PROTEIN-PROTEIN INTERACTION TEST IN CANDIDA ALBICANS

描述(由申请人提供)：我们的目标是开发一种简单、系统的白色念珠菌蛋白质相互作用分析方法。这种生物是人类的主要侵袭性真菌病原体，在分子和基因组技术和资源的进步的推动下，已经成为日益机械化的研究的焦点。越来越多的证据表明，许多独特的调控途径控制着白色念珠菌生长和致病性的关键方面。蛋白质-蛋白质相互作用研究对其他生物体的途径分析至关重要，但对白色念珠菌的相关方法学一直落后于其他发展。我们开发了一种新的蛋白质-蛋白质相互作用试验，即囊泡捕获相互作用(VCI)试验，它使用了高度保守的真核生物机制和最小的遗传操作。通过将荧光融合蛋白定向定位到内体表面，产生点状荧光信号来检测相互作用。通过计算方法对图像进行评估，以限制解释中的观察者偏见。VCI检测在模式酵母酿酒酵母和白色念珠菌中两个蛋白质对之间的相互作用。我们建议开发一个广泛适用的白念珠菌VCI检测平台，包括菌株、载体、分析软件和对控制检测的基因表达参数的基本了解。对白色念珠菌调控途径的VCI分析将为治疗的发展和对致病性的机制理解开辟新的途径。所有已知真核生物的检测成分的保守性表明，VCI检测也可能被引入到其他真核病原体中。公共卫生相关性：白色念珠菌是人类的主要侵袭性真菌病原体，可导致致命的深层组织感染和严重的粘膜感染。该项目将实施一种新的方法来识别控制感染和药物敏感性的白色念珠菌基因。这一成果将扩大新的抗真菌药物开发和测试的方式。

DESCRIPTION (provided by applicant): Our goal is to develop a simple, systematic protein-protein interaction assay in Candida albicans. This organism is the major invasive fungal pathogen of humans, and has been the focus of increasingly mechanistic studies, driven by advances in molecular and genomic technologies and resources. There is growing evidence that numerous unique regulatory pathways govern vital aspects of C. albicans growth and pathogenicity. Protein-protein interaction studies have been critical for pathway analysis in other organisms, but the relevant methodology for C. albicans has lagged behind other developments. We have developed a new protein-protein interaction test, the Vesicle Capture Interaction (VCI) assay that employs highly conserved eukaryotic machinery and minimal genetic manipulation. Interaction is detected through directed localization of fluorescent fusion proteins to endosome surfaces, yielding a punctate fluorescent signal. Images are evaluated through computational methods in order to limit observer bias in interpretation. The VCI assay detects interactions between two protein pairs in both the model yeast Saccharomyces cerevisiae and in C. albicans. We propose to develop a broadly applicable C. albicans VCI assay platform, including strains, vectors, analytical software, and a basic understanding of the gene expression parameters that govern detection. VCI analysis of C. albicans regulatory pathways will open new avenues toward development of therapeutics and a mechanistic understanding of pathogenicity. Conservation of assay components in all known eukaryote suggests that the VCI assay may be imported into other eukaryotic pathogens as well. PUBLIC HEALTH RELEVANCE: Candida albicans is the major invasive fungal pathogen of humans, causing lethal deep tissue infections and severe mucosal infections. This project will implement a new method to identify C. albicans genes that govern infection and drug sensitivity. The outcome will expand the ways in which new antifungal drugs can be developed and tested.