Engineering redox sensitive biosensor for real time detection of reactive sulfur species (RSS) in Mycobacterium tuberculosis (Mtb)

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global health crisis, with Mtb’s ability to persist in the host by modulat ing its metabolism and redox homeostasis. Reactive sulfur species (RSS), such as hydrogen sulfide (H2S) and persulfides, play a critical role in Mtb persistence by regulating redox balance, metabolism, and antibiotic tol erance. However, conventional approaches fail to discriminate between host and bacterial H2S, precluding real-time analysis of H2S levels in Mtb inside infected host cells such as macrophages and neutrophils. This study focuses on developing a genetically encoded biosensor based on redox-sensitive green flourescent protein (roGFP2) to detect RSS in M. tuberculosis. The biosensor was engineered to respond to intracellular RSS levels and integrated into mycobacterial strains. We validated its ability to detect dynamic changes in RSS under varying physiological conditions using fluorescence-based assays and flow cytometry. Our findings establish roGFP2 as a powerful, non-invasive tool for real time monitoring of RSS dynamics in Mtb, opening avenues to explore how sulfur metabolism influences bacterial persistence and drug resistance. By bridging the gap in our understanding of RSS in Mtb, this work contributes to ongoing efforts to combat TB more effectively.