Profiling bovine antibody responses to Mycobacterium avium subsp. paratuberculosis infection by using protein arrays

Abstract

With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.

FIG. 1.

SDS-PAGE analysis of purified recombinant fusion proteins. Two gels stained with GelCode Blue demonstrate the purity of 25 M. avium subsp. paratuberculosis recombinant fusion proteins plus the MBP-LacZ control. Most proteins show a single discrete band with limited contamination of E. coli host proteins. The MBP affinity tag is 42 kDa in size and the largest M. avium subsp. paratuberculosis protein, MAP1643, is 85 kDa in size. Therefore, this fusion protein migrates at the expected size of 127 kDa (lane 19). Protein size standards are indicated in kilodaltons in the left margin, and the migration position of the MBP-LacZ control is indicated in the right margin. Lane assignments: 1, protein size standards; 2, MBP-MAP1174c; 3, MBP-MAP1121c; 4, MBP-MAP3131; 5, MBP-MAP4198; 6, MBP-MAP2657; 7, MBP-MAP3121; 8, MBP-MAP1204; 9, MBP-MAP0961c; 10, MBP-MAP0853; 11, MBP-MAP3734c; 12, MBP-MAP2360; 13, MBP-MAP1388; 14, MBP-MAP0075; 15, MBP-LacZ; 16 and 17, protein size standards; 18, MBP-MAP3743; 19, MBP-MAP1643; 20, MBP-MAP3735c; 21, MBP-MAP3902c; 22, MBP-MAP0736; 23, MBP-MAP1609c; 24, MBP-MAP1087; 25, MBP-MAP1233; 26, MBP-MAP3761c; 27, MBP-MAP2740; 28, MBP-MAP0855; 29, MBP-MAP3751; 30, MBP-LacZ.

FIG. 2.

Controls to test the performance of the 96-dot array. Shown are the dot assignments for the protein array (A) and two dot array (B) experiments. The dot assignments are consistent for all experiments in this study. The monoclonal antibody or serum sample used is indicated beneath each array. All MBP fusion proteins were detected by the MBP monoclonal antibody (MBP mAb), whereas the serum from uninfected cow 111 showed little to no reactivity with most of the proteins. Because background reactivity was so low with all three uninfected cow sera, a control strip (shown on the right side of the array) containing serial dilutions of unconjugated bovine antibody was processed in parallel with all arrays exposed to uninfected cow sera to ensure the experiment worked correctly. A whole-cell lysate representing a majority of the proteins produced by M. avium subsp. paratuberculosis is spotted in positions 12E and 12H for all dot arrays. Note that the MBP monoclonal antibody does not detect the three His-tagged proteins spotted in positions 12A, 12B, and 12C.

FIG. 3.

Cross-reactive antibodies from cattle infected with mycobacteria other than M. avium subsp. paratuberculosis. Each array was exposed to serum from an infected cow as indicated beneath the image. Antibodies from the M. avium subsp. avium (M. avium)-infected cow were observed to be more cross-reactive than those from the M. bovis-infected cow. Dot assignments are as shown in Fig. 2A.

FIG. 4.

Protein arrays used to assess the humoral immune response to M. avium subsp. paratuberculosis. The protein arrays were processed by immunoblot analysis with various primary sera, as indicated in the margins. (A) Arrays probed with sera from a rabbit exposed to M. avium subsp. paratuberculosis and anti-MBP antibody. mAb, monoclonal antibody. (B) Antibody profiles from one experimentally infected cow (5903) and three clinical cattle (509, 3235, and 3494). Sera from cow 5903 were collected at 321 days post-intratonsillar infection with M. avium subsp. paratuberculosis and represent an early stage of disease. Sera were collected from the other three naturally infected cows during the appearance of clinical Johne's disease. Antibody profiles are generally similar among all four cattle despite the representation of distinct infection routes and disease stages. Dot assignments are as shown in Fig. 2A.