Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

Authors

Fernando L. Leite, Iowa State University, AmesFollow
Timothy A. Reinhardt, USDA, ARS, National Animal Disease CenterFollow
J. P. Bannantine, USDA-ARS, National Animal Disease CenterFollow
Judith R. Stabel, USDA-ARS, National Animal Disease CenterFollow

Document Type

Article

Date of this Version

2015

Citation

Veterinary Microbiology 175 (2015) 275–285

http://dx.doi.org/10.1016/j.vetmic.2014.11.009

Comments

U.S. government works are not subject to copyright.

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 Mycobacterium 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-g) release assay as well as immunoblots. 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 was characterized.