Stress-induced transposon mobilization in the human fungal pathogen Cryptococcus

项目摘要 隐球菌是一种环境真菌，主要在免疫功能低下的人群中致病， 包括一种致命的隐球菌性脑膜炎，占艾滋病毒/艾滋病相关死亡的15%。吸入时 进入肺部，这些真菌必须迅速适应，以在人类宿主中遇到的各种压力下生存下来， 包括高温应激、pH变化和氧化应激。在持续性疾病的情况下， 隐球菌必须逃避宿主的免疫防御，并抵抗抗真菌药物的治疗。适应性基因组 已知可在感染期间增强毒力或导致抗药性的隐球菌的变化包括碱基 替换、小插入/小缺失和非整倍体。转座元件(TES)是一种可移动的小DNA 大多数真核生物基因组中存在的能够引起重要基因组的元素 变化和表型变异。TES在隐球菌和其他病原真菌中的潜在作用 (念珠菌和曲霉菌)在促进真菌发病或耐药方面的作用在很大程度上尚不清楚。我们 最近发现齿状隐球菌中TE动员是小鼠突变的重要原因 感染模式。耐药报告基因中TES的突变在高水平显著增加 温度(与宿主相关的温度37°)，表明热应激刺激TE的迁移率 隐球菌基因组。此外，我们还证实TE插入是临床耐药的一个原因。 体外抗真菌药物雷帕霉素/FK506和5-氟胞嘧啶。我们的研究是第一次发现 在致病真菌的感染过程中，作为突变原因的动员。此外，Te的诱变作用 对热应激的反应以前没有在任何模式酵母物种中描述过。值得注意的是，炎热- 在假单胞菌中鉴定的响应性TES包括DNA转座子和反转录转座子，每个转座子 不同的动员模式和基因组整合的首选位置。在拟议的研究中，我们寻求 1)确定热应激是否是去甲藻期间TE动员增加的主要原因 感染，2)确定热应激诱导齿状芽孢杆菌TE突变的调节因子，3)确定 TES是否在体外或在宿主感染期间在其他隐球菌种(新生隐球菌或加蒂隐球菌属)中移动。 阐明适应性变化增强真菌致病作用或使其产生耐药性的机制 在开发和维持有效的抗真菌治疗方面至关重要。这项研究将加深我们对 在可能导致疾病的隐球菌感染期间出现的应激诱导突变的类型 患者临床结果的持续性和变异性。此外，我们的研究将突出一套 可用于识别和量化的实验方法、感染模型和测序工具 广泛致病和非致病物种的基因突变(TE和非TE)。

Project Abstract Cryptococcus species are environmental fungi that cause disease primarily in immunocompromised populations, including a deadly cryptococcal meningitis that contributes to 15% of HIV/AIDS-related deaths. When inhaled into the lungs, these fungi must adapt rapidly to survive a variety of stresses encountered in the human host, including high temperature stress, changes in pH and oxidative stress. In cases of persistent disease, Cryptococcus must evade host immune defenses and resist antifungal drug treatment. Adaptive genomic changes in Cryptococcus known to enhance virulence or cause drug resistance during infection include base substitutions, small insertions/deletions and aneuploidy. Transposable elements (TEs) are small, mobile DNA elements present in the genomes of most eukaryotic organisms that are capable of causing significant genomic changes and phenotypic variation. The potential role of TEs in Cryptococcus and other pathogenic fungal species (Candida and Aspergillus) in contributing to fungal pathogenesis or drug resistance is largely unexplored. We recently identified TE mobilization in Cryptococcus deneoformans as a significant cause of mutation in a murine model of infection. Mutations by TEs in reporter genes for drug resistance were dramatically elevated at high temperature (37° host-relevant temperature) in vitro, suggesting that heat stress stimulates TE mobility in the cryptococcal genome. Additionally, we demonstrated TE insertion as a cause of drug resistance to clinical antifungal agents rapamycin/FK506 and 5-fluorocytosine in vitro. Our study was the first to identify TE mobilization as a cause of mutation during infection in a pathogenic fungus. In addition, TE mutagenesis in response to heat stress had not been described previously in any model yeast species. Remarkably, the heat- responsive TEs identified in C. deneoformans include both DNA transposons and retrotransposons, each with distinct modes of mobilization and preferred sites of genomic integration. In the proposed research, we seek to 1) determine whether heat stress is the primary cause of increased TE mobilization during C. deneoformans infection, 2) identify regulators of heat stress-induced TE mutagenesis in C. deneoformans, and 3) determine whether TEs mobilize in other cryptococcal species (C. neoformans or C. gattii) in vitro or during host infection. Elucidating the mechanisms of adaptive change that enhance fungal pathogenesis or enable drug resistance is critical in developing and maintaining effective antifungal treatments. This study will further our understanding of the types of stress-induced mutations that arise during cryptococcal infection that may contribute to disease persistence and variations in clinical outcomes for patients. In addition, our study will highlight a set of experimental approaches, infection models and sequencing tools that can be used to identify and quantitate genetic mutations (TE and non-TE) in a broad range of pathogenic and non-pathogenic species.