Fig 1.
General strategy for imaging of NTR activity with Nitroreductase Caged Luciferin (NCL) probe.
Fig 2.
Evaluation of NTR-specific uncaging of NCL probe.
(A) NCL (20 μM) uncaging by NTR (0.5 μg mL-1) in the presence of NADH (100 μM) was inhibited by dicoumarol (0 to 200 μM). (B) Total luminescence over 30 min from luciferin or NCL (0.25–5 μM) with NADH, luciferase, and NTR, compared with the control (no NTR), *P < 0.001.
Fig 3.
Light production in E. coli by NTR-mediated uncaging of NCL.
(A). Light output from NCL (300 μM) after 2 h incubation in luciferase-expressing E. coli (+ luc gene) is significantly higher than in NTR mutant (NTR KO luc+) (*P < 0.001) and in wild type (- luc gene). The dashed line indicates the background. (B). Overlay of a photographic image and bioluminescence from the assay described. (C). Bioluminescence from 100 μM NCL probe and luciferin incubated with various concentrations of luciferase expressing E.coli AB1157 (102–108 bacteria mL-1) for 10 min before imaging. (D). Bioluminescence from luciferase expressing E.coli luc+ (108 bacteria mL-1) incubated with various concentrations of NCL or luciferin (1–250 μM) for 10 min before imaging.
Fig 4.
In vivo activation of NCL probe by luciferase and nitroredictase expressing E.coli in a mouse model of thigh infection.
(A). Luminescence over 4 h from E. coli luc+ infected quadriceps (5 × 104–5 × 107 bacteria) after IP injection of 0.8 mg NCL probe (200 μL of 10 mM solution in PBS). (B). Luminescence over 4 h from E. coli luc+ infected quadriceps (5 × 107 bacteria) following IP injection of 0.8 mg of probe or 0.63 mg of luciferin (200 μL of 10 mM solution in PBS). (C). Luminescence imaging of mice over 24 h bearing 5 × 107 bacteria, treated with various (0.08, 0.8 and 1.6 mg) NCL probe concentrations (200 μL of 1, 10 and 20 mM solutions of NCL in PBS). As a positive control, mice were injected with equal amounts of E. coli MG1655 expressing lux luciferase that doesn't require exogenous substrate for light production [44]. The signal was collected over 24 h, n = 3 per group.
Fig 5.
Imaging of NTR activity in cells and in in vivo cancer model with NCL.
(A) Concentration-dependent uncaging of NCL in MDA-MB-231 NTR+luc+ cancer cells in comparison with luciferin. (B) Selectivity of NTR imaging by NCL in the same cells in comparison with NTR-luc+ cells. The dashed line indicates background (cells only), *P = 0.0001. (C) In vivo imaging of NTR activity in subcutaneous NTR+ and NTR- xenografts (n = 5). Total luminescence over 1 h from IP injection of luciferin (1.5 mg) and NCL (1.9 mg). d) Representative images of mice 15 min post injection of luciferin or NCL.