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.

EGFR monoclonal antibody panitumumab inhibits chronic proliferative cholangitis by downregulating EGFR

“…Furthermore, Pani was shown to inhibit the BrdU-positive rate and activity of β-G. β-G is a potent tumor marker for cancer diagnosis and pro-drug therapies, and its elevated level in tumors is considered to be due to tumor overexpression and release from tumor tissues or tumor-infiltrating immune cells (35). Bacterial β-G, extensively present in intestinal microflora, is also involved in metabolism and detoxification in mammals, and the decreased activity of β-G induced by Pani monoclonal antibody in the present study may refer to relieved CPC (36). The Pani monoclonal antibody reduces the extent of mTOR phosphorylation by inhibiting the gene expression of EGFR, thus inhibiting CPC lesions.…”

Sex, gut microbiome, and cardiovascular disease risk

“…Gut bacteria may facilitate the reabsorption of conjugated estrogens, as antibiotic administration has been associated with a 60-fold rise in conjugated estrogen excretion in feces [94, 95]. Bacterial beta-glucuronidase is the primary enzyme involved in deconjugating estrogens for reabsorption in the intestines [96], and the genes encoding this protein are primarily found in the Firmicutes phylum [97, 98]. Though levels of Firmicutes appear to be influenced by body weight, women may harbor higher intestinal Firmicutes compared to men irrespective of BMI [35].…”

Vancomycin Relieves Mycophenolate Mofetil-Induced Gastrointestinal Toxicity by Eliminating Gut Bacterial β-Glucuronidase Activity

“…We performed ex vivo tissue imaging of intestinal bacterial GUS activity through quantification of fluorescence following hydrolysis of fluorescein di-b-D-glucuronide (FDGlcU, Sigma-Aldrich) to fluorescein (65). Three hours prior to imaging, each animal was gavaged with FDGlcU (7.3 μmol/kg).…”

“…based upon activation of a fluorescent substrate after GUS hydrolysis(39), demonstrated an increase in overall bacterial GUS activity in the proximal colon, but not the cecum, of MMF-treated mice as compared to controls that was abolished by treatment with vancomycin. Evaluation of bacterial populations in the proximal colon specifically may confirm the identity of the bacteria that are responsible for MPAG catabolism and the liberation of free MPA.We assessed the effect of a vancomycin-induced reduction in bacterial GUS activity on the hydrolysis of MPAG to MPA and GA in the GI tract of MMF-treated mice using mass spectrometry.…”