Forward genetics-based discovery of Histoplasma virulence genes

描述（由申请人提供）：目前对组织胞浆菌发病机制的分子机制的理解仍然有限。与条件致病菌不同，真菌病原体荚膜组织胞浆菌甚至可以通过寄生宿主的吞噬细胞在免疫活性宿主中引起疾病。迄今为止，只有少数毒力因子被鉴定和表征。在这个建议中，我们将使用正向遗传学方法来发现毒力因子，使组织浆菌破坏巨噬细胞的防御，组织浆菌的主要宿主细胞。将使用插入诱变产生组织胞浆菌酵母菌的随机突变体。将使用转基因巨噬细胞系和组织胞浆菌菌株筛选巨噬细胞中毒性降低的突变体，该菌株已被荧光工程化以提供高通量筛选能力。通过巨噬细胞内生长动力学分析，根据组织胞浆菌发病机制被阻断的阶段对突变体进行分类。将通过突变作图鉴定每个减毒突变体代表的毒力基因。毒力缺陷突变体的最终集合将根据其损伤的严重程度、其致病缺陷的分类以及毒力基因身份的身份进行排序。这些排名将用于在未来的研究中优先进一步表征所发现的毒力因子，以确定其在促进宿主巨噬细胞中组织胞浆菌存活和生长中的作用。

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.