Molecular Mechanisms that Regulate Phase Transition in Dimorphic Fungi

描述（由申请人提供）：地方性二态真菌是临床实践中越来越重要的病原体，在免疫功能受损的人群中造成大量死亡。这些病原体必须从霉菌转化为酵母才能获得毒力并逃脱宿主的防御。尽管形态发生在这些感染的发病机制中起着关键作用，但调控相变的机制尚不清楚。我的研究目标是利用皮炎芽孢菌作为模型，揭示和表征调节二态真菌相变的基因。我假设涉及环境感知、细胞内信号、细胞分裂和形态的基因控制相变。为了研究这个问题，我提出了两个互补的目标，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转移产生的皮炎芽孢杆菌相变缺陷突变体中基因的身份和功能；2)研究皮炎芽孢杆菌基因组中两个被认为与真菌相变有关的同源物——septin和磷酸二酯酶——通过RNA干扰抑制其表达，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，并在小鼠肺部感染模型中测试它们对毒力的影响。我的研究结果将为二形真菌相变背后的分子机制提供基本的新见解。建议的研究培训将使我能够磨练我作为一名实验室科学家所需的技能，以推进分子医学真菌学领域的发展。该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打下坚实的知识基础，同时满足硕士学位的要求。一个在真菌发病机理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展。整个培训计划将使我能够建立一个当代研究计划，研究二形真菌形态发生的分子调控。拟议的研究与公共卫生有关，因为它将提供真菌引起疾病的机制的见解，并可能导致新的抗真菌药物靶点的鉴定。

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.