Biochemical, Molecular &Immunological Characterization of the Mycoplasma pneumon

哮喘是一种常见的炎症性疾病，会导致呼吸道狭窄和喘息，影响人们。 所有年龄段(十分之一的成年人和四分之一的儿童)。肺炎支原体是引起肺炎的重要原因 呼吸道疾病，越来越多的证据表明肺炎支原体参与了肺炎支原体的启动、加重 和慢性哮喘。然而，肺炎支原体感染导致的变化的机制 肺功能、呼吸道阻塞和高反应性尚不清楚。此外，不足之处 在肺炎支原体的诊断中，加上缺乏可以直接与之相关的已知毒力决定因素 肺炎支原体/AE介导的病理学阻碍了我们目前对其真实流行率和 致病潜力。最近，我们发现了一种表面活性蛋白-A结合，ADP-核糖化和 肺炎支原体空泡化细胞毒素(见初步结果)被指定为获得性群落 呼吸窘迫综合征毒素(卡片TX)。重组卡TX本身就能够 复制促炎细胞因子/趋化因子反应、相关的组织病理学和 肺炎支原体感染时观察到的呼吸道高反应性(见项目的初步结果 1-3)。此外，酶联免疫吸附试验和聚合酶链式反应检测结果显示肺炎支原体和卡介苗毒素与哮喘有关。 发展和进步(项目3和4)。考虑到这些发现，我们假设卡片 TX是肺炎支原体/阿司匹林介导的哮喘的急性、慢性和加重的原因。我们进一步 假设针对卡介苗Tx的抗体的存在和效价以及 卡介苗TX基因聚合酶链式反应阳性是判断病情的关键指标。此外，抗原的发展(即， 卡片Tx)捕获方法将进一步将卡片Tx与哮喘和相关症状联系起来。 为了验证这些假设，我们将：1.鉴定CARDS TX介导的ADP-核糖基转移酶(ART) 通过检测CARDS TX最小结构域(S)和酶活性所必需的氨基酸来检测酶的活性； 并鉴定被CADS TX ADP核糖化的哺乳动物蛋白。2.执行转录和 卡介苗Tx野生型和肺炎支原体卡介苗Tx的蛋白质组学分析 在不同环境条件下与绿色荧光蛋白和荧光素酶融合的启动子(与 项目1和2)。3.作为抗原性和诊断决定簇的MAP卡片TX表位 人类(项目3)和小鼠(项目1和2)；并识别能够诱导 中和抗体。我们相信，这个项目是创新的，应该会产生有效的战略，以 了解肺炎支原体感染和卡介苗Tx在哮喘发生发展中的作用。 我们的长期目标是开发有效的策略来诊断、治疗和预防哮喘和相关的 儿童和成人中有相当一部分人患有呼吸道疾病。

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.