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

 描述(申请人提供)：在吸入性炭疽病期间，大量证据表明，肺泡巨噬细胞(mφS)通过将休眠的炭疽杆菌(BA)孢子运送到血液中，促进播散性感染和疾病的发展。虽然孢子吸收mφS的最初步骤已经被广泛研究，但一个主要的知识空白是细胞内BA逃逸感染mφS的机制，这是细胞外传播之前的必要步骤，这是R21探索性应用的重点。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死亡必须先于向外部环境释放BA。相反，我们的初步数据表明，在休眠孢子被含芽孢杆菌空泡(BCV)吞噬后，很大一部分细胞内的BA在没有mφ杀伤的情况下逃逸。由于我们没有发现巴将BCV送入感染的φS的胞浆中的证据，所以我们认为这些细胞内的空泡代表着对巴逃逸很重要的特殊生态位。为了支持这一观点，感染BA促进了溶酶体内容物到细胞表面的运输，并诱导BCV与膜泡融合蛋白synaptopagmin-7的时间依赖地浓缩，这与胞外过程一致。基于这些初步数据，我们将评估一个总体假设，即细胞内BA从感染的mφS的非溶血性逃逸需要BCV从降解的隔室重塑为胞外隔室。我们将确定BCV相关蛋白在BA感染过程中的变化，并表征它们在BCV成熟成与细胞内BA出口回到细胞外环境相关的间隔中的功能重要性。这些研究将首次详细描述BaB孢子占据的细胞内生态位，并有助于我们理解吸入性炭疽病的基本生物学基础。

 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.