Systematic Genetic Analysis of Fungal Virulence

描述（由申请人提供）：我们试图利用完整的基因组序列来了解宿主-病原体相互作用，这些相互作用决定真菌病原体在哺乳动物宿主中引起疾病的能力。机会致病菌新型隐球菌是T淋巴细胞缺陷个体的主要死因，估计是每年由艾滋病引起的300万例死亡中5-10%死亡的近因。C. neoformans由于其分子可操作性、单倍体遗传学和可靠的动物感染模型而成为了解真菌毒力的极好模式生物。我们对C.新形虫基因组血清型A基因组序列，通过基因靶向约1200个基因（对应于约20%的基因组）产生标记标记的敲除突变体，构建并测试全基因组寡核苷酸DMA微阵列，并在体外和小鼠体内感染模型中启动毒力因子的遗传筛选。我们寻求继续开发和利用这些工具，以开发一个全面的图片病原体基因的毒力所必需的，并确定和启动更详细的研究亚组参与入侵中枢神经系统的C。新人类为了实现这些目标，我们1）完成了C。新生动物敲除收集物，2）进行敲除收集物的体外表征，3）使用小鼠模型筛选敲除收集物的毒力突变体，和4）鉴定和表征在侵袭中枢神经系统方面有缺陷的突变体。通过系统鉴定C.通过了解它们的功能，我们希望为发现治疗真菌感染的新疗法奠定基础。

DESCRIPTION (provided by the applicant): We seek to harness complete genome sequences to understand host-pathogen interactions that determine the ability of a fungal pathogen to cause disease in a mammalian host. The opportunistic pathogen Cryptococcus neoformans is a major cause of death in individuals with T lymphocyte defects, and is estimated to be the proximal cause of mortality in 5-10% of the 3 million deaths; caused by AIDS annually. C. neoformans is an excellent model organism for understanding fungal virulence because of its molecular manipulability, haploid genetics and faithful animal infection models. We have annotated the C. neoformans genome serotype A genome sequence, generated signature-tagged knockout mutants by gene targeting for -1200 genes (corresponding to about 20% of the genome), constructed and tested a whole-genome oligonucleotide DMA microarray, and initiated genetic screens for virulence factors in vitro and in a mouse in vivo infection model. We seek to continue to develop and utilize these tools to develop a comprehensive picture of pathogen genes necessary for virulence and to identify and to initiate more detailed studies of the subset involved in invasion of the central nervous system by C. neoformans. To achieve these goals we 1) complete construction of the C. neoformans knockout collection, 2) perform in vitro characterization of the knockout collection, 3) screen knockout collection for virulence mutants using a mouse model, and 4) identify and characterize mutants defective in invasion of the central nervous system. By systematically identifying molecules produced by C. neoformans important for virulence and by understanding how they function, we hope to lay the groundwork for the discovery of new therapies to treat fungal infections.