Rewiring Redox-Sensitive GFP for Dynamic Imaging of Mycothiol Redox Potential in Mycobacterium Tuberculosis during Infection.

Abstract

based signaling associated with Mycobacterium tuberculosis (Mtb) infection are poorly characterized. The mycothiol redox couple (MSH/MSSM) controls redox homeostasis in Mtb, yet we lack non-invasive techniques for its real-time imaging. Here, we demonstrated that the coupling of Mtb mycoredoxin-1 (Rv3198A) to redox-sensitive GFP (roGFP2) allowed real-time imaging of dynamic changes in the intra-mycobacterial mycothiol redox potential (E MSH) with unprecedented sensitivity and specificity. The biosensor facilitated first quantitative mapping of E MSH in diverse mycobacterial species, genetic mutants, clinical isolates including multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains in vitro, inside macrophages, and in sub-vacuolar compartments. Using this biosensor, we showed that macrophage environment induces oscillatory changes in E MSH that are distinctly strain-and compartment-specific. Remarkably, disruption of Mycothiol signaling uniformly inhibited the growth of drug-sensitive and MDR/XDR Mtb. Unexpectedly, Mrx1-roGFP2 revealed that environments inside sub-vacuolar compartments provide niche for evolution of redox heterogeneities in Mtb populations during infection.