Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing.

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.