Biochemical, Molecular &Immunological Characterization of the Mycoplasma pneumon

生化、分子

• 批准号: 7686482
• 负责人: JOEL Barry BASEMAN
• 金额: $ 26.31万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686482
• 关键词: ADP ribosylation; Acute; Adult; Affect; Age; Antibodies; Antigens; Asthma; Bacterial Toxins; Binding; Biochemical; Biological Assay; Cells; Child; Chronic; Collaborations; Community Acquired Respiratory Distress Syndrome Toxin; Condition; Cytotoxin; Defect; Development; Diagnosis; Diagnostic; Disabled Persons; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Essential Amino Acids; Frequencies; Goals; Green Fluorescent Proteins; Histopathology; Human; In Vitro; Individual; Infection; Inflammatory; Intervention; Lead; Length; Link; Luciferases; Mammalian Cell; Maps; Mediating; Methodology; Molecular; Monitor; Mus; Mycoplasma; Mycoplasma pneumoniae; Passive Immunization; Pathogenesis; Pathogenicity; Pathology; Pertussis Toxin; Phenotype; Pneumonia; Polymerase Chain Reaction; Population; Prevalence; Property; Proteins; Proteomics; Pulmonary Surfactant-Associated Protein A; Purpose; Recombinants; Relative (related person); Role; Site-Directed Mutagenesis; Testing; Tissues; Transferase; Virulence; Wheezing; airway hyperresponsiveness; airway obstruction; base; chemokine; cytokine; gene therapy; handicapping condition; in vivo; innovation; insight; neutralizing antibody; polyclonal antibody; prevent; programs; promoter; pulmonary function; response; therapeutic vaccine

Asthma is a common inflammatory disease that results in airway narrowing and wheezing and affects people of all ages (one in ten adults and one in four children). Mycoplasma pneumoniae is an important cause of airway disorders, and a growing body of evidence implicates M. pneumoniae in the initiation, exacerbation and chronicity of asthma. However, the mechanisms by which M. pneumoniae infection leads to changes in pulmonary function and airway obstruction and hyper-reactivity are not understood. In addition, deficiencies in diagnosis of M. pneumoniae, plus the lack of known virulence determinants that can be directly linked to M. pneumon/ae-mediated pathologies handicap our current understanding of its true prevalence and pathogenic potential. Recently, we discovered a surfactant protein-A binding, ADP-ribosylating and vacuolating cytotoxin of M. pneumoniae (see Preliminary results) designated Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Recombinant CARDS TX by itself is capable of replicating the proinflammatory cytokine/chemokine responses, associated histopathology, and changes in airway hyper-responsiveness observed with M. pneumoniae infections (see Preliminary results of Projects 1-3). In addition, results from ELISA and PCR assays implicate M. pneumoniae and CARDS TX in asthma development and progression (Projects 3 and 4). Considering these findings, we hypothesize that CARDS TX is responsible for acute, chronic, and exacerbation of M. pneumon/ae-mediated asthma. We further hypothesize that the presence and titer of antibodies reactive against CARDS TX and the frequency of cards tx gene PCR positivity are key indicators of disease status. Also, the development of antigen (i.e., CARDS TX) capture methodologies will further link CARDS TX to asthma and associated symptomatology. To test these hypotheses, we will: 1. Characterize CARDS TX-mediated ADP-ribosyl transferase (ART) activity through detecting CARDS TX minimal domain(s) and amino acids essential for enzymatic activity; and identify the mammalian proteins that are ADP-ribosylated by CARDS TX. 2. Perform transcriptional and proteomic analysis of CARDS TX in wild type strains and in M. pneumoniae strains having their CARDS TX promoter fused to GFP and luciferase under different environmental conditions (in collaboration with Projects 1 and 2). 3. Map CARDS TX epitopes that serve as antigenic and diagnostic determinants in humans (Project 3) and mice (Projects 1 and 2); and identify epitopes of CARDS TX capable of inducing neutralizing antibodies. We believe that this project is innovative and should lead to effective strategies to understand the role of M. pneumoniae infection and CARDS TX in asthma development and progression. Our long-term goal is to develop effective strategies to diagnose, treat and prevent asthma and related airway diseases in a substantial population of both children and adults.

哮喘是一种常见的炎症性疾病，导致气道狭窄和喘息，并影响人们 所有年龄段（每十个成人中有一个，每四个儿童中有一个）。肺炎支原体是一个重要的原因， 气道疾病，越来越多的证据表明M。肺炎，在开始，加重 和慢性哮喘。然而，M.肺炎感染会导致 肺功能和气道阻塞以及高反应性尚不清楚。此外，缺陷 在诊断M.肺炎，加上缺乏已知的毒力决定因素，可以直接联系到 M.肺炎/AE介导的病理阻碍了我们目前对其真实患病率的理解， 致病潜力最近，我们发现了一种表面活性蛋白A结合，ADP-核糖基化， M.空泡毒素肺炎（见初步结果）指定为社区获得性 呼吸窘迫综合征毒素（RESTATE DISTRESS SYNDROME TOXIN）重组MTX本身能够 复制促炎性细胞因子/趋化因子反应、相关组织病理学和 用M.肺炎感染（见项目的初步结果 第1-3段）。此外，ELISA和PCR检测结果表明M.哮喘中的肺炎和肺炎支原体 发展和进步（项目3和4）。考虑到这些发现，我们假设， TX与M的急性、慢性和加重有关。肺炎/AE介导的哮喘。我们进一步 我假设，抗ESTTX抗体的存在和滴度以及 卡tx基因PCR阳性是疾病状态的关键指标。此外，抗原的发展（即， 捕获方法学将进一步将哮喘和相关的哮喘学联系起来。 为了验证这些假设，我们将：1。表征MTX介导的ADP-核糖基转移酶（ART） 通过检测酶活性所必需的一个或多个MTX最小结构域和氨基酸来检测酶活性; 并鉴定被ADP-TX核糖基化的哺乳动物蛋白质。2.进行转录和 野生型菌株和M.肺炎菌株，其具有它们的 在不同环境条件下与GFP和荧光素酶融合的启动子（与 项目1和2）。3.绘制作为抗原决定簇和诊断决定簇的HLA-TX表位， 人（项目3）和小鼠（项目1和2）;并鉴定能够诱导 中和抗体我们认为，这一项目具有创新性，应导致制定有效的战略， 理解M的作用。肺炎杆菌感染和肺炎克雷伯氏菌毒素在哮喘发生和发展中的作用。 我们的长期目标是制定有效的策略来诊断、治疗和预防哮喘及相关疾病。 在儿童和成人的大量人群中存在气道疾病。