Comparative Genomics of Histoplasma and Blastomyces

描述(由申请人提供)：温度诱导从腐生霉菌形态到寄生酵母形态的转变对于二相性真菌建立疾病是必不可少的，但调节这一转变并解释酵母毒力的基因知之甚少。组织胞浆菌和皮炎芽孢杆菌是在世界范围内引起疾病的二相性真菌中亲缘关系最密切的两种真菌，代表着日益严重的人类健康问题。分子遗传学工具的发展使这两种真菌在遗传上易于处理，并为酵母阶段上调基因在致病力中的作用提供了第一个确凿的证据-囊状芽孢杆菌中的CBP1和皮炎芽孢杆菌中的BAD1。我们建议从这两种制剂中鉴定和比较所有酵母阶段上调的基因，然后探索这些基因中选定的一个子集的功能作用。我们假设这两种真菌在进化上既有共同的基因，又有不同的基因来调节形态发生和致病性。一个由三个领先实验室组成的联盟将结合他们在大规模基因组学、分子遗传学和真菌发病机制方面的经验；由此产生的合作将使用基于微阵列的方法来识别和分析潜在的毒力相关基因。 我们的具体目标是：1.对皮炎杆菌基因组进行测序，将其覆盖范围约为3倍，以便与囊状芽孢杆菌进行比较。使用微阵列和信息学鉴定酵母阶段上调的基因，并优先选择毒力相关研究的基因。III.通过在动物和细胞培养感染模型中创建用于研究的等基因零突变，利用LacZ和GFP报告器来监测基因的调控和表达，并使用微阵列来确定其他受协调调控的基因，从而从功能上表征囊状芽孢杆菌和皮炎假单胞菌16至20个优先基因。 这项工作的发现将为系统性真菌疾病的生物学和发病机制提供实质性的新知识，并可能在保守基因中识别许多潜在的新药靶点。

DESCRIPTION (provided by applicant): The temperature-induced transition from the saprophytic mold form to the parasitic yeast form is essential for the establishment of disease by dimorphic fungi, but the genes that regulate this switch and account for yeast virulence are poorly understood. Histoplasma capsulatum and Blastomyces dermatitidis are phylogenetically the most closely related among the dimorphic fungi that cause disease worldwide and represent a growing human health problem. Development of molecular genetic tools has rendered both these fungi genetically tractable and provided the first firm evidence for the role of yeast phase-upregulated genes in virulence - CBP1 in H. capsulatum and BAD1 in B. dermatitidis. We propose here to identify and compare all of the yeast phase-upregulated genes from these two agents, then explore the functional roles of a selected subset of these genes. We hypothesize that these two fungi will exhibit both evolutionarily shared and distinct genes that regulate morphogenesis and pathogenicity. A consortium of three leading labs will combine their experience with large-scale genomics, molecular genetics, and fungal pathogenesis; the resulting collaboration will use a microarray-based approach to identify and analyze potential virulence-associated genes. Our specific aims are to: I. Sequence the B. dermatitidis genome to approximately 3-fold coverage for comparson to H. capsulatum. II. Identify yeast phase-upregulated genes using microarrays and informatics, and prioritize genes for virulence-related studies. III. Functionally characterize in H. capsulatum and B. dermatitidis 16 to 20 of the prioritized genes by creating isogenic null mutants for investigation in animal and cell culture models of infection, exploiting lacZ and gfp reporters to monitor regulation and expression of the genes, and employing microarrays to identify other genes that are coordinately regulated. Findings from this work will provide substantial new knowledge about the biology and pathogenesis of systemic fungal diseases and may identify among conserved genes many potential new drug targets.