The ALS Gene Family of Candida albicans

描述（由申请人提供）：该提案描述了我们正在进行的研究，以表征白色念珠菌的8个ALS（凝集素样序列）基因，并确定其编码蛋白的功能。在之前的资助期内，我们生成了一组alsA/alsA突变株，并在检测中评估了它们与宿主细胞和分子的粘附性，以及它们在疾病模型中造成损伤的能力。数据显示，一些Als蛋白有助于白色念珠菌与宿主表面的粘附，并提出了Als蛋白的其他有趣作用。这种竞争性的延续应用包括利用具有良好特征的Als蛋白在分子水平上定义粘附功能的实验，以及对Als家族多功能性质的研究。在Aim 1中，利用AlsSp的强粘附功能分离其结合的宿主细胞蛋白；还描述了证明相互作用特异性的实验。表面等离子体共振被用来进一步表征这些分子相互作用，随机诱变被用来定义粘附功能所需的氨基酸。Aim 2的重点是根据Als2p对宿主细胞粘附的相对贡献对Als家族中的Als2p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中的作用。Aim 3扩展了先前的实验，表明诱变ALS5、ALS6或ALS7导致白色念珠菌对宿主表面的粘附增加。先前的微阵列分析显示，这些基因的缺失导致其他可能具有粘附功能的白色念珠菌细胞壁蛋白的上调。Aim 4利用我们大量收集的Als相关菌株和构建物来生产Als蛋白特异性单克隆抗体。这些抗体将用于确定Als蛋白在各种白色念珠菌形态表面、细胞交配过程和各种动物疾病模型真菌细胞上的定位。我们还将使用抗体来支持我们关于Als蛋白在白色念珠菌粘附宿主表面中的作用的结论。来自这些研究的数据是在分子水平上理解Als粘附相互作用和理解Als家族功能广度的关键。

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.