Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection

PLoS Pathogens

Kate S. Carroll¹  Devayani Bhave¹  Dhiraj Kumar²  Manbeena Chawla²  Pallavi Chandra²  Amit Singh²  Ashima Bhaskar²  Mansi Mehta²  Pankti Parikh² 

[1] Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, United States of America;International Centre for Genetic Engineering and Biotechnology, New Delhi, India

关键词: Mycobacterium tuberculosis;    Oxidation-reduction reactions;    Macrophages;    Antibiotics;    Flow cytometry;    Biosensors;    Oxidation;    Oxidative stress;   

DOI  :  10.1371/journal.ppat.1003902

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (EMSH) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial EMSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of EMSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in EMSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in EMSH, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied EMSH during infection and show that subpopulation with higher EMSH are susceptible to clinically relevant antibiotics, whereas lower EMSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence.

【 授权许可】

CC BY