Systematic Genetic Analysis of Fungal Virulence

描述（由申请人提供）：我们寻求利用完整的基因组序列来了解宿主-病原体相互作用，这些相互作用决定了真菌病原体在哺乳动物宿主中引起疾病的能力。机会致病菌新生隐球菌是T淋巴细胞缺陷患者死亡的主要原因，估计在300万死亡病例中占5-10%的近距离死亡原因；每年都会因艾滋病而死亡。由于其分子可操控性、单倍体遗传和忠实的动物感染模型，新生C. neoformans是了解真菌毒力的良好模式生物。我们已经注释了新形式C.基因组血清型A基因组序列，通过基因靶向对-1200个基因（约占基因组的20%）产生了标记标记的敲除突变体，构建并测试了全基因组寡核苷酸DMA微阵列，并在体外和小鼠体内感染模型中启动了毒力因子的遗传筛选。我们寻求继续开发和利用这些工具，以开发毒性所需的病原体基因的全面图片，并确定和启动更详细的研究，涉及由新生C.入侵中枢神经系统的亚群。为了实现这些目标，我们1)完成了新生C.敲除集合的构建，2)对敲除集合进行了体外表征，3)使用小鼠模型筛选敲除集合的毒力突变体，以及4)鉴定和表征入侵中枢神经系统缺陷的突变体。通过系统地识别新生C.产生的对毒力重要的分子，并了解它们的功能，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.