Bacillus-containing vacuole-mediated interactions of Bacillus anthracis with macrophages

 描述（由申请方提供）：在吸入性炭疽期间，大量证据表明肺泡巨噬细胞（mφs）通过转运休眠的B促进播散性感染和疾病的发展。炭疽（Ba）孢子，先前吸入肺的肺泡腔，到血液。虽然已经广泛研究了孢子摄取到Mφ中的初始步骤，但是知识上的一个主要空白（这是该探索性R21应用的焦点）是细胞内Ba逃逸感染Mφ的机制，这是细胞外播散之前的一个必要步骤。在这里，我们提出了挑战现有模型的研究，该模型预测，类似于几种不同的细胞内病原体，mφ死亡必须先于Ba释放到外部环境。相反，我们的初步数据表明，在细胞吞噬休眠孢子进入含芽孢杆菌的液泡（BCV）后，在没有mφ杀死的情况下，很大一部分细胞内Ba逸出。因为我们没有发现Ba离开BCVs进入感染的Mφ的胞质溶胶的证据，所以我们认为这些细胞内空泡代表了对Ba逃逸重要的特化小生境。为了支持这一观点，感染Ba增强了溶酶体内容物向细胞表面的转运，并诱导了具有膜囊泡融合蛋白synaptotagmin-7的PCV的时间依赖性富集，这与胞吐过程一致。基于这些初步数据，我们将评估以下总体假设：感染的mφ细胞内Ba的非溶解性逃逸需要将BCV从降解区室重塑为胞吐区室。我们将确定在Ba感染的过程中，与BCV相关的蛋白质的变化，并表征其功能的重要性，成熟的BCV进入与细胞内Ba的出口回到细胞外环境相关的隔间。这些研究将提供Ba孢子占据的细胞内生态位的第一个详细特征，并有助于我们了解吸入性炭疽的基本生物学。

 DESCRIPTION (provided by applicant): During inhalational anthrax, considerable evidence indicates that alveolar macrophages (mφs) contribute to the development of disseminated infection and disease by transporting dormant B. anthracis (Ba) spores, previously inhaled into the alveolar spaces of the lungs, to the bloodstream. Although the initial steps of spore uptake into mφs have been extensively studied, a major gap in knowledge, which is the focus of this exploratory R21 application, is the mechanism by which intracellular Ba escape infected mφs as a requisite step prior to extracellular dissemination. Here, we propose studies that challenge an existing model that predicts, analogous to several different intracellular pathogens, mφ death must precede Ba release to the external environment. Instead, our preliminary data indicate that subsequent to phagocytic uptake of dormant spores into Bacillus-containing vacuoles (BCVs), a substantial fraction of intracellular Ba escape in the absence of mφ killing. Because we find no evidence that Ba depart BCVs into the cytosol of infected mφs, we propose that these intracellular vacuoles represent specialized niches that are important for Ba escape. In support of this idea, infection with Ba enhances transport of lysosomal contents to the cell surface, and, induces the time-dependent enrichment of BCVs with the membrane vesicle fusion protein, synaptotagmin-7, consistent with an exocytic process. Based on these preliminary data, we will evaluate the overall hypothesis that non-lytic escape of intracellular Ba from infected mφs requires remodeling of BCVs from degradative to exocytic compartments. We will identify changes in BCV-associated proteins during the course of Ba infection, and, characterize their functional importance for the maturation of BCVs into compartments associated with egress of intracellular Ba back to the extracellular environment. These studies will provide the first detailed characterization of the intracellular niche occupied by Ba spores, and contribute to our understanding of the fundamental biology underlying inhalational anthrax.