Dissection of Macrophage Antifungal Activity against Aspergillus fumigatus

描述（由申请人提供）：空气传播的分生孢子（孢子）代表传染性繁殖体，负责传播主要的人类真菌病。人类每天吸入无处不在的烟曲霉分生孢子。孢子萌发成组织侵袭性菌丝导致侵袭性曲霉病（IA），这是呼吸先天免疫功能受损患者传染性发病率和死亡率的破坏性原因。尽管肺泡巨噬细胞吞噬和杀死分生孢子是呼吸道感染过程中肺泡巨噬细胞（AM）功能的标志，但这些细胞的抗真菌效应机制仍然不明确，因为测量肺泡巨噬细胞在肺部分生孢子活性的测定很难实现。为了解剖AM的分生孢子活性，我们开发了一种荧光曲霉报告菌（FLARE）菌株，该菌株在分生孢子摄取时，用荧光标记AM。分生孢子杀伤诱导荧光信号的变化，使我们能够在肺和试管中观察和量化细胞类型特异性的分生孢子杀伤。我们利用FLARE菌株证明了am具有独特的分生机制，并且在这一过程中涉及基质金属蛋白酶12 （MMP12）。通过这种方法，我们研究了AM功能模型，该模型将基质金属蛋白酶12整合为一个重要的细胞毒性效应机制，以实现杀真菌活性。这一假说的基础是am特异性因子——包括MMP12——在呼吸道真菌感染的早期阶段控制烟曲霉孢子的萌发。目的将：(1)研究MMP12作为AM分生孢子活性的主要影响因子；(2)使用基于功能性rnai的方法整合AM分生孢子的替代机制。实验设计结合候选基因（MMP12）和系统的发现方法来解剖AM抗真菌活性。这些研究将确定基质金属蛋白水解活性作为一种潜在的新型抗真菌效应机制，并将确定调控AM分生孢子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.