Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein

Abstract

Background: The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.

Results: MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-gamma production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-gamma after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows.

Conclusions: Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-gamma production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.

Figure 1

Amino acid sequence comparison of the mycobacterial 19 kDa protein. Non-conserved residues are shaded in black and gaps in amino acid sequence are indicated by hyphens (-). The predicted signal peptidase cleavage site for M. tuberculosis is indicated by a single arrow (↓), while the site for M. avium subsp. paratuberculosis is indicated by a double arrow (⇓). The GenBank accession number and abbreviations are M. avium subsp. paratuberculosis (M. paratb; AAS02578), M. avium subsp. avium (M. avium; AAB25888), M. intracellulare (M. intracel; AAB25885), M. bovis (M. bovis; S11234) and M. tuberculosis (M. tuberc; NP_218280).

Figure 2

SDS-PAGE and immunoblot analysis of the MBP-19 kDa fusion protein. (A) An SDS-PAGE gel showing the noninduced and IPTG-induced E. coli protein lysates. Lanes: 1- protein size markers; 2- noninduced E. coli MBP-19 kDa; 3- IPTG induced E. coli MBP-19 kDa; 4- Affinity purified MBP-19 kDa. (B) Immunoblot probed with a monoclonal antibody to MBP. Lane 1- purified MBP; Lane 2- noninduced E. coli MBP-19 kDa; Lane 3- IPTG induced E. coli MBP-19 kDa.

Figure 3

The recombinant 19-kDa protein is recognized by serum from infected cattle. Affinity purified MBP (lane 1) and MBP-19 kDa (lane 2) were transferred onto membranes in equal amounts and probed with sera from three clinically infected cattle: (A) animal 107; (B) animal 116; (C) animal 167. Reactivity was observed for the 19-kDa protein but not the MBP protein. Size standards are indicated in kilodaltons in the left margin.