Real-time imaging of intestinal bacterial β-glucuronidase activity by hydrolysis of a fluorescent probe

Abstract

Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit βG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial βG activity in nude mice. In vitro cell-based assays showed that the detection limit is 10⁴ colony-forming units/well of βG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for βG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial βG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to deplete intestinal bacteria, the FDGlcU-based images showed that the βG activity was decreased by 8.5-fold on day 4 and then gradually increased after treatment stopped. The results suggested that FDGlcU-based imaging revealed the in vitro and in vivo activity of intestinal bacterial βG, which would facilitate pharmacodynamic studies of specific bacterial βG inhibitors in animal studies.

Figure 1

Hydrolysis of FDGlcU in cell-based assays and inhibition by the eβG inhibitor. (A) Fluorescence intensity of 0.5 μg/mL FDGlcU hydrolyzed by various amounts (CFU/well) of BL21 cells after 1-hour (red dots) or 12-hour (blue dots) incubations. Dashed lines indicate the absence of BL21 cells in the 1-hour (red line) or the 12-hour (blue line) incubation. (B) Fluorescence intensity of various concentrations of FDGlcU hydrolyzed by 10⁷ CFU/well of BL21 cells after 1-hour (●) and 12-hour (■) incubations. (☐) and (○) indicate the absence of BL21 cells. (C) Fluorescence intensity of 1 μg/mL of FDGlcU hydrolyzed by 6.25 × 10⁵ CFU/well of BL21 cells in the presence of the eβG inhibitor at various concentrations (μM) after the 12-hour incubation. The dashed line indicates the absence of the eβG inhibitor. Error bars indicate SEM.

Figure 2

In vivo optical imaging of βG activity by FDGlcU. (A) Serial whole-body images at indicated time points after oral gavage with DDW (top), FDGlcU (middle), and fluorescein (bottom). Green arrows indicate intestine and red arrows indicate bladder. (B) Total flux in abdomens of mice gavaged with FDGlcU (●) (n = 5) or fluorescein (○) (n = 5). (C) The regions of interest in different organs and tissues after oral administration of FDGlcU (n = 3) or fluorescein (n = 3) for 3 hours were analyzed with Living Image software. Error bars indicate SEM. NS, no significant difference. *P value < 0.05. **P value < 0.01. ***P value < 0.001.

Figure 3

Optical imaging of inhibition of intestinal bacterial βG by the eβG inhibitor. (A) Mice were gavaged with the eβG inhibitor (0 μg, 2 μg, or 20 μg) 4 hours before FDGlcU administration, then whole-body imaging was performed 3 hours later. Control mice were gavaged with vehicle solution. (B) The fluorescent signal from the murine abdomens of each group (n = 6) was measured. (C) Imaging of excised large intestines after whole-body imaging at the 3-hour time point. (D) The fluorescent signal from the intestines of each group (n = 6) was measured. Error bars indicate SEM. **P value < 0.01. ***P value < 0.001.

Figure 4

Reduction of intestinal bacterial βG by an antibiotic treatment. Upper panel: Experimental schedule for a 4-day antibiotic treatment and whole-body imaging by the IVIS^® imaging system (arrow). Whole-body imaging was performed every 2 days. Antibiotics were orally administered twice daily for 4 days. Middle panel: Imaging of mice at day 0, 2, 4, 6, and 8. Lower panel: Total flux in abdomens of mice (n = 5). Error bars indicate SEM. *P value < 0.05. ***P value < 0.001.