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Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection
Ashima Bhaskar1, Manbeena Chawla1, Mansi Mehta1
1International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Plos Pathogens
|February 6, 2014
Summary
Mycobacterium tuberculosis (Mtb) uses mycothiol (MSH) to combat oxidative stress. A new biosensor, Mrx1-roGFP2, reveals MSH redox potential changes within Mtb during infection and treatment, impacting antibiotic effectiveness.
Area of Science:
- Microbiology
- Redox Biology
- Infectious Diseases
Background:
- Mycobacterium tuberculosis (Mtb) endures harsh oxidative conditions within host phagocytes.
- Mycothiol (MSH) is crucial for Mtb's cytoplasmic redox buffering and survival.
- Understanding Mtb's redox homeostasis is key to developing effective tuberculosis treatments.
Purpose of the Study:
- To develop and validate a novel real-time biosensor for measuring mycothiol redox potential (EMSH) within Mtb.
- To investigate the dynamics of EMSH in Mtb during host infection and in response to anti-tuberculosis drugs.
- To explore the relationship between EMSH, membrane integrity, and antibiotic susceptibility in Mtb.
Main Methods:
- Development of a fusion protein (Mrx1-roGFP2) linking Mtb's mycoredoxin-1 (Mrx1) to a redox-sensitive green fluorescent protein (roGFP2).
- Real-time imaging and quantification of intramycobacterial EMSH in Mtb during infection of macrophages.
- Analysis of EMSH heterogeneity in diverse Mtb species, mutants, and drug-resistant isolates.
- Assessment of Mtb membrane integrity and antibiotic susceptibility correlated with EMSH levels.
Main Results:
- The Mrx1-roGFP2 biosensor enables sensitive and specific real-time measurement of dynamic EMSH changes in Mtb.
- Intramacrophage environments induce heterogeneous EMSH levels within the Mtb population.
- Anti-tuberculosis drug treatment shifts Mtb's EMSH towards an oxidative state, indicating disrupted MSH homeostasis.
- Mtb subpopulations with higher EMSH exhibit increased susceptibility to antibiotics, while lower EMSH correlates with antibiotic tolerance.
Conclusions:
- MSH redox signaling plays a critical role in modulating Mtb survival and antibiotic response during infection.
- The Mrx1-roGFP2 biosensor provides a powerful tool for studying Mtb redox biology and identifying novel therapeutic targets.
- Targeting Mtb's redox metabolism offers a promising strategy for controlling persistent infections and overcoming drug resistance.
