Article Figures & Data
Figures
- Fig. 1.
F. prausnitzii metabolizes tacrolimus. (A) F. prausnitzii (OD600 2.6) cultured in YCFA media was incubated with tacrolimus (100 μg/ml) anaerobically at 37°C for 48 hours. The mixture was analyzed by using HPLC/UV. (B) Time profiles of tacrolimus disappearance and M1 appearance upon anaerobic incubation of tacrolimus (100 μg/ml) with F. prausnitzii.
- Fig. 2.
Chemical structures of tacrolimus and F. prausnitzii–derived metabolite M1. M1 structure was identified using mass spectrometry and nuclear magnetic resonance spectroscopy.
- Fig. 3.
M1 is less potent than tacrolimus as an immunosuppressant and antifungal agent. (A) Immunosuppressant activities of tacrolimus and M1 were examined in PBMCs by measuring cell proliferation after treatment with a T-lymphocyte mitogen in the presence of tacrolimus or M1. (B) Antifungal activities of tacrolimus and M1 were examined using Malassezia sympodialis. The yeast was inoculated on a modified Dixon agar plate. After 1 hour incubation, an aliquot of tacrolimus or M1 at different concentrations was placed on the plate, as shown in the left panel, and incubated at 37°C for 2 days.
- Fig. 4.
Multiple commensal gut bacteria convert tacrolimus to M1. (A) Representative chromatograms of bacteria incubated with tacrolimus. M1 nonproducer (Bifidobacterium longum) or producer (Clostridium aldenense, Clostridium citroniae, and Erysipelotrichaceae sp.) cultured overnight in YCFA media was incubated with tacrolimus (100 μg/ml) anaerobically at 37°C for 48 hours. The mixture was analyzed by using HPLC/UV at 210 nm. (B) Mouse or human hepatic microsomes [(HMs); 3 mg microsomal protein/ml] were incubated with tacrolimus (100 μg/ml) at 37°C for 2 hours aerobically. The mixture was analyzed by using HPLC/UV.
- Fig. 5.
Human gut microbiota convert tacrolimus to M1. Tacrolimus (100 μg/ml) was incubated anaerobically with human stool samples from two different subjects (100 mg wet weight/ml) for 48 hours at 37°C. A separate set of samples was boiled for 10 minutes before incubation with tacrolimus. The incubation mixtures were analyzed by HPLC/UV.
- Fig. 6.
M1 formation by small intestinal bacteria may be extensive. (A) Tacrolimus (10 μg/ml) was incubated anaerobically with F. prausnitzii (6.3 × 107 cells/ml) for varying amounts of time. (B) Tacrolimus (10 μg/ml) was incubated with varying amount of F. parusnitzii in PBS at 37°C for 2 hours. (C) Tacrolimus at varying concentrations was incubated with F. prausnitzii (6.3 × 107 cells/ml) for 1 hour. M1 concentrations in the reaction mixtures were analyzed by liquid chromatography–MS/MS.
Tables
Order Bacterium OD600 M1 Production Detected Bifidobacteriales Bifidobacterium longum 1.8 No Bacteroidales Bacteroides cellulosilyticus 0.6 Yesa Bacteroides finegoldii 3.4 Yesa Bacteroides ovatus 4.2 Yesa Parabacteroides merdae 2.7 Yesa Parabacteroides johnsonii 3.6 Yesa Parabacteroides goldsteinii 3.3 Yesa Clostridiales Ruminococcaceae sp. 0.5 Yes Clostridium innocuum 3.4 Yes Anaerostipes sp. 2.7 Yes Dorea formicigenerans 2.4 Yes Clostridium clostridioforme 3.0 Yes Clostridium hathewayi 2.6 Yes Blautia sp. 4.7 Yes Clostridium aldenense 1.4 Yes Clostridium symbiosum 2.5 Yes Clostridium citroniae 1.7 Yes Coprococcus sp. 2.4 Yes Clostridium bolteae 3.6 Yes Clostridium cadaveris 1.4 Yes Ruminococcus gnavus 3.4 Yes Erysipelotrichales Erysipelotrichaceae sp. 3.8 Yes ↵a M1 production observed only when using sensitive HPLC-MS/MS for detection.
