Novel Mycoplasma pneumoniae CARDS Toxin as Mediator of Airway Dysfuntion in Mice

描述（由申请人提供）： 圣安东尼奥/达拉斯哮喘和过敏性疾病合作研究中心代表了一项综合、协作和创新的多学科努力，旨在研究独特的肺炎支原体毒素在哮喘和相关气道疾病中的作用。这种毒素被称为社区获得性呼吸窘迫综合征毒素 (CARDS TX)，显着复制了肺炎支原体感染所伴随的促炎细胞因子/趋化因子谱和组织病理学。德克萨斯大学圣安东尼奥健康科学中心和德克萨斯大学达拉斯西南医学院之间的联盟将 4 个项目（重点关注基础、临床和动物建模策略）与 2 个支持核心（管理和病理学）结合起来，为定义肺炎支原体与哮喘发病机制之间的关系带来了全新的方法。三十五年来积累的大量文献将肺炎支原体与哮喘的发作、恶化和慢性联系起来，但没有任何一种支原体毒力决定因素或支原体分子被证明是症状和相关病理的介质。缺乏可定义的肺炎支原体致病因子极大地阻碍了对肺炎支原体如何影响气道疾病的发生和进展的理解。这在哮喘等复杂疾病中尤其具有挑战性，因为遗传、免疫、感染和环境变量似乎会影响疾病的发生和进展。 AADCRC 的一个主要重点是将 ADP 核糖基化空泡肺炎支原体 CARDS TX（项目 4）的生化、分子和免疫学特性与哮喘患者的诊断和治疗直接联系起来（项目 3 和 4）。通过这样做，我们希望证明 CARDS TX 是人类（项目 3）和实验感染或中毒小鼠（项目 1 和 2）中哮喘相关病理学的关键介质。因此，我们打算 (a) 使用患者的鼻腔灌洗液、痰液和血清样本，通过新颖有效的 CARDS TX 靶向诊断评估（ELISA、免疫组织化学、抗原捕获和 PCR 方法），将 CARDS TX 与哮喘发病机制直接联系起来； (b) 使用肺炎支原体感染和 CARDS TX 中毒的小鼠模型来检查哮喘的急性和慢性阶段和治疗干预措施以及 CARDS TX 对气道高反应性的影响； (c) 进一步表征 CARDS TX 的 ADP-核糖基化活性，并开发有效且快速的诊断方法，以协助治疗和控制哮喘及相关病理。每个项目和核心的主要研究人员在哮喘、气道相关病理学、免疫发病机制、肺炎支原体生物学和毒力方面拥有丰富的跟踪记录和专业知识，以及合作和共同发表的历史。 项目 1：新型肺炎支原体卡毒素作为小鼠气道功能障碍的介质 (Hardy, R.) 描述（由申请人提供）： 越来越多的证据表明，肺炎支原体呼吸道感染与一部分哮喘患者的哮喘发病、恶化和慢性化有关。然而，这一联系所涉及的致病微生物机制尚未得到很好的表征。具有重要意义，博士。 Baseman 和 Kannan 现在已经鉴定出一种新型肺炎支原体毒素，CARDS TX。我们的研究人员联盟（Baseman、Coalson、Dube、Kannan、Peters 和 Hardy 博士）初步证据表明 CARDS TX 在与呼吸道肺炎支原体感染相关的气道炎症、气道阻塞、气道高反应性中发挥致病作用。拟议研究的假设是，CARDS TX 介导肺炎支原体诱导哮喘急性加重的能力，并导致支原体呼吸道感染的有害长期影响。此外，我们假设针对 CARDS TX 的治疗干预将改善肺炎支原体相关的反应性气道疾病和哮喘。简而言之，具体目标是 1) 了解活性 CARDS TX 对肺炎支原体呼吸道感染中观察到的气道阻塞、高反应性和炎症的具体贡献，2) 确定对 CARDS TX 的宿主免疫反应是否对肺炎支原体感染的呼吸道表现具有保护作用，以及 3) 确定细菌蛋白合成抑制剂治疗对肺炎支原体呼吸道感染中 CARDS TX 蛋白产生的影响。这些研究的长期目标是制定疾病调节策略来治疗患有支原体相关反应性气道疾病和哮喘的儿童和成人。该项目的重点是在我们建立的肺炎支原体呼吸道感染的急性和慢性小鼠模型中研究新型肺炎支原体毒素 CARDS TX，该模型中的气道炎症、气道阻塞和气道高反应性已由我们的实验室进行了表征。将 BALB/c 小鼠暴露于肺炎支原体（野生型和 CARDS TX 无效突变体）或重组 CARDS TX，以确定 CARDS TX 对肺炎支原体感染的气道表现的贡献。此外，将在我们的小鼠模型中评估针对 CARDS TX 的治疗干预措施，目的是转化适用于治疗儿童和成人与肺炎支原体相关的反应性气道疾病和哮喘。

DESCRIPTION (provided by applicant): The San Antonio/Dallas Asthma and Allergic Diseases Cooperative Research Center represents an integrative, collaborative and innovative multidisciplinary effort to investigate the role of a unique Mycoplasma pneumoniae toxin in asthma and related airway diseases. This toxin, designated Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX) remarkably replicates the proinflammatory cytokine/chemokine profiles and histopathology that accompany M. pneumoniae infection. This consortium between The University of Texas Health Science Center at San Antonio and The University of Texas Southwestern Medical School in Dallas combines 4 projects, which focus on basic, clinical and animal modeling strategies, with 2 support cores (administrative and pathology) to bring a totally new approach to defining the relationship between M. pneumoniae and the pathogenesis of asthma. A substantial literature, which has accumulated over thirty-five years, connects M. pneumoniae to onset, exacerbation, and chronicity of asthma, yet no single mycoplasma virulence determinant, or mycoplasma molecule for that matter, has been shown to be a mediator of symptoms and associated pathologies. This lack of definable M. pneumoniae pathogenic factors has greatly hampered an understanding of how M. pneumoniae influences the development and progression of airway diseases. This is especially challenging in complex diseases like asthma, where genetic, immunologic, infectious and environmental variables appear to affect disease development and progression. A major focus of the AADCRC is to directly link the biochemical, molecular and immunological properties of the ADP-ribosylating, vacuolating M. pneumoniae CARDS TX (Project 4), to diagnosis and treatment of asthmatic patients (Projects 3 and 4). By so doing, we hope to demonstrate