Polarity Establishment Mechanisms in Candida albicans

描述（由申请人提供）：白色念珠菌菌丝尖端生长过程中极性建立和维持的分子机制在很大程度上仍然未知，这方面的知识差距限制了菌丝如何侵入组织以及如何预防这种情况的模型的发展。与本研究项目相关的长期目标是了解菌丝尖端的极性是如何建立的（菌丝引导），以及菌丝引导蛋白如何介导白色念珠菌的接触感应（嗜黑性）和侵入行为。这些目标将在本提案中通过追求以下三个具体目标来实现：1)鉴定对菌丝尖端极性建立重要的蛋白质；2)确定尖端定位极性建立蛋白参与趋血性和侵袭的程度；3)建立已知菌丝引导基因GTPase Rsrlp控制菌丝引导的机制。在第一个目标下，将使用正向遗传筛选来鉴定皮质定位的极性建立蛋白（通过GFP融合的定位），并通过分析缺乏这些尖端定位候选蛋白的菌株的极性建立表型。在第二个目标下，将确定菌丝引导蛋白在响应脊和上皮细胞损伤时介导菌丝重定向的能力。在第三个目标下，Rsrlp与菌丝尖端独特极性蛋白之间的遗传和生化相互作用将通过a)缺乏Rsrlp活性的菌株的尖端极性结构（极化体和Spitzenkorper）的定位，b) Rsrlp与尖端极性蛋白之间物理相互作用的共免疫沉淀分析，以及c)缺乏Rsrlp和第二极性建立蛋白的菌株的趋近性和侵袭能力的上位性分析来确定。拟议的研究意义重大，因为它将提供了解菌丝如何定向生长以及这与临床相关过程-宿主组织入侵之间的关系所需的知识。同时，对菌丝极性建立的基础性新认识有望推动其他丝状真菌和其他极化真核细胞系统极性响应的研究。这是白色念珠菌生物学中一个重要且研究不足的领域，它适用于理解免疫功能低下患者全身白色念珠菌感染期间组织侵袭的发病机制。

DESCRIPTION (provided by applicant): The molecular mechanism by which polarity is established and maintained during Candida albicans hyphal tip growth remains largely unknown and this gap in knowledge has limited the development of models for how hyphae invade tissues how this can be prevented. The long-term goal associated with this research program is to understand how polarity is established at hyphal tips (hyphal guidance) and how hyphal guidance proteins mediate C. albicans contact sensing (thigmotropism) and invasive behavior. These objectives will be achieved in this proposal by pursuing the following three Specific Aims: 1) Identify proteins that are important for polarity establishment at hyphal tips; 2) Determine the extent to which tip-localized polarity establishment proteins are involved in thigmotropism and invasion; and 3) Establish the mechanism by which the known hyphal guidance GTPase, Rsrlp, controls hyphal guidance. Under the first aim, a forward genetic screen will be used to identify cortically-localized polarity establishment proteins, (by localization of GFP fusions) and by analysis of polarity establishment phenotypes in strains lacking these tip-localized candidate proteins. Under the second aim, the ability of hyphal guidance proteins to mediate hyphal re-orientation in response to a ridge and epithelial cell damage will be determined. Under the third aim, genetic and biochemical interactions between Rsrlp and unique polarity proteins at the hyphal tip will be determined by a) localization of tip polarity structures (polarisome and Spitzenkorper) in strains lacking Rsrlp activity, b) co-immunoprecipitation analysis of physical interactions among Rsrlp and tip polarity proteins, and c) epistasis analysis of thigmotropic and invasive ability of strains lacking both Rsrlp and a second polarity establishment protein. The proposed research is significant because it will provide the knowledge needed to understand how hyphae grow in a directional manner and how this is related to a clinically relevant process - host tissue invasion. At the same time, the fundamental new knowledge obtained about polarity establishment in hyphae is expected to advance the general field of polarity responses in other filamentous fungi and in other polarized eukaryotic cell systems. This is an important and under-investigated area of C. albicans biology that has applicability to understanding the pathogenesis of tissue invasion during systemic C. albicans infections in immunocompromised patients.