Comparative and functional genomics of C. neoformans

新型隐球菌的比较和功能基因组学

• 批准号: 6850675
• 负责人: JAMES W KRONSTAD
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850675
• 关键词: Cryptococcus neoformans; artificial chromosomes; comparative genomic hybridization; enzyme activity; functional /structural genomics; fungal genetics; fungal proteins; gene mutation; genetic mapping; genetic strain; genetic transcription; laboratory mouse; microarray technology; serial analysis of gene expression; serotyping; temperature sensitive mutant; virulence

DESCRIPTION (provided by applicant): The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections in AIDS patients and this fungus therefore poses a major threat to the estimated 40 million people worldwide who are infected with HIV. In this context, our long-term objectives are to develop a detailed understanding of virulence in C. neoformans through a combination of genomic and molecular genetic approaches and to contribute the knowledge that we acquire to strategies to combat fungal infections. Our first specific aim is to determine the gene content in the two capsular serotypes of C. neoformans (A and D) that represent the global population of isolates that infect AIDS patients. To achieve this goal, we will perform comparative DNA hybridization experiments with whole genome arrays of bacterial artificial chromosome (BAC) clones. The arrays will be developed from physical maps of BAC clones generated by a fingerprinting method; maps have already been constructed with this method for three isolates of C. neoformans. Comparative hybridization will identify regions of difference between strains and these regions will be sequenced and compared to existing genomic sequence information to develop a view of variability in gene content within and between serotypes. We will also examine the conservation of a gene cluster with a putative role in iron acquisition. The second specific aim is to characterize the transcriptomes of two strains of C. neoformans representing the A and D serotypes during pulmonary infection in a murine model and after phagocytosis by a macrophage-like cell line. We will use serial analysis of gene expression (SAGE) to identify the relative abundance of transcripts on a genomic scale. The new data that we obtain will be compared between the serotypes and compared with SAGE data that we have already collected from C. neoformans cells that are responding to different temperatures (25 degrees C vs. 37 degrees C) and iron levels. The third specific aim is to perform a molecular genetic analysis of the cluster of iron-regulated genes that was identified by our SAGE analysis in a serotype D strain. These genes encode a putative multicopper oxidase (CnFET3) and a putative iron permease (CnFTR1). We will compare the organization and transcriptional regulation of these genes (and a third iron-regulated gene CnURF1) between serotypes. We will also disrupt the genes to examine their role in virulence.

描述（由申请人提供）：真菌病原体新型隐球菌在艾滋病患者中导致危及生命的感染发生率很高，因此这种真菌对全世界估计有 4000 万艾滋病毒感染者构成了重大威胁。在这种情况下，我们的长期目标是通过基因组和分子遗传学方法相结合，详细了解新型隐球菌的毒力，并将我们获得的知识贡献给对抗真菌感染的策略。我们的第一个具体目标是确定代表感染艾滋病患者的全球分离株群体的两种新型隐球菌荚膜血清型（A 和 D）的基因含量。为了实现这一目标，我们将利用细菌人工染色体 (BAC) 克隆的全基因组阵列进行比较 DNA 杂交实验。该阵列将根据指纹识别方法生成的 BAC 克隆的物理图谱开发；已经用这种方法为三种新型隐球菌分离株构建了图谱。比较杂交将鉴定菌株之间的差异区域，并对这些区域进行测序并与现有的基因组序列信息进行比较，以了解血清型内和血清型之间基因内容的变异性。我们还将研究在铁获取中具有推定作用的基因簇的保守性。第二个具体目标是表征小鼠模型肺部感染期间和巨噬细胞样细胞系吞噬后代表 A 和 D 血清型的两种新型隐球菌菌株的转录组。我们将使用基因表达系列分析 (SAGE) 来确定基因组规模上转录本的相对丰度。我们获得的新数据将在血清型之间进行比较，并与我们已经从对不同温度（25 摄氏度与 37 摄氏度）和铁水平做出反应的新型隐球菌细胞收集的 SAGE 数据进行比较。第三个具体目标是对我们的 SAGE 分析在 D 血清型菌株中鉴定出的铁调节基因簇进行分子遗传学分析。这些基因编码假定的多铜氧化酶 (CnFET3) 和假定的铁渗透酶 (CnFTR1)。我们将比较这些基因（以及第三个铁调节基因 CnURF1）在血清型之间的组织和转录调控。我们还将破坏基因以检查它们在毒力中的作用。