Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals

Abstract

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5' and 3' regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

FIG. 1.

Genomic map of PE, PPE, and UPF genes in M. avium subsp. paratuberculosis K-10 (MAP). Genes encoding PE (shown as bars inside of the circular chromosome), PPE (bars crossing the circular chromosome), and UPF (bars outside the circle) protein family members are shown. No PGRS protein-coding sequences were found. (Inset) MAP1152-MAP1156 genomic region, indicating genes encoding PPE proteins MAP1152, MAP1153, and MAP1155 (black boxes), UPF protein MAP1156 (gray box), hypothetical protein MAP1154 (dark patterned box), and MAP1150c and MAP1151c (light patterned boxes). Arrowed boxes indicate the direction of transcription.

FIG. 2.

SDS-PAGE and immunoblot analysis of recombinant M. avium subsp. paratuberculosis MAP1152 and MAP1156 proteins. Shown is a 12% SDS-PAGE gel, stained with GelCode blue, along with three corresponding immunoblots containing purified recombinant fusion proteins. Antibody or serum samples used to probe the immunoblot are indicated beneath the panels: MBP MAb, monoclonal antibody against the maltose-binding protein; mouse 160, serum derived from a mouse immunized with live M. avium subsp. paratuberculosis cells; rabbit 273, serum derived from a rabbit immunized with live M. avium subsp. paratuberculosis cells. Size standards, reported in kDa, are indicated to the left. Assignments for the gel and blots were as follows: lane 1, protein size standards; lane 2, MAP1152; lane 3, MAP1156; lane 4, LacZ.

FIG. 3.

Western blot analysis of antibody responses to MAP1152 and MAP1156 in naturally infected cattle. Immunoblots containing MAP1152 and MAP1156 were probed with sera from five cows (183, 2075, 84, 805, and 45) naturally infected with M. avium subsp. paratuberculosis (A) or from two additional cows experimentally infected with M. avium or M. bovis (B). Size standards, reported in kDa, are indicated to the left. Assignments for the blots were as follows: lane 1, protein size standards; lane 2, MAP1152; lane 3, MAP1156; lane 4, LacZ.

FIG. 4.

Western blot analysis of antibody responses to MAP1152 and MAP1156 during the course of JD. Immunoblots of MAP1152 (upper gel), MAP1156 (middle gel), and K-10 whole-cell extract (lower gel) were probed with serum samples withdrawn from a naturally infected cow (cow 47) at various times during the course of the experiment: lane 1, first bleed, subclinical infection; lane 2, 12 months after first bleed, borderline clinical/subclinical infection; lane 3, 26 months after first bleed, clinical infection; lane 4, 35 months after first bleed, advanced clinical infection; lane 5, anti-MBP monoclonal antibody control. Size standards (in kDa) are indicated to the left.

FIG. 5.

Seroreactivities of MAP1152 and MAP1156 recombinant proteins to infected and noninfected cattle. Antigen (0.010 mg) reactivity was evaluated by ELISA against sera from cattle naturally infected with M. avium subsp. paratuberculosis (filled bars) or sera from culture-negative cattle (open bars). Each serum sample was diluted 1:25 in Idexx dilution buffer. Each column represents mean absorbance (from triplicate measurements against LacZ; averaged from measurements on three different days in duplicate or triplicate for other antigens [see Table S2 of the supplemental material]) per antigen, ± standard errors of the means from noninfected cattle (left columns) or infected cattle (right columns), as evidenced from the original classification of serum samples. Significance levels are indicated as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 6.

Individual responses of serum samples from infected and noninfected cattle to recombinant antigens. Antigen (0.010 mg) reactivity was evaluated by ELISA against sera from cattle naturally infected with M. avium subsp. paratuberculosis and sera from culture-negative cattle. Each serum was diluted 1:25 in Idexx dilution buffer. Each column represents mean absorbance (triplicate measurements against LacZ; average measurements on three different days in duplicate or triplicate for other antigens, as indicated in Table S2 of the supplemental material) per antigen, ± standard errors of the means from noninfected (−) or infected (+) cattle, as evidenced from the original classification of serum samples. The reaction to each antigen is indicated (see inset for pattern key).