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Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

ABSTRACT: 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.

SUBMITTER: Bhaskar A

PROVIDER: S-EPMC3907381 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

Bhaskar Ashima A Chawla Manbeena M Mehta Mansi M Parikh Pankti P Chandra Pallavi P Bhave Devayani D Kumar Dhiraj D Carroll Kate S KS Singh Amit A

PLoS pathogens 20140130 1

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-sensit ...[more]

PMID: 24497832

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Project description:RationaleConversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability.ObjectivesTo improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion.MethodsSources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis.Measurements and main resultsIndividuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection.ConclusionsImplementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

Project description:Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The Mycobacterium tuberculosis iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The ideR gene is essential in M. tuberculosis and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and loxP mediated recombination in Mycobacterium smegmatis. Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene mshA results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mshA mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCEMycobacterium smegmatis is a tractable organism to study mycobacterial gene regulation. We used M. smegmatis to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene mshA is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by Mycobacterium tuberculosis to overcome temporary or local changes in iron availability during infection.

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Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

Publications

Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.

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