Bioenergetics and Metabolism as a New Target Space to Treat Persistent Mycobacterium tuberculosis

Abstract

Most chemotherapeutics currently used in the treatment of Mycobacterium tuberculosis (Mtb) target conventional systems of anti-microbial therapy, like cell wall and DNA biosynthesis. While targeting pathways essential for proliferation is sufficient to cure most bacterial infections, these approaches require at least six months to treat tuberculosis (TB) in order to clear persistent cell populations that are metabolically dormant and phenotypically tolerant to antibiotics. Importantly, there is a disturbing lack of drugs in our arsenal that can effectively target and sterilize these phenotypically tolerant populations. The severity of the TB epidemic mandates the development of drugs that target pathways essential during persistence. Several systems have been proposed to be important in Mtb's transition to and maintenance of persistence, including the respiratory chain and amino acid metabolism.

Mtb can effectively survive adverse conditions such as hypoxia, host defenses and chemotherapy. These are redox stressors that threaten the equilibrium of essential cellular processes and induce the transition to persistence. Maintenance of redox homeostasis is managed in large part by the respiratory chain which includes the alternative terminal oxidase, cytochrome bd oxidase (Cyt-bd). This enzyme is highly upregulated in response to redox stress. Here, we probe the role of Cyt-bd in adaptation of Mtb to adverse conditions that result in redox disruptions. We demonstrate that inactivation of Cyt-bd results in enhanced susceptibility to redox stress and existing drugs via Cyt-bd-mediated modulation of respiration. Furthermore, we find that alterations in respiratory rates in Mtb can antagonize front-line drugs like isoniazid. Our findings indicate that respiratory chain flexibility, in particular Cyt-bd, are important components of persistence and are valuable drug targets to develop novel therapies to treat persistent Mtb.

The "aspartate pathway" in Mtb is responsible for the biosynthesis of essential amino acids (lysine, threonine, isoleucine, methionine). Previous work demonstrated that methionine auxotrophy was bactericidal. We now investigate the requirement of three other enzymes in the aspartate pathway during persistence and characterize the lethal mechanism of action of auxotrophy. Using a genetic inducible knockdown system, we found that the aspartate pathway was required for both acute and chronic infection in mice. Transcriptomic and metabolomic characterization of inhibition revealed a novel killing mechanism by which threonine auxotrophy causes a toxic accumulation of cytosolic lysine. This was found to occur through a loss of threonine-mediated feedback regulation of the pathway and caused the upregulation of novel lysine degradation and export mechanisms. These data strongly indicate that the aspartate pathway is a valuable and target rich space for drug development with novel mechanisms of action that promises effective treatment of TB.

The respiratory chain and the aspartate pathway are clearly required for persistence in M. tuberculosis and are therefore promising targets to shorten the length of TB chemotherapy. Reduction in treatment timelines promises to increase cure rates, reduce drug resistance acquisition, and move society closer to eradicating this terrible disease.