Homology Between the MPB70 and MPB83 Proteins of Mycobacterium bovis BCG

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.     

Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.     

Immunochemical Characterization of the MPB70/80 and MPB83 Proteins of Mycobacterium bovis

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.     

Cloning and Sequencing of an MPB70 Homologue Corresponding to MPB83 from Mycobacterium bovis BCG

MPB70 is secreted in high concentrations by Mycobacterium bovis BCG substrain Tokyo (BCG Tokyo), but little by substrains Pasteur (BCG Pasteur) and M. tuberculosis . The gene encoding a MPB70 homologue secreted by BCG Tokyo was found at the upstream region of the gene encoding MPB70, with approximately 2.3 kilobase pairs (kbp) spacing: the same gene was also found in BCG Pasteur. This gene was cloned and sequenced from BCG Tokyo. The DNA sequence which contained a 663 base pair (bp) open reading frame beginning at position 1 and ending with a TAA codon at position 661 was found. Its theoretical molecular mass was calculated to be 22.068 kDa. This gene was highly homologous to the coding region of mpb70 and the deduced amino acid sequence was very similar to MPB83 reported by Harboe et al . It was speculated that the gene the authors characterized probably corresponded to the mpb 83 gene.     

Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG.

The gene for immunogenic protein MPB64 found in culture filtrates of only Mycobacterium tuberculosis and some strains of Mycobacterium bovis BCG was cloned by using a single-probe method and was sequenced. The gene analysis revealed that the structural gene for MPB64 consisted of 618 base pairs, and its deduced molecular weight was 22,400. Twenty-two amino acids for a putative signal peptide and 205 amino acids for the MPB64 protein were observed. In the coding region, the third letter of the codon showed a biased codon and a high G+C content (78.5%). The gene was expressed in Escherichia coli by using an E. coli expression vector. The product showed migration similar to that of the authentic MPB64 protein by electrophoresis and reacted with the polyclonal and the monoclonal antibodies raised against the MPB64 protein. The strict specificity of MPB64 could be applied to immunodiagnosis of tuberculosis.     

Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG.

Substrains of Mycobacterium bovis BCG have been divided in two major groups, high producers and low producers of the secreted proteins MPB64 and MPB70. Of these, Mycobacterium tuberculosis secretes only the analog MPT64 during growth on Sauton medium. It has been confirmed that high-producer and low-producer substrains of BCG as well as M. tuberculosis contain the gene for the MPB/MPT70 protein. By contrast, polymerase chain reaction and hybridization experiments are reported here which indicate that the MPB64 gene is absent in the BCG substrains Copenhagen, Pasteur, Glaxo, and Tice, in which previous methods did not permit distinction between secretion of small amounts or absence of the protein in culture fluids.     

MPB59, a widely cross-reacting protein of Mycobacterium bovis BCG

The MPB59 protein of Mycobacterium bovis BCG was purified to homogeneity from culture fluid of BCG substrain Tokyo, and characterized by biochemical and immunological techniques. The molecular weight was 28,000, determined by SDS-polyacrylamide gel electrophoresis, and the pI value was 5.3. The N-terminal amino acid sequence was determined for 32 steps and showed no significant homology with MPB64, MPB70 or MPB80. By crossed immunoelectrophoresis, MPB59 was found to belong to the BCG antigen 85 complex and identified as corresponding to the 85B component of this complex. The protein cross-reacted extensively with other species of mycobacteria, and induced a marked humoral immune response in armadillos and monkeys during development of systemic mycobacterial infection after inoculation with Mycobacterium leprae.     

