CAPSULE REGULATION AND VIRULENCE IN CRYPTOCOCCUS NEOFORMANS

描述（由申请人提供）：包封的新型隐球菌真菌可导致危及生命的疾病，特别是在免疫力低下的情况下，目前的治疗方法尚不充分。新形梭菌的主要毒力因子是广泛存在的多糖胶囊。胶囊多糖的结构是已知的，并且已经研究了胶囊结构的各个方面，但是对胶囊合成的事件是如何调节的了解有限。我们的长期目标是详细了解胶囊合成，以便我们可以针对这一过程进行抗真菌治疗。在这个应用中，我们建议使用分子和计算技术的强大组合来重建控制胶囊合成的调节网络。当前应用程序的目的一是系统地识别参与胶囊调节的基因。Aim II旨在通过计算重建胶囊调控网络，从而对胶囊调控中的事件进行定量建模，并确定相互作用的优先级，以便进一步研究。Aim III提出通过调节关键转录因子的表达水平来验证选定的调控关系，并表征这些转录因子的结合位点。方法包括基因缺失、基因表达分析、显微镜、自动网络重建、定量建模、RNA干扰、四环素调控、染色质免疫沉淀与测序。这一系列研究将通过两个具有互补技能的实验室的协同努力得以实现，这种强有力的结合将在这一重要研究领域取得重大进展。这一创新的技术组合的实施将使人们对隐球菌生物学和发病机制有更深入的了解，确定抗真菌药物发现的靶点，并为今后对这种和其他真核病原体的研究产生有价值的数据集和实验方法。

DESCRIPTION (provided by applicant): The encapsulated fungus Cryptococcus neoformans is responsible for life-threatening disease, particularly in the context of compromised immunity, and current therapy is not adequate. The main virulence factor of C. neoformans is an extensive polysaccharide capsule. The structures of the capsule polysaccharides are known and aspects of capsule construction have been studied, but there is only limited understanding of how the events of capsule synthesis are regulated. Our long-term goal is to understand capsule synthesis in detail, so that we can target this process for antifungal therapy. In this application we propose to use a powerful combination of molecular and computational techniques to reconstruct the regulatory network that controls capsule synthesis. Aim I of the current application is designed to systematically identify genes involved in capsule regulation. Aim II is designed to computationally reconstruct the capsule regulatory network, enabling quantitative modeling of events in capsule regulation and prioritization of interactions for further study. Aim III proposes to validate selected regulatory relationships by modulating the expression levels of key transcription factors and to characterize the binding sites of these transcription factors. Methods will include gene deletion, gene expression analysis, microscopy, automated network reconstruction, quantitative modeling, RNA interference, tetracycline regulation, and chromatin immunoprecipitation coupled with sequencing. This range of studies will be enabled by the synergistic efforts of two labs with complementary skill sets, a potent combination that will generate significant progress in this important research field. Implementation of this innovative combination of techniques will yield greater understanding of cryptococcal biology and pathogenesis, identify targets for anti-fungal drug discovery, and generate valuable data sets and experimental approaches for future work on this and other eukaryotic pathogens.