs will be defined during intra-abdominal candidiasis of mice by using RNA-Seq, a largely unbiased method that comprehensively quantitates gene expression. The project employs a series of innovative techniques to study a novel pathway that is relevant to antifungal drug resistance and the pathogenesis of candidiasis. Therefore, it is likely to yield clinically useful insights that wouldnot be obtained through other studies. Our findings will be significant because they will explain how the PI (4, 5) P2-septin pathway governs cell wall integrity, echinocandin susceptibility and resistance, and pathogenesis, and place the pathway within the context of other regulators of these processes. The project will open new avenues of investigation that will define, in detail, the molecular and cellular mechanisms by which the PI (4, 5) P2-septin pathway contributes to diverse types of candidiasis, and the impact of cell wall regulation on interactions with the host.