Patient Age Gender Post-Transplant Day Tacrolimus Oral Dosea Fecal Abundance Baseline Level in Stool Samples M1 Production upon Tacrolimus Incubation F. prausnitzii Clostridiales Tacrolimus M1 yr mg/day % % ng/mg stool ng/mg stool ng/mg stool 1 45 Female 31 9 46 86 0.88 0.38 5.1 2 56 Male 18 3 39 89 BQLb BQLb 3.5 3 61 Male 20 5 32 71 0.63 BQLb 4.5 4 59 Female 12 6 27 76 0.71 0.12 2.9 5 50 Male 32 10 26 79 0.37 0.41 6.4 6 52 Female 28 6 ND 15 0.29 BQLb 3.5 7 57 Male 15 3 ND 44 0.85 BQLb 4.1 8 71 Male 18 4 ND 95 BQLb 0.60 7.1 9 25 Male 27 4 ND 74 0.49 BQLb 12.6 10 52 Male 32 6 ND 95 0.14 BQLb 11.0
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - Different strains of F. prausnitzii (i.e., A2-165, ATCC 27766 and ATCC 27768) were cultured overnight in YCFA media, followed by anaerobic incubation with tacrolimus (100 μg/ml) at 37°C for 48 h
Supplemental Figure 2 - MS2 spectra of tacrolimus and M1 (IT-TOF)
Supplemental Figure 3 - MS2 fragments of tacrolimus and M1 obtained in IT-TOF and QTOF systems
Supplemental Figure 4 - Tacrolimus (50 mg) was incubated with F. prausnitzii A2-165 (OD ~2; 500 ml) for 96 h, followed by ethyl acetate extraction of the mixture
Supplemental Figure 5 - IR spectra of a) tacrolimus, b) M1, c) overlay of tacrolimus (red) and M1 (blue) spectra
Supplemental Figure 6 - 1H NMR spectrum (900 MHz, CDCl3) of M1
Supplemental Figure 7 - DEPTQ spectrum (226 MHz, CDCl3) of M1
Supplemental Figure 8 - COSY spectrum (900 MHz, CDCl3) of M1
Supplemental Figure 9 - HSQC spectrum (900 MHz, CDCl3) of M1
Supplemental Figure 10 - HMBC spectrum (900 MHz, CDCl3) of M1
Supplemental Figure 11 - 1H-13C HSQC-TOCSY spectrum (900 MHz, CDCl3, 90 ms mixing time) of M1
Supplemental Figure 12 - Key 2D NMR correlations of M1 (Isomer I)
Supplemental Figure 13 - Expansions of the DEPTQ spectra (226 MHz, CDCl3) of M1 (three major isomers assigned) and tacrolimus (two isomers)
Supplemental Figure 14 - Bacteria cultured overnight in YCFA media were incubated with tacrolimus (100μg/ml) or vehicle control anaerobically at 37°C for 48 h
Supplemental Figure 15 - Tacrolimus (10 μg/ml) was incubated with F. prausnitzii (6.28×107 CFU/ml) anaerobically at 37°C up to 24 h
Supplemental Table 1 - List of commensal gut bacteria obtained from Biodefense and Emerging Infections (BEI) Research Resources Repository
Supplemental Table 2 - Demographic information of kidney transplant patients whose stool samples were analyzed
Supplemental Table 3 - NMR spectroscopic data of M1 (Isomer I), CDCl3a
Supplemental Table 4 - NMR Spectroscopic Data of M1 (Isomer II), CDCl3a
- Supplemental Data -
In this issue
Tacrolimus Metabolism by Gut Bacteria
