The Role of Lung Epithelium in B. anthracis Pathogenesis

描述(申请人提供)：我们最近证明，炭疽芽孢杆菌的孢子进入小鼠肺后不久，在肺上皮细胞内发现了大量这样的孢子。在这里，我们假设这种细胞内的存在在炭疽杆菌的发病机制中起着重要作用。具体地说，我们提出了一个模型，其中孢子在感染的早期阶段进入肺上皮细胞，它们要么萌发成营养杆菌并跨上皮细胞转运，要么以孢子的形式在上皮细胞内存活，即细胞内萌发是开启“移位途径”的先决条件。吸入性炭疽病的传播和萌发的关键重要性已得到充分证实。几十年来，人们也注意到了肺部的持续孢子。然而，在体内维持、萌发和传播的机制还知之甚少或未知。我们的假设是，肺上皮细胞的细胞内环境为所有这些事件提供了场所，这是一个新的概念，并为经典的巨噬细胞摄取和扩散模型提供了一种替代途径。最近有证据表明，炭疽杆菌在体外和体内都容易被巨噬细胞吞噬杀死，这一点更加令人信服。因此，这种新的模型在细菌的存活率方面具有显著的优势。因此，这一应用的结果将对了解炭疽杆菌的致病机理产生重大影响。从更广泛的角度来看，传播和持久性是涉及许多微生物感染的两个重要问题。尽管这一点很重要，但仍有许多事情需要阐明，包括肺上皮在感染过程中可以扮演什么角色。因此，这一应用将对一般细菌的发病机制具有广泛的意义，并加深我们对微生物挑战下肺上皮功能的理解。我们的新模型进一步得到了以下数据的支持。炭疽杆菌孢子被培养的上皮细胞系和原代肺上皮细胞内化。炭疽杆菌可以在肺上皮细胞内存活，并可以通过跨细胞途径从肺尖到基底外侧穿过体外模型的肺泡上皮。即使在不允许孢子在细胞外萌发的测试条件下，也只能从上皮的基侧回收萌发的孢子/营养细菌，这表明萌发发生在上皮细胞内，是离开这些细胞的先决条件。在这一应用中，我们提出了三个特定的目标来确定肺上皮细胞是否是体内扩散、持续和萌发的场所。在未来的研究中，我们将研究这些事件背后的分子基础以及这些事件之间的动态平衡。与公共卫生相关：炭疽芽孢杆菌是炭疽病的病原体，如何在宿主体内传播并在肺部持续存在尚不清楚。我们的研究表明，肺上皮细胞可能在这些过程中发挥关键作用。对这种可能性的研究将提高目前对这种细菌如何致病的理解，并可能揭示新的治疗策略。

DESCRIPTION (provided by applicant): We recently demonstrated that soon after Bacillus anthracis spores are delivered into mouse lungs significant quantities of these spores are found inside the lung epithelial cells. Here we hypothesize that this intracellular presence plays an important role in B. anthracis pathogenesis. Specifically, we propose a model where spores enter lung epithelial cells at the early stages of infection and they either germinate into vegetative bacilli and are translocated across the epithelial cells, or, they persist inside them as spores, i.e., intracellular germination is a prerequisite to switching on a "translocation pathway". The crucial importance of dissemination and germination in inhalational anthrax is well established. Persistence of spores in the lung has also been noted for decades. However, the mechanisms permitting persistence, germination and dissemination in vivo are poorly understood or unknown. Our hypothesis that the intracellular environment of the lung epithelial cells provides a venue for all these events is a novel concept and provides an alternative pathway to the classical model of macrophage uptake and dissemination. This is made more compelling by recent evidence that B. anthracis bacilli are susceptible to phagocytic killing by macrophages both in vitro and in vivo. Therefore, this new model has significant advantages for the bacterium in terms of survivability. Consequently results from this application will have a significant impact on understanding the pathogenic mechanisms of B. anthracis. From a broader point of view, dissemination and persistence are two important issues concerning many microbial infections. Despite the importance, much remains to be elucidated including what roles the lung epithelium can play during an infection. Therefore, this application will have broad implications to bacterial pathogenesis in general as well as enhancing our understanding of the function of the lung epithelium under microbial challenge. Our new model is further supported by the following data. B. anthracis spores are internalized by cultured epithelial cell lines and primary lung epithelial cells. B. anthracis can survive inside lung epithelial cells and can cross an in vitro model of the alveolar epithelium from the apical to the basolateral side via a transcellular route. Only germinated spores/vegetative bacilli are recovered from the basolateral side of the epithelium even under assay conditions that do not permit extracellular germination of spores, suggesting that germination occurs inside epithelial cells and is a prerequisite for exiting these cells. In this application, we propose three specific aims to determine if lung epithelial cells are a site for dissemination, persistence and germination in vivo. In future studies, we will investigate the molecular basis underlying these events and the dynamic balances between these events. PUBLIC HEALTH RELEVANCE: How Bacillus anthracis, the causative agent of anthrax, spreads in the host body and persists in the lung is not well understood. Our studies suggest that the lung epithelium may play a critical role in these processes. Investigation of this possibility will improve the current understanding of how this bacterium causes disease and may reveal new therapeutic strategies.