Control of Aspergillus fumigatus polarized growth through regulation of Ras prote

描述(由申请人提供)：我目前是一名高级博士后研究员，正在过渡到一名独立研究员。我的近期目标是完成我目前在斯坦巴赫实验室的研究项目，这将为我过渡到一个独立的学术职位奠定基础。我的长期职业目标是建立一个国际公认的实验室，研究导致人类疾病的真菌，重点是丝状真菌病原体。K22职业奖对于建立一个充满活力的、由外部资金资助的研究实验室将特别重要。烟曲霉是一种常见的分离、机会性霉菌。烟曲霉的分生孢子被吸入并萌发，让位于持续的极化生长。这种高度极化的尖端生长使烟曲霉菌能够侵入肺组织。尽管目前有抗真菌治疗，但由烟曲霉引起的侵袭性曲霉病(IA)在免疫功能低下的患者中仍有很高的发病率和死亡率。尽管极化生长对烟曲霉菌的发育具有本质的影响，但人们对烟曲霉极化生长的分子机制知之甚少。在此之前，我发现烟曲霉菌的蛋白对于正常的极化生长和菌丝形态的形成是必需的。这一提议将检验两个主要假设：(1)通过rasa蛋白的棕榈酸化，rasa活性的质膜定位是极化生长所必需的，因此，在烟曲霉中是毒力所必需的；(2)在我的初步数据中确定的真菌特异性rasa结构域，是支持持续极化生长所需的rasa GTP酶水平所必需的。在目标1中，将通过测定棕榈酰化缺陷的RASA蛋白的形态效应来分析棕榈酰化介导的RASA活性的调节。为了确定质膜定位的重要性，将对表达缺乏适当棕榈酰化基序的突变rasa等位基因的菌株进行rasa定位、整个发育过程中的菌丝形态发生和毒力的检查。在目标2中，我将分析一个新的真菌特异性蛋白结构域对调节rasa GTP酶活性的重要性。此外，将使用体外结合分析来探索真菌特定的RASA结构域在与调节蛋白相互作用中的作用。这一目标的完成将确定真菌特异性RAS结构域对烟曲霉生长和毒力的贡献。在这项提议完成后，我将确定RAS蛋白区隔对菌丝形态发生和毒力的重要性，并将确定这一过程的调控机制。这将使我能够开发更详细的模型，在特定的亚细胞位置差异RAS蛋白功能，描述RAS活性的区隔如何调节极化生长。此外，我还将确定一种新的真菌特异性蛋白结构域对RASA功能的重要性。由于RAS蛋白在真菌形态发生中的重要作用，鉴定和鉴定RAS蛋白的真菌特异性特性具有发现新的抗真菌治疗方法的潜力。

DESCRIPTION (provided by applicant): I am currently a senior post-doctoral fellow transitioning to an independent investigator. My immediate objective is to complete my current research projects in the Steinbach lab that will lay the foundation for my transition to an independent academic position. My long term career objectives are to establish an internationally-recognized laboratory investigating fungi that cause human disease, with an emphasis on filamentous fungal pathogens. The K22 career award will be particularly important for establishing a vibrant, extramurally funded research laboratory. Aspergillus fumigatus is a commonly isolated, opportunistic mould. The conidia of A. fumigatus are inhaled and undergo germination, giving way to sustained polarized growth. This highly polarized tip growth allows A. fumigatus to invade lung tissues. Despite current antifungal therapy, invasive aspergillosis (IA) caused by A. fumigatus continues to have high morbidity and mortality in immunocompromised patients. Despite the essential nature of polarized growth to the development of IA, little is known about the molecular mechanisms of polarized growth in A. fumigatus. Previously, I identified that the A. fumigatus RasA protein is necessary for proper polarized growth and hyphal morphogenesis. This proposal will test two main hypotheses: (1) Plasma membrane localization of RasA activity, through palmitoylation of the RasA protein, is required for polarized growth and, therefore, virulence in A. fumigatus, and (2) a fungal-specific RasA domain, identified in my preliminary data, is required for RasA GTPase levels needed to support sustained polarized growth. In Aim 1, palmitoylation-mediated regulation of RasA activity will be analyzed by determining the morphological effects of palmitoylation deficient RasA proteins. To define the importance of plasma membrane localization, strains expressing mutant rasA alleles lacking appropriate palmitoylation motifs will be examined for RasA localization, hyphal morphogenesis throughout development, and virulence. In Aim 2, I will analyze the importance of a novel, fungal-specific protein domain to regulation of RasA GTPase activity. In addition, the role of the fungal-specific RasA domain in interaction with regulatory proteins will be explored using in vitro binding assays. Completion of this aim will define the contribution of the fungal-specific Ras domain to A. fumigatus growth and virulence. Upon completion of this proposal, I will have defined the importance of Ras protein compartmentalization to hyphal morphogenesis and virulence and will also have identified regulatory mechanisms of this process. This will enable me to develop more detailed models of differential Ras protein function at specific sub-cellular sites, describing how compartmentalization of Ras activity modulates polarized growth. In addition, I will define the importance of a novel, fungal-specific protein domain to RasA function. Due to the essential nature of Ras proteins to fungal morphogenesis, the identification and characterization of fungal-specific properties of Ras proteins carries the potential of uncovering novel anti-fungal therapies.