Signal transduction in C. neoformans

描述（由申请人提供）：C。新生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非常重要，特别是因为它们是已成功用作抗真菌治疗靶点的重要细胞器。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（PKC 1）途径影响C细胞壁的完整性和毒力。新形式，并已鉴定出具有弱化的细胞壁和无毒或超强毒的突变体。此外，我们已经表明，PKC 1在响应氧化和亚硝化应激中发挥作用。在本申请中，我们建议进一步表征PKC 1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，以确定与PKC 1通路相互作用的蛋白质在C.新形式的，以确定下游基因的影响，扰动的途径，并确定蛋白质的感觉细胞壁损伤。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扰动对通路的下游影响。我们将鉴定突变体中的失调基因，目的是能够确定突变体细胞壁中的特定缺陷。在第二个目标中，我们将剖析的作用，上游调控的途径，特别是Rho蛋白和鸟嘌呤交换因子和GT3激活蛋白。我们的初步数据表明，这部分途径不同于S。啤酒。在第三个目标中，我们将采用遗传筛选来寻找作为PKC 1通路传感器的蛋白质，重点是氧化和亚硝化应激，并触发通路的激活。在本申请中，我们提出分析真菌病原体C.新人类该途径对于细胞生长、对细胞壁破坏剂的抗性和毒力至关重要，并且在抗真菌剂的应答中被诱导。由于这些原因，至关重要的是描绘通路的组成部分，确定它们如何相互作用，通路具有什么样的细胞效应，以及通路如何从环境中接收输入。这些研究应该阐明真菌细胞用于调节其细胞壁生物合成的机制，其长期目标是确定抗真菌治疗的新靶点。 公共卫生相关性：近年来，由于艾滋病、器官移植和化疗导致的免疫功能低下患者人数增加，真菌感染变得更加普遍。全身性真菌感染是严重的健康威胁，安全，高效的抗真菌治疗不可用。真菌细胞壁的生物合成是抗真菌治疗的一个有吸引力的靶点，因为细胞壁是人类宿主中不存在的重要细胞器，该项目将描述决定真菌病原体新型隐球菌细胞壁生物合成的信号转导。

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.