Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains.

SETTING: Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression. OBJECTIVES: To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains. DESIGN: Plasmids were constructed using mycobacterial promoters (hsp60, 19-kDa antigen, 85A antigen--from the Mycobacterium tuberculosis complex--and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes. RESULTS: The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product. Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters. BCG Moreau appeared to be more susceptible to deletions than other BCG strains. CONCLUSIONS: The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product. Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation. With intact plasmid there was no marked difference in expression between BCG strains.

Activity of mycobacterial promoters during intracellular and extracellular growth.

pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.