Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection
Figure 2
Mrx1 catalyzes specific equilibration between mycothiol redox system and roGFP2 in vitro and in vivo.
(A) Pre-reduced roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) were exposed to 50 µM of MSSM for 10 min and ratiometric sensor response was measured. (B) Pre-reduced Mrx1-roGFP2 was treated with 1 µM of MSSM, GSSG, cystine (Cys2) or 2-hydroxyethyl disulfide (HED) and ratiometric sensor response was measured at various time points. (C) Molecular mechanism showing the reduction of oxidized Mrx1-roGFP2 by MSH/Mtr/NADPH pathway. (D) Oxidized Mrx1-roGFP2 was added as a substrate to the MSH/Mtr/NADPH redox pathway and ratiometric sensor response was measured over time. A control reaction in the absence of MSH was performed in parallel. (E) Reduction of oxidized roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) by MSH/Mtr/NADPH redox pathway. Maximum ratio change after 150 min of incubation with MSH/Mtr/NADPH reaction mixture is shown. (F) Mrx1-roGFP2 is extremely sensitive towards small changes in OxDMSH. Reduced uncoupled roGFP2 and Mrx1-roGFP2 proteins were incubated with mycothiol solutions (1 mM total) containing increasing fractions of MSSM for a maximum of 30 sec and ratiometric sensor response was measured. Note that the response of Mrx1-roGFP2 becomes exceedingly linear in the window between 10% to 90% oxidation, suggesting that the biosensor can effectively measure changes in EMSH within this range of probe oxidation. (G) Excitation spectra of Msm expressing Mrx1-roGFP2 upon treatment with 0.4 mM of diamide (oxidant) or 10 mM of DTT (reductant) for 5 min. (H) Msm expressing Mrx1-roGFP2 was either left untreated (control) or exposed to 50 µM dequalinium, cisplatin and 5-methoxyindole-2-carboxylic acid (MICA) and ratiometric sensor response was measured after 2 h and 24 h post-exposure. p-values shown in the panel were calculated by comparing untreated group and dequalinium treated group. (I) Percentage of OxDMrx1-roGFP2 in exponentially grown Msm, MsmΔmshA, MsmΔmshD, and MsmΔsigH was calculated (see Materials and Methods for mathematical definition). Note that biosensor is completely oxidized (∼95%) in the absence of MSH reducing system in MsmΔmshA. p-values shown in the panel were calculated by independently comparing MsmΔmshA and MsmΔmshD groups with the Msm group. Error bars represent standard deviations from the mean. * p<0.01, ** p<0.001. Data shown is the representative of at least three independent experiments.