that CARDS TX is a key mediator of asthma-associated pathobiology in humans (Project 3) and in experimentally infected or intoxicated mice (Projects 1 and 2). Therefore, we intend to (a) directly connect CARDS TX to asthma pathogenesis through novel and effective CARDS TX-targeted diagnostic assessments (ELISA, immunohistochemistry, antigen capture and PCR methodologies) using patient's nasal lavage, sputum and serum samples; (b) use mouse models of M. pneumoniae infection and CARDS TX intoxication to examine both acute and chronic stages of asthma and therapeutic interventions as well as the impact of CARDS TX on airway hyper-reactivity; and (c) further characterize ADP-ribosylating activities of CARDS TX and develop effective and rapid diagnostics to assist in the treatment and control of asthma and related pathologies. The key investigators of each project and core have strong track records and expertise in asthma, airway-related pathologies, immunopathogenesis and M. pneumoniae biology and virulence as well as a history of collaboration and co-publication. PROJECT 1: Novel Mycoplasma pneumoniae CARDS Toxin as Mediator of Airway Dysfunction in Mice (Hardy, R.) DESCRIPTION (provided by applicant): There is growing evidence linking M. pneumoniae respiratory infection and the inception, exacerbation, and chronicity of asthma in a subset of asthmatics. However, the pathogenic microbiologic mechanisms involved in this link have not been well characterized. Of great significance, Drs. Baseman and Kannan have now identified a novel M. pneumoniae toxin, CARDS TX. Our consortium of researchers (Drs. Baseman, Coalson, Dube, Kannan, Peters, and Hardy) has preliminary evidence of CARDS TX playing a pathogenic role in the airway inflammation, airway obstruction, airway hyper-reactivity associated with respiratory M. pneumoniae infection. The hypothesis for the proposed research is that CARDS TX mediates the ability of M. pneumoniae to induce acute asthma exacerbations and is responsible for the deleterious long-term effects of mycoplasma respiratory tract infection. In addition, we hypothesize that therapeutic interventions directed against CARDS TX will ameliorate M. pneumoniae-associated reactive airway disease and asthma. Briefly, the Specific aims are to 1) understand the specific contribution of active CARDS TX to the airway obstruction, hyper-reactivity, and inflammation observed in M. pneumoniae respiratory infection, 2) determine if the host immune response to CARDS TX is protective against the respiratory manifestations of M. pneumoniae infection, and 3) determine the effect of bacterial protein synthesis inhibitor therapy on CARDS TX protein production in M. pneumoniae respiratory infection. The long-term goal of these investigations is to develop disease modifying strategies to treat children and adults with mycoplasma-associated reactive airway disease and asthma. This project focuses on investigating the novel M. pneumoniae toxin, CARDS TX, in our established acute and chronic murine model of M. pneumoniae respiratory infection in which airway inflammation, airway obstruction, and airway hyper-reactivity have been previously characterized by our laboratory. BALB/c mice will be exposed to M. pneumoniae (wild-type and CARDS TX null mutant) or recombinant CARDS TX to determine the contribution of CARDS TX to the airway manifestations of M. pneumoniae infection. In addition, therapeutic interventions directed against CARDS TX will be assessed in our murine model with the goal of translational applicability to the treatment of reactive airway disease and asthma associated with M. pneumoniae in children and adults.