Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity |
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| Affiliation: | 1. Iowa State University, Department of Veterinary Microbiology and Preventive Medicine, 2180 Veterinary Medicine, Iowa State University, Ames, IA 50011, USA;2. USDA-ARS, National Animal Disease Center, 1920 Dayton Avenue, Ames, IA 50010, USA;1. University of Vienna, Division of Drug Design and Medicinal Chemistry, Department of Pharmaceutical Chemistry, Pharmacoinformatics Research Group, Althanstrasse 14, A-1090 Vienna, Austria;2. Swiss Institute of Bioinformatics, CALIPHO Group, CMU - Rue Michel-Servet 1, 1211 Geneva 4, Switzerland;3. Discovery Sciences, Chemistry Innovation Center, AstraZeneca R&D, Mölndal, Sweden;4. European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom;1. Department of Clinical Laboratory, Kunshan First People''s Hospital, Affiliated to JiangSu University, Kunshan 215300, China;2. Department of Clinical Laboratory, The Second Xiangya Hospital of Central South University, Changsha 410078, China;3. Department of Pathology, ChongQing Cancer Institute, ChongQing 404100, China;1. Pan Genome Systems, Madison, WI 53719, USA;2. Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA;3. Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt;1. Istituto Zooprofilattico Sperimentale dell’Emilia Romagna e della Lombardia, Sezione di Piacenza-Gariga, Strada della Faggiola 1, 29027, National Reference Centre for Paratuberculosis, Gariga di Podenzano, (PC), Italy;2. Istituto Zooprofilattico Sperimentale dell’Emilia Romagna e della Lombardia, Sezione di Brescia, Via Bianchi 7/9, 25124, Brescia, Italy;3. Istituto Zooprofilattico Sperimentale dell’Emilia Romagna e della Lombardia, Sezione di Sondrio, Via Bormio 30, 23100, Sondrio, Italy;4. Istituto Zooprofilattico Sperimentale delle Venezie, Sezione di Bolzano, Via Laura Conti 4, 39100, Bolzano, Italy;5. Department of Veterinary Sciences, University of Pisa, Viale delle Piagge, 2, 56124 Pisa, Italy |
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| 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. |
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| Keywords: | Protein complexes Antigens |
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