Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli.

Pathogenomic evidence suggests that Mycobacterium tuberculosis evolved from an environmental ancestor similar to Mycobacterium canettii, a rare human pathogen. However, the adaptations responsible for the transition from an environmental mycobacterium to an obligate human pathogen are poorly characterised. The ability to persist in the human host appears to be one important trait. Here we set out to identify the adaptations that contribute to the evolution of persistence in M. tuberculosis. By using experimental evolution of 8 M. canettii populations in mice, we selected mutants with enhanced persistence in vivo when compared to parental strains, which were thus phenotypically closer to M. tuberculosis. Genome sequencing of 140 M. canettii mutants and complementation analysis revealed that mutations in two loci were responsible for the enhanced persistence phenotypes. Most of the tested mutants were more resistant than their parental strains to nitric oxide, which is an important effector of immunity against M. tuberculosis infection. This resistance was common to modern M. tuberculosis strains but not to M. canettii strains. Our findings demonstrate phenotypic convergence during the experimental evolution of M. canettii, which mirrors natural evolution of M. tuberculosis. Furthermore, they indicate that the ability to withstand hostinduced stresses, such as nitric oxide, was key for the emergence of persistent M. tuberculosis.