Candida albicans responses to antifungals and cell wall stress

念珠菌病，美国第四种最常见的血液感染，以及其他形式的系统性感染 念珠菌病与40%或更高的死亡率有关，尽管接受了抗真菌药物的治疗。这个 白念珠菌细胞壁是白念珠菌病发病机制的核心，但连接细胞壁的机制 人们对监管和毒性的理解才刚刚开始。最近，我们证明了白色念珠菌 快速离域磷脂酰肌醇-(4，5)-二磷酸(PI(4，5)P2)和葡聚糖苷作为天然的一部分 对胞壁活性抗真菌卡泊芬净的反应。此外，我们还鉴定了一种新的白色念珠菌 PI(4，5)P2-Septin途径，调节念珠菌病小鼠的细胞壁完整性和毒力。我们 假设激活或下调PI(4，5)P2-Septin途径的能力由 环境(即平衡调节)是白念珠菌对细胞壁压力的最佳反应所必需的 药物暴露或侵袭性念珠菌病。这个项目的目标是证明我们的均衡监管 假设，验证提出的PI(4，5)P2-Septin途径，并确定其输出。我们将追求三个目标 明确的目标。第一个目标是证明平衡的PI(4，5)P2调节与保护性 对卡泊芬净的反应。PI(4，5)P2的动态反应将与细胞存活率相关 调节突变体和卡泊芬净敏感和耐药白念珠菌菌株。第二个目标是 卡泊芬净过程中PI(4，5)P2与其他PI(4，5)P2-Septin途径成分的相互作用 暴露和侵袭性念珠菌病。将通过在延时期间跟踪组件来评估交互 活细胞成像，演示物理相互作用，并通过荧光可视化细胞内的相互作用 共振能量转移(FRET)。突变体的PI(4，5)P2水平与PKC- MAPK细胞壁完整性通路激活。第三个目的是将转录因子与PI(4，5)P2-Septin联系起来 途径，并确定参与卡泊芬净应答的转录产物和途径靶点 发病机制。在小鼠的腹内念珠菌感染期间，将通过使用 RNA-Seq，一种全面量化基因表达的基本无偏见的方法。 该项目使用了一系列创新技术来研究与抗真菌相关的新途径 耐药性与念珠菌病的发病机制。因此，它可能会产生临床上有用的见解， 不会通过其他研究获得。我们的发现将具有重要意义，因为它们将解释 PI(4，5)P2-Septin途径调控细胞壁完整性、棘球蛋白敏感性和耐药性，以及 发病机制，并将该途径置于这些过程的其他调节因子的背景下。该项目将 开辟新的研究途径，详细定义分子和细胞机制， PI(4，5)P2-Septin途径在不同类型的念珠菌病中起作用，以及细胞壁调节的影响 关于与主持人的互动。

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)P2-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 would not 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.