Immunogenicity of Proteome-DeterminedMycobacterium aviumsubsp.paratuberculosis-Specific Proteins in Sheep with Paratuberculosis

Abstract

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionizationtime of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.

Surface Proteome of “Mycobacterium avium subsp. hominissuis” during the Early Stages of Macrophage Infection

“…Although putative surface and/or secreted proteins are a small fraction of the mycobacterial proteome, they represent a large portion of known bacterial antigens (3,37,38,45,87). This is largely because the surface-exposed molecules of the bacterium are primarily what the immune system sees when it targets a pathogen.…”

“…Effective cellular and humoral immune responses depend on targeting accessible molecules, which tend to be on the surface of a pathogen (87). With respect to mycobacteria, putative and known surface-exposed proteins comprise a substantial proportion of antigens observed in comprehensive screens using sera from mycobacterium-infected hosts (37,42,45,85). The characterization of these proteins improves our understanding of bacterial pathogenesis and host immunity, providing insights that can lead to new diagnostic tools and vaccines (38,87).…”

In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

“…paratuberculosis proteins (MAP1297, MAP2872c, MAP3567, and MAP3651c [17], as well as MAP1168c [23]). However, none of these proteins were identified by in silico analysis for the purpose of assessing immunogenic potential, and only one of these proteins (MAP1297) was evaluated by antibody assay to confirm a reaction with sera obtained from sheep infected with M. avium subsp.…”

Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

“…Reaction buffer (100 l) was added to the mixture, which was vortex mixed briefly and incubated for 16 to 40 h at room temperature (RT) (22 to 24°C). Fifteen-microliter aliquots were collected after 16,18,20,25,30,35, and 40 h of incubation for SDS-PAGE analysis. The optimal cleavage time was determined for each protein based on the appearance of two bands that corresponded to the expected molecular masses of MBP and the relevant M. avium subsp.…”

“…paratuberculosis antigens have been investigated for their diagnostic potential in cell-and antibody-mediated assays (11,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Some of these M. avium subsp.…”