T-Cell Recognition of Mycobacterial Proteins MPB70 and MPB64 in Cattle Immunized with Antigen and Infected with Mycobacterium bovis

Defined antigenic reagents and knowledge of T-cell responses are required for the design of improved diagnostic tests for bovine tuberculosis. The limited species distribution of Mycobacterium bovis antigens MPB70 and MPB64 has indicated their potential for inclusion in future tests. The strategy adopted in this study was to define bovine T-cell responses to these antigens at the epitope level, using cattle immunized with recombinant forms of the antigens, and to compare these responses with cattle which had been experimentally infected with M. bovis . Panels of synthetic peptides (20-mers with 10-residue overlaps) were used and five epitopes were identified and found to be powerful stimulators of T-cell responses in both types of animal (residues 81–100 and 174–190 for MPB70; and residues 1–20, 41–60 and 181–200 for MPB64). Further investigation in larger numbers of cattle ( n  = 14) of mixed breeds from tuberculosis-infected herds confirmed that each peptide produced response in several of the cattle, but no single peptide was recognized by all animals. However, the limited numbers of animals in this study suggest that peptide reagents may identify as many positive animals as the intact antigenic protein and could form components of a future diagnostic test. The use of cattle immunized with the proteins of interest has proved to be an interesting model for studying the nature of bovine T-cell responses to defined mycobacterial proteins.     

Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70.

A protein, isolated and purified from the unheated culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172) and designated MPB70, elicited a delayed skin reaction in guinea pigs sensitized with viable cells of BCG but not in those sensitized with heat-killed cells. The skin reaction reached the maximum 4 to 8 weeks after the inoculation of the BCG and then decreased gradually, resulting in conversion to negative after 20 weeks, whereas the skin reaction to purified protein derivative (PPD) continued to be positive. Guinea pigs immunized with viable cells of various substrains of BCG were skin tested with MPB70 and PPD. Guinea pigs immunized with the BCG substrain Tokyo 172 and the substrain Moreau (Brazil) showed strong delayed skin reactions to both MPB70 and PPD. On the other hand, guinea pigs immunized with the Pasteur substrain 1173P2, the Glaxo substrain 1077, the Copenhagen substrain 1331, the Tice substrain, or the Beijing substrain 64-42 showed negative skin reactions to MPB70, whereas they were strongly positive to PPD. In a two-dimensional acrylamide gel electrophoretic analysis of proteins from the culture filtrates of the BCG substrains, the culture filtrates of the Tokyo and Moreau substrains showed the spot of MPB70 on the gel slabs, whereas those of the other BCG substrains did not.     

Nucleotide sequence of MPB63 gene in Mycobacterium bovis BCG Tokyo

Mycobacterium bovis BCG has been used for the prevention of tuberculosis and as therapy for bladder tumor. MPB63 in M. bovis BCG is one of the immunogenic proteins and is secreted in large quantities. Therefore, it is of interest that the MPB63 gene be examined for the determination of its nucleotide sequence. A fragment of 820 base pairs (bp) including the MPB63 gene was prepared by amplification using polymerase chain reaction (PCR) employing M. bovis BCG Tokyo chromosomal DNA as a template and its nucleotide sequence was determined. The nucleotide sequence of mpb63 in M. bovis BCG was then compared with that of mpt63 in M. tuberculosis. The result indicated that the nucleotide sequences between two protein genes were quite agreeable in the genes' structural and upstream regions, except that one base change from C in mpt63 to G in mpb63 was detected in the downstream trailer sequence. This suggests that the genetic information of M. bovis BCG is not entirely identical to that of M. tuberculosis, although the characteristics of both microorganism are very similar to each other.     

Relationship of secretion pattern and MPB70 homology with osteoblast-specific factor 2 to osteitis following Mycobacterium bovis BCG vaccination.

Significant homology was found between MPB70 and each of four repeat domains of osteoblast-specific factor 2 (OSF-2). Two internal homology regions within each repeat domain of OSF-2 presumed to be related to the active site(s) of this bone adhesion molecule showed the highest homology. A literature search concerning osteitis after Mycobacterium bovis BCG vaccination in neonates revealed that MPB70-high-producer substrains were associated with an increased incidence of osteitis following vaccination. These observations indicate that the function of MPB70 is related to the interaction between bacilli and the host following vaccination or infection with mycobacteria.     

