Signal transduction in C. neoformans

描述（由申请人提供）：新生梭状菌是一种重要的真菌病原体，特别是在免疫功能低下的患者中。了解真菌细胞壁的生物合成和调控是很重要的，特别是因为它们是必不可少的细胞器，已经成功地用作抗真菌治疗的靶点。在我们之前的资助期间，我们已经产生了重要的初步数据，表明蛋白激酶C （PKC1）途径影响新生C的细胞壁完整性和毒力，并已经确定了细胞壁减弱、无毒或高毒的突变体。此外，我们已经证明PKC1在氧化和亚硝化应激反应中起作用。在这个应用中，我们建议进一步表征PKC1通路，以确定哪些基因对信号传导和细胞壁完整性是重要的，定义在新生C.中与PKC1通路相互作用的蛋白质，确定受通路扰动影响的下游基因，并确定感知细胞壁损伤的蛋白质。有三个具体目标。首先，我们将使用微阵列和生化分析来确定该途径中扰动的下游效应。我们将识别突变体中被错误调控的基因，目标是能够在突变体的细胞壁中建立特定的缺陷。在第二个目标中，我们将剖析该途径上游调控因子的作用，特别是Rho蛋白、鸟嘌呤交换因子和GTPase激活蛋白。我们的初步数据表明，这部分途径与酿酒酵母不同。在第三个目标中，我们将采用遗传筛选来寻找作为PKC1途径传感器的蛋白质，重点是氧化和亚硝化应激，并触发该途径的激活。在这个应用中，我们建议分析真菌病原体C. neoformans中的PKC1通路。这一途径对细胞生长、抵抗细胞壁破坏剂和毒力至关重要，并在抗真菌剂的作用下被诱导。由于这些原因，描述该通路的组成部分，确定它们如何相互作用，该通路具有什么细胞效应，以及该通路如何接收来自环境的输入是至关重要的。这些研究应该阐明真菌细胞用来调节其细胞壁生物合成的机制，并以确定抗真菌治疗的新靶点为长期目标。

DESCRIPTION (provided by applicant): C. neoformans is a significant fungal pathogen, particularly in immunocompromised patients. It is important to understand the biosynthesis and regulation of fungal cell wall, especially because they are essential organelles that have been successfully used as targets for antifungal therapy. In our previous funding period we have generated significant preliminary data to suggest that the protein kinase C (PKC1) pathway impacts cell wall integrity and virulence in C. neoformans and have identified mutants that have weakened cell walls and that are avirulent or are hypervirulent. Additionally, we have shown that PKC1 has a role in response to oxidative and nitrosative stresses. In this application, we propose to further characterize the PKC1 pathway to determine which genes are important for signaling and cell wall integrity, to define the proteins that interact with the PKC1 pathway in C. neoformans, to determine the downstream genes that are affected by perturbations in the pathway, and to identify the proteins that sense cell wall damage. There are three specific aims. In the first, we will determine the downstream effects of perturmations in the pathway using micro-arrays and biochemical analysis. We will identify genes that are miss-regulated in our mutants, with the goal of being able to establish the specific defects in the cell walls of the mutants. In the second aim, we will dissect the role of the upstream regulators of the pathway, specifically the Rho proteins and the guanine exchange factors and the GTPase activating proteins. Our preliminary data indicates that this part of the pathway is distinct from S. cerevisiae. In the third aim, we will employ a genetic screen to find the proteins that act as sensors for the PKC1 pathway with an emphasis on oxidative and nitrosative stress, and trigger activation of the pathway. In this application we have proposed to analyze the PKC1 pathway in the fungal pathogen, C. neoformans. This pathway is critical for cell growth, resistance to cell wall damaging agents and for virulence, and is induced in response to antifungal agents. For these reasons, it is critical to delineate the components of the pathway, determine how they interact, what cellular effects the pathway has, and how the pathway receives input from the environment. These studies should elucidate the mechanisms that fungal cells use to regulate biosynthesis of their walls, with the long-term goal of identifying novel targets for antifungal therapy. PUBLIC HEALTH RELEVANCE: Fungal infections have become more prevalent in recent years due to the increase in the immunocompromised patient population from AIDS, organ transplants and chemotherapies. Systemic fungal infections are serious health threats, and safe, highly effective antifungal therapies are not available. Biosynthesis of the fungal cell wall is an attractive target for antifungal therapies because the cell wall is an essential organelle that is not present in the human host, and this project will delineate signal transduction that determines the biosynthesis of the cell wall of a fungal pathogen, Cryptococcus neoformans.