Phosphoenolpyruvate depletion mediates both growth arrest and drug tolerance of Mycobacterium tuberculosis in hypoxia

Abstract

Significance Nonreplicating (NR) Mycobacterium tuberculosis (Mtb) was thought to be drug tolerant due to its low metabolic activity. However, drug tolerance often gradually increases by exposure to antibiotics, inferring the role of an adaptive strategy. This study established that drug tolerance of NR Mtb is associated with phosphoenolpyruvate (PEP) anabolic down-regulation. PEP is a substrate of multiple pathways needed for replication; in NR Mtb, their functions were abolished due to PEP depletion. Intriguingly, PEP supplementation partly restored drug sensitivity and prevented the emergence of drug resistance (DR). The results expand our knowledge of how NR Mtb remodels its metabolic networks and propose potential Mtb metabolites as a source of therapeutic adjuvants to synthetically kill NR Mtb and prevent development of DR. Mycobacterium tuberculosis (Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.