Molecular Mechanisms that Regulate Phase Transition in Dimorphic Fungi

描述(申请人提供)：地方性二相性真菌在临床实践中是日益重要的病原体，并在免疫力受损的人中导致大量死亡。这些病原体必须从霉菌转化为酵母菌，才能获得毒力并逃避宿主防御。尽管形态发生在这些感染的发病机制中起着关键作用，但调节相变的机制尚不清楚。我的研究目标是以皮炎芽孢霉菌为模型，揭示和表征调节二相性真菌相变的基因。我假设，参与环境感知、细胞内信号传递、胞质分裂和形态的基因控制着相变。为了研究这一点，我提出了两个相互补充的方法，使用正向和反向遗传策略：1)阐明我已经通过农杆菌介导的DNA转移产生的相变缺陷皮炎假单胞菌突变株中基因的身份和功能，以及2)通过RNA干扰抑制它们的表达，研究皮炎假单胞菌基因组中两个可能参与真菌相变的同源基因--Septin和磷酸二酯酶--在形态发生中的作用。我的实验方法将包括使用尖端的分子生物学工具来发现新的基因，辨别它们在形态发生中的作用，并在肺部感染的小鼠模型中测试它们对毒力的影响。我的研究结果将为了解二相性真菌相变背后的分子机制提供基本的新见解。拟议的研究培训将使我磨练作为一名板凳科学家所需的技能，以推进分子医学真菌学领域的发展。这门课程将为我在分子遗传学、微生物发病机制和医学真菌学方面奠定坚实的知识基础，同时满足硕士学位的要求。一个在真菌发病机制方面具有专业知识的指导委员会将指导我在实验室和课堂上的进步。整个培训计划将使我能够建立一个当代的研究计划，研究二相性真菌形态发生的分子调控。拟议的研究与公共健康有关，因为它将提供对真菌致病机制的洞察，并可能导致确定新的抗真菌药物靶标。

DESCRIPTION (provided by applicant): The endemic dimorphic fungi are increasingly significant pathogens in clinical practice and cause substantial mortality in those with impaired immunity. These agents must convert from mold to yeast to acquire virulence and escape host defenses. Despite the pivotal role of morphogenesis in pathogenesis of these infections, the mechanisms that regulate the phase transition are poorly understood. The goal of my research is to uncover and characterize genes that regulate phase transition in dimorphic fungi using Blastomyces dermatitidis as a model. I hypothesize that genes involved in environmental sensing, intracellular signaling, cytokinesis, and morphology govern phase transition. To investigate this, I propose two aims with complementary approaches, using forward and reverse genetic strategies: 1) Elucidate the identity and function of genes in B. dermatitidis mutants defective in phase transition that I have already generated by Agrobacterium-mediated DNA transfer, and 2) Investigate the role in morphogenesis of two orthologs in the B. dermatitidis genome postulated to be involved in fungal phase transition - septin and phosphodiesterase - by knocking down their expression with RNA interference. My experimental approach will involve the use of cutting-edge molecular biology tools to discover new genes, discern their role in morphogenesis, and test their impact on virulence in a murine model of pulmonary infection. The results of my research will provide fundamental new insight about the molecular mechanisms behind phase transition in dimorphic fungi. The research training proposed will allow me to hone the skills I will need as a bench scientist to advance the field of molecular medical mycology. The coursework will give me a solid intellectual foundation in molecular genetics, microbial pathogenesis and medical mycology, while fulfilling the requirements for a Masters Degree. A mentoring committee with expertise in fungal pathogenesis will guide my progress in the lab and classroom. The overall training program will allow me to establish a contemporary research program investigating molecular regulation of morphogenesis in dimorphic fungi. The research proposed is relevant to public health because it will provide insight into the mechanisms by which fungi cause disease and may lead to the identification of new antifungal drug targets.