Biological Activity in Sensitized Guinea Pigs of MPB 70, a Protein Specific for Some Strains of Mycobacterium bovis BCG

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.     

Immunological properties of ribosomal proteins from Mycobacterium bovis BCG.

Two proteins with molecular mass 65 kDa, a heat shock protein, and an S1-like protein were found in a 30S ribosomal subunit from Mycobacterium bovis BCG. The 17-kDa protein in the 30S subunit was homologous to alpha-crystallin heat shock protein, and the 16-kDa protein in the 50S subunit was homologous to the L7/L12 protein. The latter provoked a strong delayed-type hypersensitivity reaction in the sensitized guinea pigs. The GroES-like protein (12 kDa) loosely associated with ribosomes.     

Heterogenous Expression of the Related MBP70 and MPB83 Proteins Distinguish Various Substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv

MPB70 and MPB83 are homologous cross-reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high- and low-producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low-producing strains are also MPB83 low-producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high-producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS-PAGE at 1–2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross-reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis     

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.     

PCR identification of Mycobacterium bovis BCG.

The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG.     

Effect of Mycobacterium bovis BCG vaccination upon Mycobacterium lepraemurium infection.

Mice were infected with 10(8) Mycobacterium lepraemurium in the footpad (unsuppressed mice), and some of these animals were concurrently given 10(9) heat-killed M. lepraemurium intravenously (suppressed mice). These groups of mice were preimmunized with 10(7) viable organisms of Mycobacterium bovis BCG by several routes. BCG inhibited the proliferation of M. lepraemurium in the unsuppressed mice, but not in the suppressed mice. In effect, the intravenous administration of heat-killed M. lepraemurium suppressed the immunity to M. lepraemurium that BCG vaccination had engendered. BCG did not protect normal mice against intravenous infection with M. lepraemurium. It appears that normal mice against intravenous infection with M. lepraemurium. It appears that the inhibitory effect of BCG vaccination upon M. lepraemurium infection is due to cross-reactive immunity rather than to nonspecific immunity or immunopotentiation. Thus, the route of BCG vaccination was immaterial, and vaccination 12 weeks before M. lepraemurium infection was as beneficial as vaccination 4 weeks before infection. Moreover, spleen cells from M. lepraemurium-immunized mice conferred adoptive immunity to BCG. The implications of this study for the use of BCG as a prophylactic and therapeutic agent in human leprosy are discussed.     

Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG.

Fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major FN-binding molecule. In 21-day-old supernatants, FN bound to a double protein band of 30 and 31 kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa). FN-binding molecules in this size range, but not of 30 to 31 kDa, were also found in sonicates. We showed that the 31- and 30-kDa FN-binding bands correspond to components A and B of the BCG85 complex, previously shown to be abundant in culture supernatants of Mycobacterium bovis BCG. Thus, a polyclonal antibody to the BCG85 complex bound to the 30- and 31-kDa antigens and inhibited binding of FN to them on immunoblots of the culture filtrates. Similarly, FN bound to the purified components of the BCG85 complex, and this binding was blocked by the antibody. A monoclonal antibody, HYT27, also bound both to the BCG85 components A and B and to the 30- and 31-kDa FN-binding molecules of M. tuberculosis, but it did not block the binding of FN. Related molecules appear to be present on the surface of BCG and to mediate the binding of BCG to FN-coated plastic surfaces, since this binding could also be blocked by the polyclonal anti-BCG85 antibody and by the purified components of BCG85, particularly component A, but not by monoclonal antibody HYT27. The binding of these mycobacterial antigens to FN appears to be of very high affinity, and we suggest that this property of major secreted antigens of M. tuberculosis indicates an important role in mycobacterial disease and in the binding of BCG to tumor cells during immunotherapy of bladder cancer.