The primary mechanism of attenuation of bacillus Calmette–Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue

TLDR

Tuberculosis remains a leading cause of death worldwide, and although BCG is an attenuated Mycobacterium bovis strain, the functional basis for its attenuation has not been elucidated. Deletion of the RD1 region, which encodes the ESAT‑6–CFP‑10 operon, produces ΔRD1 mutants that are highly attenuated and lack cytolytic activity against pneumocytes and macrophages, indicating that RD1 is essential for virulence. The ΔRD1 mutants confer protection against aerosol challenge comparable to BCG, and the loss of secreted ESAT‑6–mediated cytolysis is identified as the primary mechanism of BCG attenuation, reducing tissue invasiveness.

Abstract

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette–Guérin (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 Δ RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis Δ RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the Δ 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–Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.