Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity
Fernando L Leite1, Timothy A Reinhardt2, John P Bannantine2
1Iowa State University, Department of Veterinary Microbiology and Preventive Medicine, 2180 Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
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-γ) 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.
Insights
Researchers identified protein complexes in Mycobacterium avium subsp. paratuberculosis (MAP), the cause of Johne's disease. They found key antigens like MAP2121c interacting with other proteins, revealing potential new diagnostic targets for this ruminant enteric disease.
Area of Science:
- Microbiology and Immunology
- Bacterial Pathogenesis
- Proteomics
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a significant chronic enteric illness in ruminants.
- Understanding MAP's cell envelope protein interactions is crucial for developing effective diagnostics and control strategies.
- Previous studies have focused on individual MAP proteins, but their complex formation in the envelope remains largely unexplored.
Purpose of the Study:
- To investigate the presence and composition of protein complexes within the MAP cell envelope.
- To identify specific protein interactions and their roles in MAP pathogenesis.
- To characterize the antigenicity of identified proteins for potential diagnostic applications.
Main Methods:
- Blue native PAGE and 2D SDS-PAGE were employed to separate MAP envelope protein complexes.
- Mass spectrometry (MS) was utilized for the identification of individual proteins within these complexes.
- Western blotting and interferon-gamma (IFN-γ) release assays were performed to assess protein antigenicity using animal sera and recombinant proteins.
Main Results:
- Seven putative membrane complexes were identified, including a complex of major membrane protein (MAP2121c) with cysteine desulfurase (MAP2120c).
- Other identified complexes were involved in energy metabolism and included a T cell antigen (Cfp29).
- MAP2121c was recognized by sera from clinically infected cows more than subclinical or control cows, indicating its potential as a diagnostic marker.
Conclusions:
- The study demonstrates the existence of functional protein complexes in the MAP cell envelope, highlighting protein-protein interactions.
- MAP2121c and other identified antigenic proteins represent promising candidates for developing novel diagnostic tools for Johne's disease.
- Further characterization of these complexes could offer new insights into MAP virulence and host-pathogen interactions.
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