Ion channel regulation of ATP homeostasis in mycobacteria

The intracellular pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which has been one of the most deadly infectious diseases globally for decades. TB is not on target to be eradicated, and multi drug-resistant (MDR) strains of Mtb are a growing world health problem, with 500,000 new cases of MDR-TB reported annually. An additional challenge is the resistance of dormant mycobacteria to current antibiotic treatments, leading to long treatment regimens (ranging up to 2 years). It will thus be critical to build on our understanding of the molecular physiology of mycobacteria to gain insight toward improved anti TB treatments. A strategy for new drugs to kill dormant and MDR Mtb is to target Mtb ATP synthesis pathways, and a potential class of molecular targets includes K+ channels, which can modulate membrane potential to affect ATP synthase activity and ATP production. Insertion mutagenesis screens of Mtb have implicated the putative K+ channel-encoding gene Rv3200c as a potential virulence gene (i.e. critical for Mtb survival following infection), although functional, mechanistic studies of this gene have not been pursued in detail. In preliminary experiments using the non-pathogenic model organism Mycobacterium smegmatis, we have found that deletion of the Rv3200c ortholog (Msmeg_Δ1945) leads to decreased cellular ATP levels, consistent with a role for this channel in modulating membrane potential to support ATP production. In addition, our preliminary studies using K+ channel-deleted Mtb (Mtb_ΔRv3200c) further show that MycK deletion enhances sensitivity to killing by the H+/K+ ionophore nigericin (which decreases the transmembrane H+ gradient). These preliminary results support the working hypothesis that the Rv3200c-encoded K+ channel, termed MycK, may be a key modulator of the protonmotive force to regulate mycobacterial ATP levels. To better understand fundamental mechanisms underlying modulation of the MycK channel, as well as its role in mycobacterial physiology, we propose to 1) determine the ligand activation mechanism of the MycK channel by solving its structure by single particle cryo-electron microscopy, and by identifying the gating mechanism through analysis of single-channel recordings of purified MycK channels; and 2) determine the physiological role of MycK in control of energy metabolism, by measuring ATP levels and metabolic activity in gene-targeted mycobacteria. The new structural and functional data generated in this multi-disciplinary project will contribute to the ultimate goal of developing improved therapies for TB and other mycobacterial infection.