Forward genetics-based discovery of Histoplasma virulence genes

描述(由申请人提供)：目前对组织胞浆致病的分子机制的了解仍然有限。与机会性病原体不同，真菌病原体组织胞浆菌甚至可以通过寄生宿主的吞噬细胞而在免疫活性宿主中致病。到目前为止，只有几个毒力因子被鉴定和表征。在这项提案中，我们将使用正向遗传学方法来发现使组织浆体能够颠覆巨噬细胞防御的毒力因子，巨噬细胞是组织浆体的主要宿主细胞。将使用插入突变技术创建组织胞浆酵母的随机突变株。将使用转基因巨噬细胞系和已通过荧光工程提供高通量筛选能力的组织浆体株来筛选突变株，以降低巨噬细胞的毒力。突变株将根据巨噬细胞内生长动力学分析阻止组织胞浆致病的阶段进行分类。每个减毒突变体所代表的毒力基因将通过突变图谱进行鉴定。最终收集的毒力缺陷突变体将根据其损伤的严重程度、其致病缺陷的分类和毒力基因同一性进行排序。这些排名将被用来在未来的研究中进一步确定已发现的毒力因子的特征，以确定它们在促进组织胞浆在宿主巨噬细胞中的生存和生长中的作用。

DESCRIPTION (provided by applicant): Current understanding of the molecular mechanisms that underly Histoplasma pathogenesis remains limited. Unlike opportunistic pathogens, the fungal pathogen Histoplasma capsulatum can cause disease even in immunocompetent hosts by parasitizing phagocytes of the host. Only a few virulence factors have been identified and characterized to date. In this proposal, we will use a forward genetics approach to discover the virulence factors that enable Histoplasma to subvert the defenses of the macrophage, Histoplasma's primary host cell. Random mutants of Histoplasma yeasts will be created using insertional mutagenesis. Mutants will be screened for decreased virulence in macrophages using a transgenic macrophage line and a Histoplasma strain that has been engineered with fluorescence to provide high-throughput screening capability. Mutants will be classified according to the stage at which Histoplasma pathogenesis is blocked by analysis of intramacrophage growth kinetics. The virulence genes represented by each attenuated mutant will be identified by mapping of the mutation. The final collection of virulence-defective mutants will be ranked according to the severity of their impairment, the classification of their pathogenesis defects, and the identity of the virulence gene identities. These rankings will be used to prioritize further characterization of the discovered virulence factors in future studies t define their roles in facilitating Histoplasma survival and growth in host macrophages.