Forward genetics-based discovery of Histoplasma virulence genes

基于正向遗传学的组织胞浆菌毒力基因发现

基本信息

批准号：
8681794
负责人：
Chad A Rappleye
金额：
$ 7.37万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8681794
关键词：
Acute Agrobacterium Attenuated Biological Preservation Cell Line Cells Classification Collection Cytolysis Defect Development Disease Enabling Factors Engineering Environment Exhibits Fluorescence Fungal Chromosomes Future Galactosidase Gene Expression Profile Genes Genetic Genome Goals Growth Histoplasma Histoplasma capsulatum Histoplasmosis Immune Immune system Immunocompetent Impairment Individual Infection Insertional Mutagenesis Kinetics LacZ Genes Libraries Life Lung diseases Maps Measurement Mediating Modeling Molecular Monitor Mutagenesis Mutagenicity Tests Mutagens Mutation Pathogenesis Phagocytes Phase Process Role Severities Staging Systemic disease T-DNA Techniques Testing Therapeutic Transgenic Organisms United States Virulence Virulence Factors Yeasts adaptive immunity attenuation base forward genetics high throughput screening improved macrophage mutant pathogen public health relevance screening

项目摘要

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.

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。与机会性病原体不同，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而在免疫功能正常的宿主中引起疾病。迄今为止，仅鉴定和表征了少数毒力因子。在本提案中，我们将使用正向遗传学方法来发现使组织胞浆菌能够破坏巨噬细胞（组织胞浆菌的主要宿主细胞）的防御的毒力因子。将使用插入诱变产生组织胞浆菌酵母的随机突变体。将使用转基因巨噬细胞系和经过荧光工程改造以提供高通量筛选能力的组织胞浆菌菌株来筛选巨噬细胞中毒力降低的突变体。通过分析巨噬细胞内生长动力学，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。每个减毒突变体所代表的毒力基因将通过突变图谱来鉴定。毒力缺陷突变体的最终集合将根据其损伤的严重程度、发病机制缺陷的分类以及毒力基因的身份进行排序。这些排名将用于在未来的研究中优先考虑已发现的毒力因子的进一步表征，以确定它们在促进组织胞浆菌在宿主巨噬细胞中存活和生长中的作用。