Francisella - ATII interactions in respiratory tularemia

描述（由申请人提供：ABS土拉热弗朗西丝菌（Ft）是一种革兰氏阴性兼性胞内病原体，也是土拉菌血症（一种与人类高发病率和死亡率相关的疾病）的病原体。由吸入雾化的Ft或Ft从远处感染灶到肺部的血行性传播引起的肺源性土拉菌病是该疾病的最致命形式，在不治疗的情况下导致高达50%的死亡率。Ft肺炎是一种急性炎症性感染，常导致肺损伤，其特征是肺泡上皮细胞严重破坏，肺泡和间质单核细胞浸润。肺泡巨噬细胞是感染后Ft靶向的主要细胞类型，作为细菌复制的位点和随后传播的载体。然而，Ft在吸入感染后遇到其他细胞类型，包括肺上皮细胞。最近，Ft与肺泡II型和I型（ATII和ATI）肺细胞的相互作用在小鼠肺炎土拉菌病的早期阶段。这些相互作用在Ft生命周期中的重要性尚不清楚，迄今为止还没有研究讨论Ft与人肺泡肺细胞相互作用的重要性。本提案中描述的初步研究表明，Ft在人ATII样细胞系A549中感染、复制和持续存在，即使在高感染复数下也不会诱导宿主细胞病变效应，这是一种与Ft感染其他细胞类型（包括巨噬细胞）后观察到的表型形成对比的表型，其中在24小时内观察到宿主细胞病变效应。该提案旨在通过调查Ft与原代人ATII和ATI肺细胞相互作用的方面来填补我们目前的知识空白。我们假设ATII和/或ATI肺细胞可以作为一个细胞内的水库毒性Ft内的人肺，允许一个子集的细菌被保持在一个受保护的小生境，在那里他们能够调节随后的主机响应。为了解决这一假设，提出了三个具体目标。首先，将建立Ft的原代人ATII/ATI感染模型，并用于表征细菌的内化、复制和运输。其次，将使用RNA-seq技术并通过筛选A549细胞中> 20，000个成员的Schu S4：：himar 1转座子突变体文库中的细胞毒性抑制缺陷来鉴定肺细胞内由Ft表达的细菌决定簇。最后，将使用各种体外和体内感染模型系统靶向上文鉴定的肺细胞表达基因的子集以进行破坏和表征。总的来说，这些研究有望为ATII/ATI肺细胞在Ft发病机制中的重要性提供新的见解。这些信息可能导致识别新的治疗靶点或候选疫苗以对抗Ft感染。 公共卫生相关性：本提案中描述的实验将研究土拉热弗朗西丝菌和人肺ATII/ATI肺细胞之间的相互作用。对这些相互作用的研究有望填补一个关键的知识空白，并进一步加深我们对 细菌引起的肺炎和相关的肺部炎症。该提案的完成还可能导致鉴定用于预防或对抗由这种高毒性细菌引起的感染的新的治疗靶点。

DESCRIPTION (provided by applicant : ABS Francisella tularensis (Ft) is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia, a disease associated with high morbidity and mortality in humans. Pneumonic tularemia, caused by inhalation of aerosolized Ft or hematogenous spread of Ft from distant infectious foci to the lung, is the most lethal form of the disease, resulting in up to 50% mortality without treatment. Characterized by profound disruption of the alveolar epithelium and profuse alveolar and interstitial mononuclear infiltrates, Ft pneumonia is an acute inflammatory infection often resulting in lung damage. Alveolar macrophages are the primary cell type targeted by Ft following infection, serving as both a site of bacterial replication and vehicle for subsequent dissemination. However, Ft encounters other cell types following inhalation infection, including lung epithelial cells. Recently, Ft interaction with alveolar type II and type I (ATII and ATI) pneumocytes was reported during early stages of murine pneumonic tularemia. The significance of these interactions in the lifecycle of Ft remains unclear, and no studies to date have addressed the importance of Ft interactions with human alveolar pneumocytes. Preliminary studies described in this proposal indicate that Ft infects, replicates, and persists in the human ATII-like cell lin A549 without inducing host cell cytopathic effects even at high multiplicities of infection, a phenotype that contrasts that seen following infection of Ft in other cell types including macrophages where host cytopathic effects are observed within 24 hrs. This proposal seeks to fill our current knowledge gap by investigating aspects of Ft interactions with primary human ATII and ATI pneumocytes. We hypothesize that ATII and/or ATI pneumocytes may serve as an intracellular reservoir for virulent Ft within the human lung, allowing a subset of bacteria to be maintained within a protected niche where they are able to modulate the subsequent host response. To address this hypothesis, three specific aims have been proposed. First, a primary human ATII/ATI infection model for Ft will be established and used to characterize internalization, replication, and trafficking of the bacterium. Second, bacterial determinants expressed by Ft within lung pneumocytes will be identified using RNA-seq technology and by screening a >20,000-member Schu S4::himar1 transposon mutant library in A549 cells for defects in cell cytotoxicity suppression. Finally, a subset of pneumocyte-expressed genes identified above will be targeted for disruption and characterization using various in vitro and in vivo model systems of infection. Collectively, these studies are expected to provide novel insights into the importance of ATII/ATI pneumocytes in Ft pathogenesis. This information may lead to identification of novel therapeutic targets or vaccine candidates to combat Ft infection. PUBLIC HEALTH RELEVANCE: Experiments described in this proposal will investigate interactions between Francisella tularensis and human lung ATII/ATI pneumocytes. Examination of these interactions is expected to fill a critical knowledge gap and further our understanding of bacteria-induced pneumonia and associated lung inflammation. Completion of this proposal may also led to identification of novel therapeutic targets for preventing or combating infection caused by this highly virulent bacterium.