Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E. coli-mycobacteria shuttle vector.
ABSTRACT MPB70 is known to be an immunogenic mycobacterial protein secreted in large amounts from Mycobacterium bovis BCG (BCG) Tokyo. The analogous gene for MPT70 was cloned from Mycobacterium tuberculosis H37Rv which produces this protein in only a small amount. The gene encoding 193 amino acid residues including 30 amino acids for the signal peptide, the promoter-like sequence, and the ribosome-binding site, was completely identical to that of BCG Tokyo. Computer analysis revealed that the carboxy-terminal half of MPT70 was homologous to amino acid sequences of fasciclin I, osteoblast-specific factor 2 (OSF-2), and human transforming growth factor-beta induced gene product (beta IG-H3). Escherichia coli (E. coli) -mycobacteria shuttle vectors containing mpt70 or mpb70 genes 0.7kbp upstream of the 5' end of them were able to be expressed in BCG Pasteur which is a MPB70 low-producer, but the extent of the expression was not that of a high-producer.
- SourceAvailable from: Harald Wiker[Show abstract] [Hide abstract]
ABSTRACT: Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high- and low-producing strains. By quantitative reverse transcription polymerase chain reaction (RT-PCR), we determined that transcription of the antigen-encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50-fold in low-producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low-producing strains compared with high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low-producing BCG strains. Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK fully restored MPB70 and MPB83 production. Microarray-based analysis and confirmatory RT-PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low-producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.Molecular Microbiology 07/2005; 56(5):1302-13. DOI:10.1111/j.1365-2958.2005.04618.x · 5.03 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Substrains of Mycobacterium bovis BCG (BCG) have been divided into two major groups, high and low producers, on the basis of the amount of secretion of the MPB70 protein. The antigen is produced in high concentration by BCG Tokyo, Moreau, Russia and Sweden (high-producer substrains), whereas in BCG Pasteur, Copenhagen and Tice (low-producer substrains) it is detected at 1% (w/w) or less of the concentration of BCG Tokyo. To investigate why this protein is secreted differently, the MPB70 genes of BCG Tokyo and Pasteur were cloned, sequenced and compared. The MPB70 genes in two substrains showed exactly the same sequence. Even the upstream and downstream regions of the MPB70 gene were identical. MPB70 gene expression was assessed by means of Northern hybridization analysis and reverse transcriptase polymerase chain reaction. The mRNA was clearly detected in BCG Tokyo, but a very low level in BCG Pasteur. On the basis of these results, the difference in the secretion of the MPB70 protein between BCG Tokyo and Pasteur was attributed to differential transcription efficiencies.Microbiology 08/1995; 141 ( Pt 7):1601-7. DOI:10.1099/13500872-141-7-1601 · 2.84 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum. The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.FEMS Microbiology Letters 02/1996; 135(2-3):237-43. DOI:10.1111/j.1574-6968.1996.tb07995.x · 2.72 Impact Factor