Comparative and functional genomics of C. neoformans

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引起危及生命的感染的高发，因此这种真菌对全球约4000万艾滋病毒感染者构成重大威胁。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的结合，对新生C.的毒力进行详细的了解，并为我们获得的对抗真菌感染的策略提供知识。我们的第一个具体目标是确定两种荚膜新生C. （A和D）血清型的基因含量，这两种荚膜新生C. （A和D）代表了感染艾滋病患者的全球分离菌群。为了实现这一目标，我们将与细菌人工染色体（BAC）克隆的全基因组阵列进行比较DNA杂交实验。该阵列将从指纹法生成的BAC克隆的物理图谱中开发出来；用这种方法已经为三个分离株的新生梭状芽孢杆菌构建了图谱。比较杂交将确定菌株之间的差异区域，这些区域将被测序，并与现有的基因组序列信息进行比较，以形成血清型内部和血清型之间基因含量差异的观点。我们还将研究在铁获取中具有假定作用的基因簇的保护。第二个具体目标是在小鼠模型肺部感染期间和被巨噬细胞样细胞系吞噬后，表征代表A和D血清型的两种新型C.菌株的转录组。我们将使用基因表达序列分析（SAGE）来确定基因组尺度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C. neoformans细胞收集的SAGE数据进行比较，这些数据对不同的温度（25℃vs. 37℃）和铁水平有反应。第三个具体目标是对我们的SAGE分析在血清型D菌株中鉴定的铁调控基因簇进行分子遗传学分析。这些基因编码一个假定的多铜氧化酶（CnFET3）和一个假定的铁渗透酶（CnFTR1）。我们将比较这些基因（以及第三个铁调控基因CnURF1）在不同血清型之间的组织和转录调控。我们还将破坏这些基因，以检验它们在毒力中的作用。

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.