Candida albicans responses to antifungals and cell wall stress

描述（由申请人提供）： 血吸虫病是美国第四大常见的血液感染，和其他形式的系统性念珠菌病与40%或更高的死亡率相关，尽管用抗真菌剂治疗。白念珠菌细胞壁是念珠菌病发病机制的核心，但细胞壁调节和毒力之间的联系机制才刚刚开始被理解。最近，我们证明了C。白色念珠菌迅速使磷脂酰肌醇-（4，5）-二磷酸（PI（4，5）P2）和胞隔膜蛋白离域，这是对细胞壁活性抗真菌剂卡泊芬净的天然反应的一部分。此外，我们还鉴定了一个新的C.白色念珠菌PI（4，5）P2-septin途径，调节念珠菌病小鼠细胞壁完整性和毒力。我们假设激活或下调PI（4，5）P2-septin途径的能力取决于环境（即，平衡调节）对于最佳C是必要的。白念珠菌对药物暴露或侵袭性念珠菌病期间细胞壁应激的反应。本项目的目标是证明我们的平衡调节假说，验证提出的PI（4，5）P2-septin途径，并确定其输出。我们将追求三个具体目标。第一个目的是证明平衡的PI（4，5）P2调节与对卡泊芬净的保护性应答相关。动态PI（4，5）P2响应将与PI（4，5）中的细胞活力相关。 P2调节突变体和卡泊芬净敏感和耐药C.白色念珠菌菌株。第二个目的是建立在卡泊芬净暴露和侵袭性念珠菌病期间PI（4，5）P2和其他PI（4，5）P2-septin途径组分之间的相互作用。将通过在延时活细胞成像期间跟踪组分、证明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相互作用。途径突变体中PI（4，5）P2水平将与PKC-MAPK细胞壁完整性途径活化直接相关。第三个目的是将转录因子与PI（4，5）2-septin途径联系起来，并鉴定有助于卡泊芬净反应和发病机制的转录产物和途径靶点。将通过使用RNA-Seq（一种全面定量基因表达的基本上无偏倚的方法）在小鼠腹腔内念珠菌病期间定义转录输出。该项目采用一系列创新技术来研究与抗真菌药物耐药性和念珠菌病发病机制相关的新途径。因此，它很可能产生临床上有用的见解，而这些见解是通过其他研究无法获得的。我们的研究结果将是重要的，因为他们将解释PI（4，5）P2-septin途径如何管理细胞壁的完整性，棘白菌素的敏感性和抗性，和发病机制，并将这些过程的其他监管机构的背景下的途径。该项目将开辟新的研究途径，详细定义PI（4，5）P2-septin途径导致不同类型念珠菌病的分子和细胞机制，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.