CAPSULE REGULATION AND VIRULENCE IN CRYPTOCOCCUS NEOFORMANS

描述（由申请方提供）：包囊真菌新型隐球菌是危及生命的疾病的原因，特别是在免疫力受损的情况下，目前的治疗是不够的。C. neoformans是一种广泛的多糖荚膜。胶囊多糖的结构是已知的，并且已经研究了胶囊构建的各个方面，但是对胶囊合成的事件如何被调节的理解有限。我们的长期目标是详细了解胶囊合成，以便我们可以将此过程用于抗真菌治疗。在本申请中，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控网络。本申请的目的I被设计成系统地鉴定参与包膜调控的基因。目的II旨在通过计算重建胶囊调节网络，使胶囊调节事件的定量建模和相互作用的优先级为进一步研究。目的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。方法将包括基因缺失，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。这一系列的研究将通过两个具有互补技能的实验室的协同努力来实现，这一有力的组合将在这一重要的研究领域取得重大进展。实施这种创新的技术组合将产生更好的理解隐球菌生物学和发病机制，确定抗真菌药物发现的目标，并产生有价值的数据集和实验方法，为未来的工作在这个和其他真核病原体。

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.