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、系统的白念珠菌蛋白质-蛋白质相互作用试验。这种生物体是人类的主要侵入性真菌病原体，并且由于分子和基因组技术和资源的进步，已经成为越来越多的机制研究的焦点。越来越多的证据表明，许多独特的调节途径控制着C。白色念珠菌生长和致病性。蛋白质间相互作用的研究对于其他生物的途径分析是至关重要的，但C。白色念珠菌的发展落后于其他发展。我们已经开发了一种新的蛋白质-蛋白质相互作用测试，囊泡捕获相互作用（VCI）测定，采用高度保守的真核生物机械和最小的遗传操作。通过将荧光融合蛋白定向定位于内体表面，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，以限制观察者在解释中的偏差。VCI检测检测两个蛋白质对之间的相互作用，在两个模型酵母酿酒酵母和C。白色念珠菌我们建议开发一个广泛适用的C。白念珠菌VCI检测平台，包括菌株，载体，分析软件，以及对控制检测的基因表达参数的基本理解。VCI分析表明，C.白念珠菌的调节途径将为治疗方法的发展和致病机制的理解开辟新的途径。在所有已知的真核生物中的测定组分的保守性表明VCI测定也可以输入到其他真核病原体中。公共卫生相关性：白色念珠菌是人类主要的侵袭性真菌病原体，可引起致命的深部组织感染和严重的粘膜感染。本项目将实现一种新的方法来识别C。控制感染和药物敏感性的基因。这一结果将扩大新的抗真菌药物的开发和测试方法。

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.