Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Gué rin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting ⌬RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis ⌬RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the ⌬RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Gué rin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness. B acillus Calmette-Guérin (BCG) was first isolated fromMycobacterium bovis after serial subculturing in ox bile medium (1, 2), when Drs. Calmette and Guérin set out to test the hypothesis that a bovine tubercle bacillus could transmit pulmonary tuberculosis after oral administration (1, 3, 4). However, unexpectedly after the 39th passage, the strain was unable to kill experimental animals (1, 2), and showed no reversion to virulence even after the authors had performed over 200 passages (3), which is consistent with the attenuating mutation being a deletion mutation. In proceeding studies, BCG was determined to be able to protect animals receiving a lethal challenge of virulent tubercle bacilli (5), and in 1921 was first used as an anti-tuberculous vaccine (6). Presently, an estimated 3 billion doses have been used to vaccinate the human population against tuberculosis, yet the mechanism that causes the attenuation of BCG remains unknown.Mahairas et al. (6) first compared the genomic sequences of BCG and M. bovis, by using subtractive hybridization, and found that there were three regions of difference (designated RD1, RD2, and RD3) present in the genome of M. bovis, but missing in BCG. Behr et al. (7), and others (8), later identified 16 large deletions, including RD1-RD3, which were present in the Mycobacterium tuberculosis genome but absent in BCG. Eleven of these 16 deletions were unique to M. bovis whereas the remaining 5 del...

SummarySuccessful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms -organized communities of surface-attached cells -but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.