Polarity Establishment Mechanisms in Candida albicans

描述(由申请人提供)：在白色念珠菌菌丝生长过程中，极性是如何建立和保持的仍是未知的，这种知识的空白限制了组织入侵如何发生以及如何预防的模型的发展。与这项研究计划相关的长期目标是了解菌丝中的极性是如何建立的，以及极性建立蛋白是如何影响白色念珠菌的形态发生和入侵特性的。本应用的目的是确定滑稽定位的GTP酶(Rsr1p)如何控制菌丝顶端的极性建立。Rsr1p是引导菌丝生长方向，侵入琼脂的能力，以及定位主要极性建立的GTPase CDc42p的细胞骨架靶标所必需的。这一应用的中心假设是，RSRLP的皮质GTP酶活性通过调节菌丝顶端CDC42P和/或其效应子(S)的局部激活来控制极性建立轴，从而控制菌丝生长的方向。在强大的初步数据的指导下，这一假说将通过追求三个具体目标来检验：1)确定RSRLP在菌丝极性建立中所需的功能域；2)确定RSRLP活性的效应者(S)；以及3)鉴定对菌丝极性建立重要的新的白色念珠菌基因。在第一个目标下，通过PCR介导的基因修饰和定点突变构建的缺乏预测功能结构域的Rsrlp版本将被分析极性建立、GTPase活性和定位方面的缺陷。在第二个目标下，RSR1和CDC42之间的遗传相互作用将通过a)分析合成致死性的双突变菌株和b)过表达RSR1来抑制CDc42的表型。此外，利用GFP融合技术，将在缺乏Rsr1p活性的菌株中确定CDC42p及其效应物的定位。在第三个目标下，将分析一组已发表的新的白色念珠菌ORF(它们对极化生长很重要，并且在其他生物中缺乏同源基因)，以使用GFP融合对表达的蛋白质进行皮质定位。缺乏已鉴定的皮质定位蛋白的菌株将被构建并分析菌丝生长过程中极性建立的缺陷。这项拟议的研究具有重要意义，因为它将确定可能的分子靶点，用于开发防止组织入侵的抗真菌策略。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而研究不足的领域，对于了解免疫受损患者系统性C-白念珠菌感染期间组织入侵的发病机制具有适用性。

DESCRIPTION (provided by applicant): How polarity is established and maintained during Candida albicans hyphal growth remains largely unknown and this gap in knowledge has limited the development of models for how tissue invasion occurs and how it can be prevented. The long-term goal associated with this research program is to understand how polarity is established in hyphae and how polarity establishment proteins influence C. albicans morphogenesis and invasion properties. The objective of this application is to determine how a comically-localized GTPase (Rsr1 p) controls polarity establishment specifically at hyphal tips. Rsr1 p is required for guiding the direction of hyphal growth, for the ability to invade into agar, and for the localization of cytoskeletal targets of the major polarity establishment GTPase Cdc42p. The central hypothesis of this application is that the cortical GTPase activity of Rsrlp controls the axis of polarity establishment and, hence, the direction of hyphal growth by regulating the localized activation of Cdc42p, and/or its effector(s), at hyphal tips. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify the domains of Rsrlp that are required for its function in polarity establishment in hyphae; 2) Determine the effector(s) of Rsrlp activity; and 3) Identify novel C. albicans genes that are important for polarity establishment in hyphae. Under the first aim, versions of Rsrlp lacking predicted functional domains, constructed by PCR-mediated gene modification and site-directed mutagenesis, will be analyzed for defects in polarity establishment, GTPase activity, and localization. Under the second aim, genetic interactions between RSR1 and CDC42 will be determined by a) analyzing double mutant strains for synthetic lethality and b) over-expression of RSR1 for suppression of cdc42 phenotypes. In addition, localization of Cdc42p and its effectors will be determined in strains lacking Rsr1 p activity, using GFP fusion technology. Under the third aim, a published set of novel C. albicans ORFs (that are important for polarized growth, and that lack orthologs in other organisms) will be analyzed for cortical localization of the expressed proteins using GFP fusions. Strains lacking the identified cortically-localized proteins will be constructed and analyzed for defects in polarity establishment during hyphal growth. The proposed research is significant because it will identify putative molecular targets for the development of anti-fungal strategies that prevent 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.