Role and Regulation of a Molecular Mimic in Histoplasma Pathogenesis

描述(申请人提供)：组织胞浆菌是一种典型的二相性真菌病原体，在温度诱导下从土壤中生长的霉菌形态转变为在肺巨噬细胞中建立感染的酵母菌形态。所有的二相性真菌都会在免疫功能低下的患者中引起最严重的疾病，但它们是主要的病原体，可以在缺乏任何明显免疫缺陷的宿主中引起严重问题。第一个被基因证实的致病因子是CBP，这是一种酵母相特异的分泌蛋白，对巨噬细胞的体外和体内增殖非常重要。解析CBP的三维结构显示出与皂苷B惊人的相似之处，皂苷B是一种哺乳动物蛋白质，参与膜处理和抗原呈递。虽然结构上的同源性强烈暗示CBP是第一个被发现的大而多样的皂苷样蛋白家族的真菌成员，但它在组织胞浆菌病中的确切机制作用仍未得到证实。这项赠款申请旨在评估CBP是否确实具有皂苷功能，并了解CBP1阶段特定调控的基础：具体目标1.定义CBP的皂苷样功能。这一目标的研究将首先测试CBP在结合脂质方面是否像皂苷一样发挥作用，无论是真菌还是哺乳动物，并将包括 结构-功能分析，以确定哪些CBP残基参与其中。CBP在CD1抗原递呈中的潜在作用也将被探索，类似于皂苷B的功能。特定目的2.确定参与CBP1转录调控的基因。插入突变将被用于产生一个突变体文库，该文库将被筛选出不再正常调控CBP1的突变体库。最有希望的候选调控基因随后将被用于识别协调调控的基因，潜在地解开与CBP功能或与发病机制中其他角色相关的基因网络。

DESCRIPTION (provided by applicant): Histoplasma capsulatum is one of the classic "dimorphic" fungal pathogens, undergoing a temperature-induced transition from a mold form that grows in soil to a yeast form that establishes infection in lung macrophages. All of the dimorphic fungi cause the most severe disease in immunocompromised patients, but they are primary pathogens that can cause serious problems in hosts lacking any demonstrable immune defect. The first genetically proven virulence factor of H. capsulatum was CBP, a yeast phase-specific secreted protein that is important for proliferation within macrophages in vitro and in vivo. Solving the three-dimensional structure of CBP revealed a surprising similarity to saposin B, a mammalian protein involved in membrane processing and antigen presentation. Although the structural homology is strongly suggestive that CBP is the first identified fungal member of the large and diverse saposin-like protein family, its precise mechanistic role in histoplasmosis remains unproven. This grant application is designed to evaluate whether CBP indeed functions as a saposin and to understand the basis for CBP1 phase-specific regulation: Specific Aim 1. Define the saposin-like function of CBP. The studies in this Aim will first test whether CBP functions like a saposin in terms of binding lipids, either fungal or mammalian, and will include a structure-function analysis to determine which residues of CBP are involved. The potential role of CBP in CD1 antigen presentation, analogous to the function of saposin B, will also be explored. Specific Aim 2. Identify the genes involved in CBP1 transcriptional regulation. Insertional mutagenesis will be used to generate a library of mutants that will be screened for those that no longer regulate CBP1 normally. The most promising candidate regulatory genes will subsequently be used to identify coordinately regulated genes, potentially unraveling a network of genes related to CBP function or to other roles in pathogenesis.