Research Summary Antibiotics have failed to control bacterial diseases typically due to the emergence of drug resistant (DR) mutants. Mycobacterium tuberculosis (Mtb) is one of the world’s most successful pathogens because of its capacity to develop DR mutants to withstand antibiotic effects. Treating DR-tuberculosis (TB) patients takes two years and costs nearly $393,000 per person, which is substantially more expensive than ~ $49,000 per person for treating a drug sensitive (DS)-TB patient. Despite this pressing human health problem, little is known about the mechanistic bases underlying the development of DR-TB. Given the low genomic mutation rates and slow replication of Mtb, intrinsic bacterial factors should play an important role in developing DR-TB, but they have been understudied. Accumulating evidence has shown that cyclic formation of Mtb persisters, a phenotypic variant transiently tolerant to TB antibiotics, can predispose TB patients to the emergence of permanent DR mutants. We recently reported untargeted metabolomics profiling of Mtb persisters and revealed that Mtb shifted its trehalose-mediated carbon flux towards the biosynthesis of central carbon metabolism (CCM) intermediates to avoid irreversible antibiotic damage, while decreasing its flux towards the biosynthesis of cell wall mycolyl glycolipids. This process was termed the “trehalose catalytic shift” and was identified to be essential for Mtb persister formation, viability, and drug tolerance. In this application, we hypothesize that the trehalose catalytic shift is an adaptive strategy executed by Mtb after treatment with TB antibiotics to achieve drug tolerance and also to facilitate the development of DR mutants, thus altering the TB disease course. In cross-sectional studies with 7 different clinical TB lineages, lineage 2 strains such as HN878 W-Beijing strain (HN878), have been associated with a high risk of developing multidrug resistant (MDR)-TB and high mortality. Thus, we will examine our hypothesis by demonstrating that HN878 is hypervirulent and more prone to develop drug resistance than other lineage strains because of its high level of trehalose catalytic shift activity. To this end, we will determine if the trehalose catalytic shift is an HN878 intrinsic factor responsible for its drug tolerance, DR mutation rates, and hypervirulence in vitro, ex vivo and then apply it in vivo using a TB murine model. A successful outcome of this application will aid in the development of new therapeutic interventions to cure DR-TB patients, including those infected with HN878.