Dissection of Macrophage Antifungal Activity against Aspergillus fumigatus

描述(申请人提供)：空气传播的分生孢子(孢子)代表感染性繁殖，负责传播主要的人类真菌病。人类每天都吸入随处可见的烟曲霉分生孢子。分生孢子萌发进入组织侵袭性菌丝会导致侵袭性曲霉病(IA)，这是呼吸系统固有免疫功能受损患者感染发病率和死亡率的毁灭性原因。虽然分生孢子吞噬和杀死是呼吸道感染期间肺泡巨噬细胞(AM)功能的标志，但这些细胞的抗真菌效应机制仍然不清楚，因为很难实现测量肺泡巨噬细胞分生孢子活性的分析。为了分析AM的杀分生孢子活性，我们开发了一种荧光曲霉报告菌株(FLARE)，该菌株在分生孢子摄取后，用荧光标记AM。分生孢子杀伤诱导荧光信号的变化，使我们能够观察和量化肺和试管中特定细胞类型的分生孢子杀伤。我们利用耀斑菌株来证明AM使用不同的分生孢子杀灭机制，并在这一过程中涉及基质金属蛋白酶12(MMP12)。通过这种方法，我们检验了AM的功能模型，该模型整合了基质金属蛋白酶12作为重要的细胞毒效应机制来实现杀菌活性。支持这一建议的假设是，AM特有的因子--包括MMP12--在呼吸道真菌感染的最早阶段控制烟曲霉的分生孢子萌发。这些目标将(1)研究MMP12作为AM分生孢子杀灭活性的主要效应者和(2)使用基于RNAi的功能性方法整合替代的AM分生孢子杀灭机制。实验设计结合了候选基因(MMP12)和系统发现方法来剖析AM的抗真菌活性。这些研究将把基质金属蛋白分解活性定义为一种潜在的新的抗真菌效应机制，并将确定调节AM分生孢子杀灭活性全谱的候选基因。

DESCRIPTION (provided by applicant): Airborne conidia (spores) represent infectious propagules that are responsible for transmission of major human mycoses. Humans inhale ubiquitous Aspergillus fumigatus conidia on a daily basis. Conidial germination into tissue-invasive hyphae leads to invasive aspergillosis (IA), a devastating cause of infectious morbidity and mortality in patients with impaired respiratory innate immune function. Although conidial engulfment and killing are hallmarks of alveolar macrophage (AM) function during respiratory infection, antifungal effector mechanisms of these cells remain poorly defined, since assays that measure alveolar macrophage conidiacidial activity in the lung have been difficult to achieve. To dissect AM conidiacidal activity, we developed a fluorescent Aspergillus reporter (FLARE) strain that, upon conidial uptake, tags AMs with a fluorescent signature. Conidial killing induces a change in the fluorescence signature, enabling us to observe and quantify cell type-specific conidial killing in the lung and test tube. We harness the FLARE strain to demonstrate that AMs employ distinct conidiacidal mechanisms and implicate matrix metalloprotease 12 (MMP12) in this process. With this approach, we examine a model of AM function that integrates matrix metalloprotease 12 as a significant cytotoxic effector mechanism to achieve fungicidal activity. The hypothesis that underlies this proposal is that AM-specific factors - that include MMP12 - control A. fumigatus conidial germination at the earliest stages of respiratory fungal infection. The aims will (1) investigate MMP12 as a major effector of AM conidiacidal activity and (2) integrate alternate AM conidiacidal mechanisms using a functional RNAi-based approach. The experimental design incorporates both a candidate gene (MMP12) and a systematic discovery approach to dissect AM antifungal activity. These studies will define matrix metalloproteolytic activity as a potential novel antifungal effector mechanism and will identify candidate genes that regulate the full spectrum of AM conidiacidal activity.