Polarity Establishment Mechanisms in Candida albicans

描述（由申请人提供）：在白色念珠菌菌丝生长期间如何建立和维持极性在很大程度上仍是未知的，这种知识差距限制了组织侵袭如何发生以及如何预防的模型的开发。这项研究计划的长期目标是了解极性是如何在菌丝中建立的，以及极性建立蛋白如何影响C。白色念珠菌的形态发生和侵袭特性。本申请的目的是确定一个滑稽本地化的GTdR（Rsr 1 p）控制极性建立，特别是在菌丝尖端。Rsr 1 p是指导菌丝生长的方向，侵入琼脂的能力，并为主要极性建立GTdR Cdc 42 p的细胞骨架目标的本地化所必需的。本申请的中心假设是Rsrlp的皮质GTdR活性控制极性建立的轴，并因此通过调节菌丝尖端处Cdc 42 p和/或其效应物的局部活化来控制菌丝生长的方向。在强有力的初步数据的指导下，该假设将通过追求三个具体目标来检验：1）鉴定Rsrlp在菌丝中极性建立中的功能所需的结构域; 2）确定Rsrlp活性的效应子;以及3）鉴定新的C.白色念珠菌的基因是重要的极性建立在菌丝。在第一个目标下，通过PCR介导的基因修饰和定点诱变构建的缺少预测功能结构域的Rsrlp版本，将分析极性建立、GTclase活性和定位中的缺陷。在第二个目标下，将通过a）分析双突变菌株的合成致死性和B）RSR 1的过表达以抑制cdc 42表型来确定RSR 1和CDC 42之间的遗传相互作用。此外，将使用GFP融合技术在缺乏Rsrlp活性的菌株中确定Cdc 42 p及其效应物的定位。在第三个目标下，出版了一套小说C。将使用GFP融合物分析白色念珠菌ORF（其对于极化生长是重要的，并且在其他生物体中缺乏直系同源物）的表达蛋白质的皮质定位。将构建缺乏鉴定的皮层定位蛋白的菌株，并分析菌丝生长期间极性建立的缺陷。这项研究意义重大，因为它将为预防组织侵袭的抗真菌策略的发展确定假定的分子靶点。与此同时，在菌丝中获得的关于极性建立的基本新知识有望推进其他丝状真菌和其他极化真核细胞系统中极性反应的一般领域。这是C语言中一个重要而又未被充分研究的领域。白念珠菌生物学，适用于了解免疫功能低下患者系统性白念珠菌感染期间组织侵袭的发病机制。

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.