MOLECULAR ANALYSIS OF GENETIC-DIFFERENCES BETWEEN MYCOBACTERIUM-BOVISBCG AND VIRULENT M-BOVIS

The live attenuated bacillus Calmette-Guerin (BCG) vaccine for the pre vention of disease associated with Mycobacterium tuberculosis was deri ved from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's at tenuation has never been elucidated, We employed subtractive genomic h ybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG, Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from B CG, and the precise junctions and DNA sequence of each deletion were d etermined, RD3, a 9.3-kb genomic segment present in virulent laborator y strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates, RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved id all virulent laborat ory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of al least 10 proteins and resulted in a protein expression profile almost identica l to that of virulent M. bovis and M. tuberculosis, as determined by t wo-dimensional gel electrophoresis, These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation, These findings ma y be applicable to the rational design of a new attenuated tuberculosi s vaccine and the development of new diagnostic tests to distinguish B CG vaccination from tuberculosis infection.