Molecular Characterization of MPT83: a Seroreactive Antigen of Mycobacterium tuberculosis with Homology to MPT70

Abstract

The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70.

The MPB83 Antigen from Mycobacterium bovis ContainsO-Linked Mannose and (1 → 3)-Mannobiose Moieties

“…They suggested that the increases in molecular weight, relative to the 22,000 MPB70, were attributable to glycosylation with mannose, as treatment of these antigens with ␣-mannosidase resulted in a reduction in their relative molecular weight. Subsequently, we demonstrated the presence of a gene, mpb83, which encodes a protein with 61% identity at the amino acid level to MPB70 (18). In the same study, the protein encoded for by this gene, MPB83, was shown to bind a monoclonal antibody specific for the 25/23-kDa M. bovis antigen.…”

“…The data shows that the 23-kDa form of MPB83 is generated by proteolytic cleavage immediately before Thr 48 from the mature acylated 25-kDa form (18). This raises the possibility that glycosylation may function either as a signal for cleavage or as a means of preventing amino-terminal degradation of the protein following cleavage from its acylated anchor.…”

“…Construction of PhoA Hybrids-mpb70 and mpb83 were amplified from the cosmid pA3, described previously (18), using the oligonucleotide pairs 1,2 and 3,4, respectively (Table I). Amplified fragments were purified using Wizard PCR Prep columns (Promega) and digested with the restriction endonucleases BamHI and KpnI (Promega).…”

“…bovis expresses high levels of both MPB70 and MPB83 and these proteins are strongly recognized by the immune system during M. bovis infection in cattle and badgers (19,20). Although the proteins are expressed only at low levels in M. tuberculosis when grown in vitro, they are highly immunogenic during infection with live bacteria in mice (18) and man (21). In addition, vaccination of mice with a plasmid encoding MPB83 has been shown to confer significant protection against challenge with M. bovis (22).…”

Association of Tuberculin-Boosted Antibody Responses with Pathology and Cell-Mediated Immunity in Cattle Vaccinated with Mycobacterium bovis BCG and Infected with M. bovis

“…MPB70 and MPB83 are both expressed constitutively in M. bovis yet are expressed only at very low levels in BCG strains that have lost the RD2 regions, such as BCG Pasteur, which was used in the present study (20,33). MPB70 is a secreted protein (21), whereas MPB83 is a cell wall-associated glycolipoprotein that is also found in culture filtrates (11). The proteins are highly homologous at the amino acid level (11) and are major constituents of PPD-B (K. Lyashchenko, A. O. Whelan, and H. M. Vordermeier, unpublished data), which would explain why the immune responses to these antigens were boosted by tuberculin skin testing.…”