Francisella - ATII interactions in respiratory tularemia

弗朗西斯菌 - ATII 在呼吸道兔热病中的相互作用

• 批准号: 8383387
• 负责人: THOMAS C. ZAHRT
• 金额: $ 18.76万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8383387
• 关键词: A549; Acute; Address; Alveolar; Alveolar Cell; Alveolar Macrophages; Attenuated Live Virus Vaccine; Bacteria; Biological Models; Breathing; Categories; Cell Line; Cells; Complement; Defect; Disease; Distant; Epithelial Cells; Epithelium; Francisella; Francisella tularensis; Generations; Genes; Hematogenous Spread; Human; Immune response; In Vitro; Infection; Inflammatory; Knowledge; Lead; Libraries; Lung; Lung Inflammation; Mediating; Modeling; Mononuclear; Morbidity - disease rate; Mus; Pathogenesis; Phenotype; Pneumonia; Proteins; Pulmonary alveolar structure; RNA; Reporting; Respiratory System; Respiratory tract structure; Screening procedure; Site; Staging; Surface; Technology; Testing; Tularemia; Virulent; aerosolized; alveolar epithelium; cell type; combat; comparative; cytotoxicity; in vivo Model; insight; interstitial; macrophage; member; mortality; mutant; new therapeutic target; novel; novel therapeutics; pathogen; pneumocyte; prevent; programs; research study; respiratory; response; therapeutic vaccine; trafficking; transmission process; vaccine candidate

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.

描述（由申请人提供：ABS 土拉弗朗西斯菌 (Ft) 是一种革兰氏阴性兼性细胞内病原体，也是土拉热病的病原体，土拉热病是一种与人类高发病率和死亡率相关的疾病。肺炎土拉热病是由吸入雾化 Ft 或 Ft 从远处感染灶经血行传播到肺部引起的，是最致命的形式。 如果不进行治疗，死亡率高达 50%。 Ft肺炎的特征是肺泡上皮的严重破坏以及大量的肺泡和间质单核细胞浸润，Ft肺炎是一种急性炎症感染，通常会导致肺损伤。肺泡巨噬细胞是感染后 Ft 靶向的主要细胞类型，既是细菌复制的场所，又是随后传播的载体。 然而，Ft 在吸入感染后会遇到其他细胞类型，包括肺上皮细胞。最近，在小鼠肺炎兔热病的早期阶段报道了 Ft 与肺泡 II 型和 I 型（ATII 和 ATI）肺细胞的相互作用。这些相互作用在 Ft 生命周期中的重要性仍不清楚，迄今为止还没有研究探讨 Ft 与人类肺泡相互作用的重要性 肺细胞。该提案中描述的初步研究表明，Ft 在人 ATII 样细胞 lin A549 中感染、复制和持续存在，即使在高复数感染下也不诱导宿主细胞的细胞病变效应，这一表型与 Ft 在其他细胞类型（包括巨噬细胞）感染后观察到的表型形成鲜明对比，在巨噬细胞中，在 24 小时内观察到宿主细胞病变效应。该提案旨在 通过研究 Ft 与原代人类 ATII 和 ATI 肺细胞相互作用的各个方面，填补了我们当前的知识空白。我们假设 ATII 和/或 ATI 肺细胞可能作为人肺内有毒 Ft 的细胞内储存库，使一部分细菌能够维持在受保护的生态位内，在那里它们能够调节随后的宿主反应。为了解决这个假设， 提出了三个具体目标。首先，将建立 Ft 的初级人类 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 细胞中超过 20,000 个成员的 Schu S4::himar1 转座子突变体文库来鉴定肺肺细胞内 Ft 表达的细菌决定簇 用于细胞毒性抑制的缺陷。最后，上面确定的肺细胞表达基因的子集将使用各种体外和体内感染模型系统进行破坏和表征。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。这些信息可能会导致新的治疗靶点的识别或 对抗 Ft 感染的候选疫苗。 公共卫生相关性：本提案中描述的实验将研究土拉弗朗西斯菌与人肺 ATII/ATI 肺细胞之间的相互作用。对这些相互作用的检查预计将填补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确定新的治疗靶点，以预防或对抗这种高毒力细菌引起的感染。