Dissection of Macrophage Antifungal Activity against Aspergillus fumigatus

描述（由申请方提供）：气载分生孢子（孢子）代表负责传播主要人类真菌病的感染性繁殖体。人类每天都会吸入无处不在的烟曲霉分生孢子。分生孢子萌发成组织侵入性菌丝导致侵袭性曲霉病（IA），这是呼吸道先天免疫功能受损患者感染性发病率和死亡率的毁灭性原因。虽然分生孢子吞噬和杀伤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不清楚，因为难以实现测量肺中肺泡巨噬细胞分生孢子活性的测定。为了剖析AM杀分生孢子活性，我们开发了一种荧光曲霉报告菌株（FLARE），在分生孢子摄取后，用荧光标记标记AM。杀死分生孢子会引起荧光特征的变化，使我们能够观察和量化肺和试管中细胞类型特异性分生孢子的杀死。我们利用FLARE菌株来证明AM采用不同的杀分生孢子机制，并在此过程中涉及基质金属蛋白酶12（MMP 12）。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作为一种重要的细胞毒性效应机制，以实现杀真菌活性。这一提议的基础假设是AM特异性因子--包括MMP 12--控制A。烟曲霉孢子萌发在呼吸道真菌感染的最早阶段。目的是（1）研究MMP 12作为AM杀分生孢子活性的主要效应子和（2）使用基于功能性RNAi的方法整合交替的AM杀分生孢子机制。实验设计结合了一个候选基因（MMP 12）和一个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，并将确定候选基因，调节全谱的AM杀分生孢子活性。

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.