Discovering the networks that control cell wall integrity, cell division, morphogenesis and adhesion in Candida albicans using high content microscopy assays.

高度动态的真菌细胞包膜作为抵御化学攻击的第一道防线，在形态发生和毒力方面起着重要作用。微生物使用几种策略来维持细胞壁完整性（CWI），包括信号转导级联来监测细胞壁并对环境挑战做出反应。在微生物中，启动CWI级联反应并延迟或抑制细胞分裂的特定分子线索尚未得到充分研究。在过去的十年里，我的实验室在开发工具来探测真菌和细菌的CWI策略方面取得了重大进展。我们将开发新的显微镜分析方法，以了解酵母糖基磷脂酰肌醇（GPI）锚定脂蛋白在精油成分和衍生物反应中的作用，激活CWI通路，调节形态发生和细胞分裂。从长远来看，我们将确定CWI途径、细胞分裂、形态和毒力之间的相互关系，以响应多种抗真菌药物，为农业和医学中基于精油的抗真菌制剂的开发提供信息。我们最近开发了一种新的高含量分析方法，使用相关原子力-定量成像-共聚焦激光扫描显微镜（AF-QI-CLSM）成像，分别以纳米（nm）和皮牛顿（pN）分辨率同时监测活细胞包膜重塑、细胞壁弹性和表面粘附，同时实时跟踪至少三种荧光蛋白（FP）标记分子的组织和动力学。相关数据提供了洞察信号转导跨越细胞包膜和进入细胞响应亚致死水平的外源。缺乏关键CWI受体蛋白的白色念珠菌突变株将确定对精油作出反应的途径。功能化的AFM提示将探测细胞壁蛋白的定位、聚类和构象，而FPs将跟踪参与应激反应和CWI信号传导的细胞内信号分子和细胞分裂蛋白。图像还将揭示形态变化，如酵母-菌丝过渡。同步红外-高夫纳米光谱（UofM）将以纳米精度报道表面超微结构和细胞壁生化组成。活细胞相关AFM-QI-CLSM是一种全新的分析工具，在细胞生物学研究中具有广泛的应用前景。本研究将在分子水平上进一步了解真菌CWI通路和细胞壁质量控制。细胞壁是抗真菌药物的主要目标，其适应能力允许真菌繁殖，对农业和医学造成重大损害。因此，了解精油对真菌CWI的影响将为其作为环境安全的抗真菌替代品的使用提供信息。

The highly dynamic fungal cell envelope plays an important role in morphogenesis and virulence as the first line of defense for chemical challenge. Microbes use several strategies to maintain cell wall integrity (CWI), including signal-transduction cascades to monitor the cell wall and respond to environmental challenges. The specific molecular cues that initiate CWI cascades and delay or inhibit cell division in microbes are understudied. Over the past decade my lab has made significant strides in developing tools to probe CWI strategies in both fungi and bacteria.We will develop novel microscopy assays to understand the role of yeast glycosylphosphatidylinositol (GPI)-anchored lipoproteins in response to essential oil components and derivatives, activating CWI pathways and regulating morphogenesis and cell division. Over the long-term we will identify the interrelationship between CWI pathways, cell division, morphology and virulence in response to numerous antifungals to inform the development of essential oil-based antifungal formulations for agriculture and medicine.Our recently developed novel high-content assays use correlative atomic force-quantitative imaging-confocal laser scanning microscopy (AF-QI-CLSM) imaging to simultaneously monitor live cell envelope remodeling, cell wall elasticity and surface adhesion with nanometer (nm) and picoNewton (pN) resolution, respectively, while simultaneously tracking the organization and dynamics of at least three fluorescent protein (FP)-tagged molecules in real time. The correlative data provides insight into signal transduction across the cell envelope and into the cell in response to sub-lethal levels of xenobiotics. Mutant strains of Candida albicans deficient in key CWI receptor proteins will pinpoint the pathways that respond to essential oils. Functionalized AFM tips will probe the localization, clustering and conformation of cell wall proteins, while FPs track intracellular signaling molecules and cell division proteins involved in the stress response and CWI signaling. Images will also reveal morphological changes such as yeast-hyphal transitions. Synchrotron-infrared nano-spectroscopy-AFM with Gough (UofM) will report on surface ultrastructure and cell wall biochemical composition with nm precision. Correlative AFM-QI-CLSM of live cells is a completely novel analytical tool with broad application to cell biology for studying virtually any cell type and stress. This research will further our understanding of fungal CWI pathways and cell wall quality control at the molecular level. The cell wall is a primary target for antifungal agents, and its adaptive capabilities allow propagation of fungi that are a significant detriment to agriculture and medicine. Thus, understanding the impact of essential oils on fungal CWI will inform their use as environmentally safe antifungal alternatives.