Location: Infectious Bacterial Diseases ResearchTitle: Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/12/2014
Publication Date: 6/22/2014
Citation: Leite, F.L., Reinhardt, T.A., Bannantine, J.P., Stabel, J.R. 2014. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity [abstract]. International Colloquium on Paratuberculosis. p. 32.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of M. tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n=9) and naturally infected cows in the subclinical (n = 10) and clinical (n=13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-gamma) release assay as well as immuno-blots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore, the identification of antigenic proteins with potential as diagnostic targets were characterized.