Trehalose catalytic shift is an intrinsic factor in Mycobacterium tuberculosis that enhances phenotypic heterogeneity and multidrug resistance.

Drug-resistance (DR) in many bacterial pathogens often arises from the repetitive formation of drug-tolerant bacilli, known as persisters. However, it is unclear whether (Mtb), the bacterium that causes tuberculosis (TB), undergoes a similar phenotypic transition. Recent metabolomics studies have identified that a change in trehalose metabolism is necessary for Mtb to develop persisters and plays a crucial role in metabolic networks of DR-TB strains. The present study used Mtb mutants lacking the trehalose catalytic shift and showed that the mutants exhibited a significantly lower frequency of the emergence of DR mutants compared to wildtype, due to reduced persister formation. The trehalose catalytic shift enables Mtb persisters to survive under bactericidal antibiotics by increasing metabolic heterogeneity and drug tolerance, ultimately leading to development of DR. Intriguingly, rifampicin (RIF)-resistant bacilli exhibit cross-resistance to a second antibiotic, due to a high trehalose catalytic shift activity. This phenomenon explains how the development of multidrug resistance (MDR) is facilitated by the acquisition of RIF resistance. In this context, the heightened risk of MDR-TB in the lineage 4 HN878 W-Beijing strain can be attributed to its greater trehalose catalytic shift. Genetic and pharmacological inactivation of the trehalose catalytic shift significantly reduced persister formation, subsequently decreasing the incidence of MDR-TB in HN878 W-Beijing strain. Collectively, the trehalose catalytic shift serves as an intrinsic factor of Mtb responsible for persister formation, cross-resistance to multiple antibiotics, and the emergence of MDR-TB. This study aids in the discovery of new TB therapeutics by targeting the trehalose catalytic shift of Mtb.