Antifungal Immunity and the Mechanism of Fungal Programmed Cell Death

项目摘要 人类每天都会吸入真菌分生孢子（即营养孢子）。先天呼吸能力 免疫系统阻止吸入分生孢子萌发成组织侵入性菌丝是一个关键 免疫检查点使用烟曲霉，最常见的病原体的侵袭性 曲霉病，作为人类真菌病原体的模型系统，我们发现分生孢子经历 在与先天免疫细胞的相互作用期间，具有类凋亡特征的程序性细胞死亡。这一发现 通过一种新的荧光真菌生理学报告， 真菌凋亡标志物，包括组蛋白降解，半胱天冬酶激活，和DNA片段。 我们的工作表明，A.烟曲霉分生孢子表达必需且可药用的抗凋亡蛋白， 称为Bir 1，其对抗宿主通过吞噬细胞的作用诱导的类凋亡的程序性细胞死亡 NADPH氧化酶。遗传和药理学研究表明，Bir 1的表达和活性是 分生孢子对寄主寄生虫样程序性细胞死亡的敏感性，反过来，寄主对入侵性 曲霉病这些发现表明，哺乳动物真菌免疫监视利用真菌细胞凋亡- 比如通过程序性细胞死亡途径来维持肺部的屏障免疫。 在这项与两名共同调查员的合作提案中，我们试图确定一种机制， Bir 1在真菌-宿主细胞相遇期间调节抗凋亡活性。我们的初步数据支持一个模型 其中Bir 1通过两个保守的BIR结构域发挥抗凋亡活性， 调节响应于促凋亡应激，调节候选真菌半胱天冬酶样酶作为细胞凋亡 效应子，并在人类致病真菌中表现出功能保守性。基于这些 观察，我们的模型预测，真菌性骨化样程序性细胞死亡是一个普遍的特点， 真菌-宿主细胞相遇，并对侵袭性真菌疾病的建立起重要作用。我们探索这个模型 （1）确定Bir 1的功能结构域和翻译后调控， 对宿主诱导的类凋亡样程序性细胞死亡的抗性，（2）确定Bir 1- 介导的对宿主诱导的类凋亡的程序性细胞死亡的抗性，重点是调节 的候选真菌半胱天冬酶样活性，和（3）定义类凋亡的程序性细胞死亡的作用， 构巢曲霉和白色念珠菌挑战后的Bir 1同源物。拟议的研究是 因为它们确定了一种新免疫监视机制， 高等真核生物可以利用低等真核生物的程序性细胞死亡来达到消毒的目的， 免疫力这项工作将提供一个机制的理解Bir 1的功能，在调节宿主真菌 遭遇从这些研究中获得的知识将为靶向真菌Bir 1同源物的策略提供信息， 利用真菌性类骨化症的程序性细胞死亡来获得治疗效果。

PROJECT SUMMARY Humans inhale fungal conidia (i.e, vegetative spores) on a daily basis. The ability of the respiratory innate immune system to prevent germination of inhaled conidia into tissue-invasive hyphae represents a critical immunologic checkpoint. Using Aspergillus fumigatus, the most common etiologic agent of invasive aspergillosis, as a model system for human fungal pathogens, we discovered that conidia undergo programmed cell death with apoptosis-like features during interactions with innate immune cells. This finding was facilitated by a novel fluorescent reporter of fungal physiology that enables visualization and quantitation of fungal apoptosis markers, including histone degradation, caspase activation, and DNA fragmentation. Our work demonstrates that A. fumigatus conidia express an essential and druggable anti-apoptotic protein, termed Bir1, that counters host induction of apoptosis-like programmed cell death by the action of phagocyte NADPH oxidase. Genetic and pharmacologic studies demonstrate that Bir1 expression and activity underlie conidial susceptibility to host apoptosis-like programmed cell death, and in turn, host susceptibility to invasive aspergillosis. These findings indicate that mammalian fungal immune surveillance exploits a fungal apoptosis- like programmed cell death pathway to maintain barrier immunity in the lung. In this collaborative proposal with two co-investigators, we seek to determine the mechanism through which Bir1 regulates anti-apoptotic activity during fungal-host cell encounters. Our preliminary data support a model in which Bir1 exerts anti-apoptotic activity via two conserved BIR domains, underlies post-translational regulation in response to pro-apoptotic stress, regulates candidate fungal caspase-like enzymes as apoptosis effectors, and demonstrates functional conservation across human pathogenic fungi. Based on these observations, our model predicts that fungal apoptosis-like programmed cell death is a general feature of fungal-host cell encounters and central to the establishment of invasive fungal disease. We explore this model in the following aims: (1) define the functional domains and post-translational regulation of Bir1 critical for resistance to host induction of apoptosis-like programmed cell death, (2) define the mechanism of Bir1- mediated resistance to host induction of apoptosis-like programmed cell death, with an emphasis on regulation of a candidate fungal caspase-like activity, and (3) define the role of apoptosis-like programmed cell death and Bir1 homologs following Aspergillus nidulans and Candida albicans challenge. The proposed studies are significant and innovative because they identify a novel mechanism of immune surveillance and demonstrate that higher eukaryotes can exploit programmed cell death in lower eukaryotes for the purpose of sterilizing immunity. This work will provide a mechanistic understanding of Bir1 function in regulating host-fungal encounters. Knowledge gained from these studies will inform strategies that target fungal Bir1 homologs and exploit fungal apoptosis-like programmed cell death for therapeutic gain.