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.
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