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

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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.