Research ArticleHEALTH AND MEDICINE

Noninvasive imaging and quantification of bile salt hydrolase activity: From bacteria to humans

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Science Advances  03 Feb 2021:
Vol. 7, no. 6, eaaz9857
DOI: 10.1126/sciadv.aaz9857
  • Fig. 1 Design and applications of BAL probes.

    (A) Enzymatic deconjugation mechanism of naturally occurring conjugated BAs. (B) Schematic representation of the BAL assay. The technology is based on BSH-specific uncaging of free luciferin, which is subsequently processed by the luciferase enzyme to produce bioluminescent light. Hence, the signal output is proportional to the level of BSH activity. (C) The BAL method is suitable for imaging and quantification of BSH activity in different settings, including pure in vitro enzymes, cultured bacteria, and mouse and human fecal samples, as well as noninvasive real-time imaging and quantification directly in vivo in both normal and altered physiological conditions (e.g., antibiotic/probiotic treatment). The BAL probes administered to the animals expressing luciferase (FBV + luc) via oral gavage. Immediately after gavage, the animals are imaged using a CCD camera to quantify the resulting signal as a function of time. GIT, gastrointestinal tract. (D) Structures of the BAL probes used here. The design is based on four principal BAs: two primary BAs, namely, choloyl-luciferin (CL) and chenodeoxycholoyl-luciferin (CDCL); and two secondary BAs, namely, deoxycholoyl-luciferin (DCL) and lithocholoyl-luciferin (LCL).

  • Fig. 2 Investigation of the selectivity of the BAL assay in various biological settings using bioluminescent whole-cell readout.

    (A) Normalized bioluminescent signal resulting from deconjugation of BAL probes (10 μM) incubated in the presence or absence of the choloylglycine hydrolase (CH), penicillin amidase (PA), pancreatic carboxypeptidase A (CPA) and B (CPB) enzymes (5 U/ml). (B) Normalized bioluminescent signal obtained from BAL probes incubated with L. plantarum WCFS1 and the corresponding bsh deletion mutants at 108 CFU/ml. (C) Comparison of light production from BAL probes incubated with intact conventional mouse (conv.), healthy human donor (human), germ-free (GF) mouse, and heat-inactivated (HI) conventional mouse fecal slurries (5 g/liter). In all cases, luciferase-transfected mammalian cells were used as a readout of BSH-specific deconjugation of the BAL probes. To assure that the experimental conditions did not alter the luciferase-luciferin interaction, all the data obtained for the BAL probes were normalized to the signal of the control experiment with luciferin alone (1 μM) in the same experimental conditions. Data are presented as means ± SEM. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) was calculated using a two-tailed unpaired t test. ns, not significant.

  • Fig. 3 Deconjugation level of the BAL probes in various biological settings with different levels of BSH activity.

    (A) Normalized bioluminescent signal resulting from incubation of BAL probes with different dilutions of pure CH enzyme at a concentration range of 0 to 10 U/ml. (B) Normalized bioluminescent signal resulting from incubation of BAL probes with different dilutions of L. plantarum WCFS1 slurries (0 to 1 × 108 CFU/ml). Normalized bioluminescent signal resulting from incubation of BAL probes with different dilutions of intact aqueous fecal slurries: (C) from conventional mice (0 to 20 g/liter) and (D) human (0 to 5 g/liter). In all cases, luciferase-transfected mammalian cells were used as a readout of BSH-specific deconjugation of the BAL probes. To verify that the experimental conditions did not alter the luciferase-luciferin interaction, all the data obtained for the BAL probes were normalized to the signal from luciferin alone in the same experimental conditions. Statistical significances in all data sets (P < 0.0001) were calculated using an ordinary one-way analysis of variance (ANOVA) test.

  • Fig. 4 Real-time noninvasive quantification of BSH activity in vivo using the BAL assay.

    (A) The ratio of bioluminescent signal resulting from FVB + luc mice treated with the antibiotic cocktail (AVNM) for 7 days over untreated animals. The animals received oral gavage of CL (green bar) or DCL (blue bar) probes before and after AVNM treatment (n = 5 per group). (B) The ratio of bioluminescent signal resulting from FVB + luc mice that received oral gavage of DCL probe before and after administration L. plantarum WT (BSH-high) or L. plantarum Δbsh1–4 (BSH-low) bacteria (n = 5 per group). (C) The ratio of bioluminescent signal resulting from the DCL probe administered to FVB + luc mice treated with prebiotics added in drinking water (Inulin or FOS mixture) for 20 days over untreated animals (n = 5 per group). Bar graphs represent the mean ratio of signals after and before the corresponding treatment for each animal with error bars representing ± SEM. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) calculated using a one-sample t test with a theoretical mean of 1.

  • Fig. 5 Quantification of BSH activity in clinical stool samples of patients with IBD.

    Normalized bioluminescent signal resulting from deconjugation of (A) CL probe and (B) DCL probe in feces of patients with active (n = 7) versus remission (n = 18) IBD clinical status. Bar graphs represent the mean total photon flux resulting from the corresponding probe normalized to the total photon flux from luciferin under the same conditions with error bars representing ±SEM. Statistical significance (*P < 0.05) calculated using unpaired t test.

  • Fig. 6 Noninvasive quantification of BSH activity in human and mouse GI tract.

    (A) Total photon flux resulting from the feces of mice that received AVNM antibiotic cocktail for 7 days (n = 3) versus control untreated group (n = 3). Both groups of mice received the oral gavage of 200 nM of CL probe in 200 μl of PBS probe 6 hours before feces collection and analysis. P = 0.0002, n = 9 (three samples × three replicates). (B) Total photon flux from incubation of CL probe with the feces of mice treated with AVNM antibiotic cocktail for 7 days versus control untreated group (n = 5). P < 0.0001, n = 15 (five samples × three replicates). (C) Total photon flux resulting from feces of a healthy 42-year-old female collected 12 hours after oral administration of two gelatin-based capsules containing a total of 6.8 mg of CL probe as one dose. BG, background. P = 0.0021, n = 3 (one sample × three replicates). Data are presented as means ± SEM. P values were calculated using a two-tailed unpaired t test.

Supplementary Materials

  • Supplementary Materials

    Noninvasive imaging and quantification of bile salt hydrolase activity: From bacteria to humans

    Pavlo V. Khodakivskyi, Christian L. Lauber, Aleksey Yevtodiyenko, Arkadiy A. Bazhin, Stephen Bruce, Tamar Ringel-Kulka, Yehuda Ringel, Bertrand Bétrisey, Joana Torres, Jianzhong Hu, Chieh Jason Chou, Elena A. Goun

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