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，导致气道变窄和喘息，并影响人们 在所有年龄段（十分之一的成年人和四分之一的孩子）中。支原体肺炎是 气道疾病和越来越多的证据暗感ilmoniae在启动中，加剧 和哮喘的慢性。然而，肺炎支原体感染导致变化的机制 尚不清楚肺功能和气道阻塞和高反应性。另外，缺陷 在诊断肺炎支原体中，以及缺乏可以直接相关的已知毒力决定因素 M.肺炎/AE介导的病理障碍我们当前对其真正患病率和 致病潜力。最近，我们发现了表面活性剂蛋白A的结合，ADP-核糖基化和 M.肺炎的液泡细胞毒素（请参阅初步结果）被指定为获得的社区 呼吸窘迫综合征毒素（Cards TX）。重组卡TX本身能够 复制促炎性细胞因子/趋化因子反应，相关的组织病理学以及变化 气道高反应性通过肺炎支原体感染观察到（请参阅项目的初步结果 1-3）。此外，ELISA和PCR分析的结果暗示了肺炎和卡片tx的哮喘。 发展和进步（项目3和4）。考虑到这些发现，我们假设该卡 TX负责肺炎支原体/AE介导的哮喘的急性，慢性和加剧。我们进一步 假设抗体的存在和滴度反应针对卡片Tx和频率 卡TX基因PCR阳性是疾病状况的关键指标。此外，抗原的发展（即 卡TX）捕获方法将进一步将卡片TX与哮喘和相关症状链接起来。 为了检验这些假设，我们将：1。表征卡TX介导的ADP-核糖基转移酶（ART） 通过检测卡的活性TX最小结构域和氨基酸对酶活性必不可少的； 并识别由卡片TX进行ADP-核糖基化的哺乳动物蛋白。 2。执行转录和 野生型菌株和肺炎菌株中的卡片TX的蛋白质组学分析 在不同的环境条件下与GFP和荧光素酶融合的发起人（与 项目1和2）。 3。地图卡TX表位，作为抗原和诊断决定因素 人类（项目3）和小鼠（项目1和2）；并确定能够诱导的卡片的表位 中和抗体。我们认为，该项目具有创新性，应该导致有效的策略 了解肺炎支原体感染和卡片TX在哮喘发育和进展中的作用。 我们的长期目标是制定有效的策略来诊断，治疗和预防哮喘和相关 大量儿童和成人的气道疾病。