Polarity Establishment Mechanisms in Candida albicans

描述(申请人提供)：在白色念珠菌菌丝顶端生长过程中，建立和维持极性的分子机制在很大程度上仍不清楚，这种知识上的差距限制了菌丝如何入侵组织的模型的开发，如何防止这种情况发生。与这项研究计划相关的长期目标是了解菌丝顶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知(嗜血性)和侵袭行为。本提案将通过追求以下三个具体目标来实现这些目标：1)确定对菌丝顶端的极性建立起重要作用的蛋白质；2)确定顶端定位的极性建立蛋白参与亲水性和侵袭性的程度；以及3)建立已知的菌丝引导GTP酶Rsrlp控制菌丝导向的机制。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末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状和上皮细胞损伤时介导菌丝重定向的能力。根据第三个目标，Rsrlp与菌丝顶端独特的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和TIP极性蛋白之间物理相互作用的免疫共沉淀分析，以及c)缺乏RsrLp和第二极性建立蛋白的菌株的上位性和侵袭性分析来确定。这项拟议的研究意义重大，因为它将提供所需的知识，以了解菌丝如何定向生长，以及这如何与临床相关的过程-宿主组织入侵有关。同时，关于菌丝体极性建立的基本新知识有望推动其他丝状真菌和其他极化真核细胞系统的极性反应的一般领域。这是白念珠菌生物学中一个重要且研究不足的领域，对于了解免疫受损患者系统性白念珠菌感染时组织侵袭的发病机制具有适用性。

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.