Mycobacterium tuberculosis (Mtb) has generated a global health catastrophe that has been compounded by the emergence of drug resistant Mtb strains. We used whole genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent and reactivated disease. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. Our data suggest that Mtb acquires a similar number of chromosomal mutations during latency as occurs during active disease or in a logarithmically growing culture over the same period of time despite reduced bacterial replication during latent infection. The pattern of polymorphisms suggests that the mutational burden in vivo is due to oxidative DNA damage. Thus, we demonstrate that Mtb continues to acquire mutations during latency and provide a novel explanation for the observation that isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of INH resistance1,2.

A critical question in tuberculosis control is why some strains of Mycobacterium tuberculosis are preferentially associated with multiple drug resistances. We demonstrate that M. tuberculosis strains from Lineage 2 (East Asian lineage and Beijing sublineage) acquire drug resistances in vitro more rapidly than M. tuberculosis strains from Lineage 4 (Euro-American lineage) and that this higher rate can be attributed to a higher mutation rate. Moreover, the in vitro mutation rate correlates well with the bacterial mutation rate in humans as determined by whole genome sequencing of clinical isolates. Finally, using a stochastic mathematical model, we demonstrate that the observed differences in mutation rate predict a substantially higher probability that patients infected with a drug susceptible Lineage 2 strain will harbor multidrug resistant bacteria at the time of diagnosis. These data suggest that interventions to prevent the emergence of drug resistant tuberculosis should target bacterial as well as treatment-related risk factors.

DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor −10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia.