Role and Regulation of a Molecular Mimic in Histoplasma Pathogenesis

描述（由申请方提供）：荚膜组织胞浆菌是一种典型的“二型”真菌病原体，经历温度诱导的从土壤中生长的霉菌形式转变为在肺巨噬细胞中建立感染的酵母形式。所有的二型真菌引起免疫功能低下患者的最严重的疾病，但他们是主要的病原体，可以导致严重的问题，在主机缺乏任何明显的免疫缺陷。H. capsulatum是CBP，一种酵母相特异性分泌蛋白，其对于体外和体内巨噬细胞内的增殖是重要的。解决CBP的三维结构揭示了一个令人惊讶的相似性saposin B，一种哺乳动物蛋白参与膜加工和抗原呈递。虽然结构同源性强烈暗示CBP是大型和多样的鞘脂激活蛋白样蛋白家族的第一个确定的真菌成员，但其在组织胞浆菌病中的确切机制作用仍未得到证实。该资助申请旨在评估CBP是否确实作为saposin发挥作用，并了解CBP 1阶段特异性调节的基础：具体目标1。定义CBP的saposin样功能。本目标中的研究将首先测试CBP在结合脂质方面是否像saposin一样起作用，无论是真菌还是哺乳动物，并将包括一个 结构-功能分析以确定CBP的哪些残基参与。CBP在CD 1抗原呈递中的潜在作用，类似于saposin B的功能，也将被探索。具体目标2。识别参与CBP 1转录调控的基因。插入诱变将用于产生突变体文库，所述突变体文库将筛选不再正常调节CBP 1的突变体。最有前途的候选调控基因随后将被用来确定协调调节基因，可能解开CBP功能或发病机制中的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.