Stress-induced transposon mobilization in the human fungal pathogen Cryptococcus

项目摘要 隐球菌属是主要在免疫功能低下人群中引起疾病的环境真菌， 其中包括一种致命的隐球菌脑膜炎，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在人类宿主中遇到的各种压力下生存， 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和抵抗抗真菌药物治疗。适应性基因组 已知在感染过程中增强毒力或引起耐药性的隐球菌的变化包括碱基 取代、小的插入/缺失和非整倍性。转座因子（Transposable elements，TE）是一种小的、移动的DNA 存在于大多数真核生物基因组中的元件，其能够引起显著的基因组改变， 变化和表型变异。TE在隐球菌和其他致病真菌中的潜在作用 （念珠菌属和曲霉属）在促成真菌发病机理或耐药性方面的作用在很大程度上未被探索。我们 最近发现，在新生隐球菌TE动员作为一个重要的原因突变的鼠 感染的模式。耐药性报告基因中的TE突变在高浓度时显著升高， 体外温度（37°宿主相关温度），表明热应激刺激TE迁移率 隐球菌基因组此外，我们证明TE插入是临床耐药的原因， 体外抗真菌剂雷帕霉素/FK 506和5-氟胞嘧啶。我们的研究是第一个确定TE 在致病真菌感染期间作为突变原因的移动。此外，TE诱变在 先前在任何模式酵母物种中都没有描述过对热应激的反应。令人惊讶的是，热量- 在C. deneoformans包括DNA转座子和反转录转座子，每一个都具有 不同的动员模式和基因组整合的优选位点。在拟议的研究中，我们寻求 1)确定热应激是否是C期间TE动员增加的主要原因。新形类 感染，2）鉴定C. deneoformans，和3）确定 TE是否在其他隐球菌物种中动员（C. neoformans或C. gattii）或在宿主感染期间。 阐明增强真菌致病性或使耐药性成为可能的适应性变化的机制， 在开发和维持有效的抗真菌治疗中至关重要。这项研究将进一步加深我们对 隐球菌感染期间出现的可能导致疾病的应激诱导突变类型 患者临床结局的持续性和变化。此外，我们的研究将突出一组 实验方法，感染模型和测序工具，可用于识别和定量 广泛的致病性和非致病性物种中的基因突变（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.