Fungal Ras-mediated invasive growth mechanisms

描述(申请人提供)：烟曲霉是侵袭性曲霉病的主要病原体，侵袭性曲霉病是一种严重的威胁免疫功能低下患者生命的疾病。烟曲霉菌是一种普遍存在的熟练病原体，能够迅速适应哺乳动物的肺部环境，并能有效地处理在人类宿主体内生长时遇到的压力。由于真菌生长的多因素性质，试图识别单个烟曲霉的毒力决定因素往往是不成功的。然而，先前的研究一直证明，丝状真菌病原体必须能够协调适当的菌丝形态发生，才能入侵组织并导致高死亡率。不幸的是，我们对支持持续极化生长和侵袭性疾病的建立和发展的分子机制的了解还不完整。为了阐明这些机制，必须建立更好的调控真菌形态发生的信号通路和蛋白质相互作用的模型。RAS蛋白在病原真菌中协调多种真菌形态发生过程。RAS对这些过程的调节在真菌毒力中起着至关重要的作用，使真菌RAS信号成为探索真菌发病机制和为新的治疗方法寻找新靶点的宝贵工具。尽管RAS信号通路的许多方面通常被认为过于保守，不能作为真核病原体的抗菌靶点，但我们已经确定了新的真菌特异性蛋白结构域，它们定义了真菌和人类RAS蛋白之间的根本差异。其中包括真菌RAS蛋白与人类对应蛋白H-ras的两个显著差异区域：1)不变精氨酸结构域(IRD)，这是一个在所有真菌基因组的RASA同源物中保守但在H-ras中不存在的新结构域；2)丝状真菌中的扩展高变区(HVR)。我们的初步数据表明，在菌丝生长和形态发生过程中，IRD和HVR是RAS功能所必需的，揭示了RAS信号的真菌特异性方面。这项提案的总体目标是充分定义 这些真菌特有的蛋白结构域与RAS生物学有关，使用这些结构域的突变来识别对极化形态发生和毒力至关重要的新的RAS途径相互作用。利用突变和生化分析，目标1或本建议将定义IRD和HVR对烟曲霉菌生物学的贡献。这些研究将解决IRD和HVR突变如何影响RASA GTP酶活性、激活、定位和下游效应器的相互作用。作为我们的AIM 1研究的补充，AIM 2利用无偏见的蛋白质组学筛选来确定有助于RAS介导的生长和毒力的新的、真菌特异性的RAS相互作用。因为RAS信号对真菌的毒力是必不可少的，识别和表征人类和RAS通路之间的根本差异具有发现新的抗真菌疗法的长期潜力。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the major causative agent of invasive aspergillosis, a severe and life-threatening disease of immunocompromised patients. A. fumigatus is a ubiquitous and adept pathogen, able to adapt quickly to the mammalian lung environment and to effectively handle the stresses encountered while growing within the human host. Because of the multi-factorial nature of fungal growth, attempts to identify individual A. fumigatus virulence determinants are often unsuccessful. However, previous studies have consistently proven that filamentous fungal pathogens must be able to coordinate proper hyphal morphogenesis in order to invade tissue and cause high mortality rates. Unfortunately, our knowledge of the molecular mechanisms supporting sustained polarized growth and the establishment and progression of invasive disease is incomplete. To elucidate these mechanisms, better models of the signaling pathways and protein interactions that regulate fungal morphogenesis must be developed. Ras proteins orchestrate multiple fungal morphogenetic processes in pathogenic fungi. Regulation of these processes by Ras plays an essential role in fungal virulence, making fungal Ras signaling an invaluable tool for probing fungal pathogenesis and identifying new targets for novel therapeutics. Although many aspects of Ras signaling pathways are often considered too highly conserved to serve as antimicrobial targets in eukaryotic pathogens, we have identified novel, fungal-specific protein domains that define fundamental differences between fungal and human Ras proteins. These include two areas of significant divergence between fungal Ras proteins and their human counterpart, H-ras: 1) the Invariant Arginine Domain (IRD), a novel domain conserved in the RasA homologs of every available fungal genome but not present in H-ras, and 2) an extended hypervariable region (HVR) in filamentous fungi. Our preliminary data show that the IRD and HVR are required for Ras function during hyphal growth and morphogenesis, revealing fungal-specific aspects of Ras signaling. The overall objectives of this proposal are to fully define the impact of these fungal-specific protein domains to Ras biology, using mutations of these domains to identify novel Ras pathway interactions critical for polarized morphogenesis and virulence. Using mutational and biochemical analyses, Aim 1 or this proposal will define the contribution of the IRD and HVR to A. fumigatus Ras biology. These studies will address how mutation of the IRD and HVR affect RasA GTPase activity, activation, localization and interaction downstream effectors. Complementing our Aim 1 studies, Aim 2 utilizes an unbiased proteomics screen to identify novel, fungal-specific Ras interactions contributing to Ras-mediated growth and virulence. Because Ras signaling is essential for fungal virulence, identification and characterization of fundamental differences between human and Ras pathways carries the long-term potential of uncovering novel antifungal therapies.