The ALS Gene Family of Candida albicans

描述(由申请人提供)：这项建议描述了我们正在进行的八个白色念珠菌ALS(凝集素样序列)基因的特征和确定其编码蛋白的功能的工作。在之前的资助期间，我们产生了一组ALSA/ALSA突变株，并在与宿主细胞和分子的黏附试验中评估了它们的表型，以及它们在疾病模型中造成损害的能力。数据显示，几种Als蛋白有助于白色念珠菌与宿主表面的黏附，并表明Als蛋白还有其他有趣的作用。这一竞争性的继续应用包括利用表征良好的Als蛋白在分子水平上定义黏附功能的实验，并进行研究以确定Als家族的多功能性质。在目标1中，利用AlsSp的强大黏附功能来分离与其结合的宿主细胞蛋白；还描述了证明这种相互作用的特异性的实验。表面等离子体共振被用来进一步表征这些分子相互作用，随机突变被用来定义黏附功能所需的氨基酸。目标2的重点是根据Als家族中Als2p对宿主细胞黏附的相对贡献进行排序，并进一步确定Als2p和Alslp在调节白色念珠菌细胞大小方面的作用。目的3扩展了先前的实验，这些实验表明ALS5、ALS6或ALS7的突变导致白色念珠菌对宿主表面的粘附性增加。先前的微阵列分析表明，这些基因的缺失会导致其他可能具有黏附功能的白色念珠菌细胞壁蛋白表达上调。目的4利用我们收集的大量ALS相关菌株和构建物来生产ALS蛋白特异性单抗。这些抗体将被用来确定Als蛋白在各种形态的白色念珠菌表面、细胞交配过程中以及各种动物疾病模型的真菌细胞上的定位。我们还将使用这些抗体来支持我们关于Als蛋白在白念珠菌与宿主表面黏附中的作用的结论。这些研究的数据是在分子水平上理解ALS粘附性相互作用和理解ALS家族内功能的广度的关键。

DESCRIPTION (provided by applicant): This proposal describes our ongoing efforts to characterize the eight ALS (agglutinin-like sequence) genes of Candida albicans and determine the function of their encoded proteins. In the previous funding period, we generated a set of alsA/alsA mutant strains and evaluated their phenotype in assays for adhesion to host cells and molecules, and for their ability to cause damage in disease models. Data showed that several of the Als proteins contribute to adhesion of C. albicans to host surfaces and suggested other intriguing roles for the Als proteins. This competing continuation application includes experiments that utilize well-characterized Als proteins to define adhesive function at the molecular level and also pursues investigations to define the multifunctional nature of the Als family. In Aim 1, the strong adhesive function of AlsSp is exploited to isolate the host cell proteins to which it binds; experiments to demonstrate the specificity of the interaction are also described. Surface plasmon resonance is utilized to further characterize these molecular interactions and random mutagenesis is used to define amino acids required for adhesive function. The focus of Aim 2 is ranking Als2p within the Als family for its relative contribution to adhesion to host cells and also to further define the role of Als2p and Alslp in regulation of C. albicans cell size. Aim 3 expands on previous experiments that showed that mutagenesis of ALS5, ALS6 or ALS7 resulted in increased adhesion of C. albicans to host surfaces. Previous microarray analysis showed that deletion of these genes results in up-regulation of other C. albicans cell wall proteins that might have adhesive function. Aim 4 makes use of our large collection of ALS-related strains and constructs to produce Als protein-specific monoclonal antibodies. These antibodies will be used to define Als protein localization on the surface of various C. albicans morphological forms, during cell mating, and on fungal cells from various animal disease models. We will also use the antibodies to support our conclusions about the role of Als proteins in C. albicans adhesion to host surfaces. Data from these studies are key to understanding Als adhesive interactions at the molecular level and understanding the breadth of function within the Als family.