Analysis of the Candida albicans adhesin Als5p

描述(由申请人提供)：我们的长期目标是了解真菌黏附在细胞相互作用中的机制和作用。白色念珠菌感染仍然是发病率和死亡率的一个重要原因，而且往往得不到有效的抗真菌药物的控制，因此造成了严重的健康问题。我们提出了以下具体目标：1.验证白色念珠菌粘附素AIs5p在附着底物后发生从非聚集态到聚集态的构象转变这一观点。构象位移将通过CD光谱进行监测。2.验证AIs5p介导的聚集性具有淀粉样蛋白特征的观点。CD光谱、淀粉样特异性染料的结合和电子显微镜将对可溶的AIs5p片段和聚集体进行测试3.测试AIs5p的串联重复区域介导同型结合促进聚集的想法。对可溶性蛋白片段进行非变性凝胶电泳，并与细胞表面表达的AIs5p变异体进行结合分析。4.验证AIs5p的N-末端球状Ig样结构域介导与底物黏附的想法。将进行与纯化的AIs5p类Ig蛋白片段的结合分析。这些实验将有助于解释来自其他病原体的一大群蛋白质的行为。

DESCRIPTION (provided by applicant): Our long term objective is to understand the mechanisms and role of fungal adhesions in cellular interactions. Candida albicans infections remain a significant cause of morbidity and mortality, and are often inadequately controlled with the available antifungals, thus creating a serious health concern. We propose the following specific aims: 1. Test the idea that Candida albicans adhesin AIs5p undergoes a conformational shift from a non-aggregative to aggregative state following adherence to substrate. Conformational shifts will be monitored by CD spectroscopy. 2. Test the idea that AIs5p mediated aggregation features amyloid characteristics. CD spectroscopy, the binding of amyloid specific dyes, and electron microscopy will be tested on soluble AIs5p fragments and aggregates 3. Test the idea that the tandem repeat region of AIs5p mediates homotypic binding facilitating aggregation. Non-denaturing gel electrophoresis on soluble protein fragments and binding assays with a cell-surface expressed AIs5p variant will be performed. 4. Test the idea that the N-Terminal globular Ig-like domain of AIs5p mediates adhesion to substate. Binding assays with purified AIs5p Ig -like protein fragments will be performed. These experiments will help explain the behavior of a large group of proteins from other pathogens.