Comparative and functional genomics of C. neoformans

描述(申请人提供)：真菌病原体新生隐球菌在艾滋病患者中导致威胁生命的感染的高发生率，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。在此背景下，我们的长期目标是通过基因组和分子遗传学方法的结合来详细了解新生梭菌的毒力，并为抗击真菌感染的策略贡献我们所获得的知识。我们的第一个特定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弧菌(A和D)的基因含量。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。这些阵列将从指纹方法产生的BAC克隆的物理图谱发展而来；已经用这种方法构建了三个新生链球菌分离株的图谱。比较杂交将识别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，以形成一种关于血清型内部和之间基因含量可变性的观点。我们还将研究一个基因簇的保守性，该基因簇可能在铁的获取中发挥作用。第二个特定目的是研究代表A和D血清型的两株新生葡萄球菌在小鼠肺部感染模型中和被巨噬细胞样细胞系吞噬后的转录本。我们将使用基因表达序列分析(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不同的血清型之间进行比较，并与我们已经从对不同温度(25摄氏度对37摄氏度)和铁水平做出反应的新生葡萄球菌细胞收集的SAGE数据进行比较。第三个具体目标是对我们的SAGE分析在D血清型菌株中鉴定的铁调节基因簇进行分子遗传学分析。这些基因编码一个可能的多铜氧化酶(CnFET3)和一个可能的铁渗透酶(CnFTR1)。我们将比较这些基因(以及第三个铁调节基因CnURF1)在不同血清型中的组织和转录调控。我们还将破坏基因，以检查它们在毒力中的作用。

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.