A tetR/tetO-regulated promoter system for A. fumigatus

描述(申请人提供)：烟曲霉是全世界骨髓和实体器官移植受者成功治疗的主要障碍。这种微生物是一种强有力的机会性真菌病原体，会导致严重的侵袭性感染，导致死亡率接近90%。器官移植计划的不断扩大，以及缺乏有效的抗真菌疗法来治疗侵袭性曲霉病，促使人们需要更详细地了解参与发病机制的烟曲霉菌基因。不幸的是，烟曲霉的遗传适应性落后于大多数其他真菌系统，这限制了可以在这种生物上进行的实验类型。可诱导启动子系统是真菌遗传学中最重要的工具之一，已被证明有助于阐明许多物种的基因功能。本研究的目的是开发烟曲霉菌可调控基因表达系统的技术，重点是已应用于其他真核生物的原核生物TetR/Teto系统。该项目的第一个目标是设计TetR/Teto系统，以便在四环素存在的情况下关闭烟曲霉菌中的一个基因。这将通过创建包含连接到最小TATA启动子的一个或多个拷贝的tet操纵子序列的启动子，并使用该混合启动子来驱动E.coliLacZ报告基因的表达来实现。表达盒将被转化到烟曲霉菌中，这些菌株结构性地表达由连接到单纯疱疹病毒VP16激活域的tet抑制因子TetR组成的人工反式激活剂，并且报告基因的活性将在四环素存在或不存在的情况下进行定量。第二个目的是确定该系统是否可以用来控制体内烟曲霉菌的基因表达。携带tet调节报告系统的菌株将用于侵袭性曲霉病小鼠模型的感染，并将在四环素存在和不存在的情况下确定报告基因在小鼠组织中的表达。在体内操纵基因表达的能力将提供一个独特的机会来评估特定的烟曲霉菌基因产物在曲霉病发病机制中的作用。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is a major obstacle to the successful treatment of bone marrow and solid organ transplant recipients worldwide. The organism is a potent opportunistic fungal pathogen, causing severe invasive infections that result in mortality rates that approach 90 percent. The continued expansion of organ transplantation programs, and the lack of effective antifungal therapy to treat invasive aspergillosis, is driving the need for a more detailed understanding of the A. fumigatus genes that contribute to pathogenesis. Unfortunately, the genetic tractability of A. fumigatus has lagged behind most other fungal systems, which limits the type of experiments that can be performed on this organism. Inducible promoter systems are one of the most important tools in fungal genetics and have proven to be instrumental for the elucidation of gene function in a number of species. The purpose of this proposal is to develop the technology for a regulatable gene expression system in A. fumigatus, focusing on the prokaryotic tetR/tetO system that has been applied to other eukaryotes. The first aim of the project is to engineer the tetR/tetO system so that a gene can be switched off in A. fumigatus in the presence of tetracycline. This will be accomplished by creating a promoter containing one or more copies of the tet operator sequence linked to a minimal TATA-promoter, and using this hybrid promoter to drive expression of an E. coli lacZ reporter gene. The expression cassette will be transformed into strains of A. fumigatus that constitutively express an artificial transactivator comprised of the tet repressor tetR linked to the Herpes simplex VP16 activator domain, and the activity of the reporter gene will be quantitated in the presence or absence of tetracycline. The second aim seeks to determine whether this system can be used to manipulate A. fumigatus gene expression in vivo. Strains carrying the tet-regulated reporter system will be used for infection in a mouse model of invasive aspergillosis, and the expression of the reporter gene in mouse tissues will be determined in the presence and absence of tetracycline. The ability to manipulate gene expression in vivo would provide a unique opportunity to assess the contribution of a specific A. fumigatus gene product to the pathogenesis of aspergillosis.