Fig. 1
In vitro formation and in vivo presence of the EntF* metabolite. a Sequences of the enterocin induction factor pro-peptide, mature quorum sensing peptide EntF, and its metabolite EntF*. b In vitro formation rate of EntF* from EntF in colon (n = 7) and feces (n = 4) homogenates. Bars represent the mean formation rate ± SEM from independent experiments. Statistically significant differences were determined by a Mann-Whitney U test with indicated p-values. c Apparent permeability coefficients (Papp) of PapRIV, EntF*, and EDF-analog in Caco-2 cells. Bars represent mean Papp values ± SEM (n = 6 independent experiments); the shaded area represents the limit of detection. d Flow chart displaying the experimental design stages, from serum sampling to peptide detection and further confirmation of EntF* presence in vivo. Different LC-MS methods: LC1-MS1, reversed-phase ultra-high-performance liquid chromatography (RP-UPLC) using triple quadrupole (TQ) in MRM mode; LC1-MS2, high-resolution quadrupole time-of-flight; LC1-MS3, high-resolution quadrupole-orbitrap; LC2-MS1, HILIC-amide UPLC using TQ in MRM mode. qPCR was performed on feces sample of mice from the same set to demonstrate the presence of EntF-encoding DNA sequences from E. faecium. e Chromatographic profiles of (1) negative serum sample, (2) positive serum sample, (3) serum sample from EntF*-treated mice. Chromatographic profiles were obtained using RP-UPLC with detection by electrospray ionization mass spectrometry (ESI-MS) using TQ in MRM mode (m/z = 865 ➔ 202.08 + 315.17). f Chromatographic profiles of (1) negative serum sample, (2) positive serum sample, (3) serum sample from EntF*-treated mice. Chromatographic profiles were obtained using HILIC amide UPLC with detection by ESI-MS using TQ in MRM mode (m/z = 865 ➔ 202.08 + 315.17). g Isotopic distribution of the double charged EntF* measured in a positive serum sample using RP-UPLC with detection by ESI-MS using quadrupole-orbitrap. h High-resolution tandem mass spectrum of EntF* with characteristic fragments, using RP-UPLC with detection by Q-TOF. i In vivo presence of EntF* in gnotobiotic mice treated with EntF-producing bacterial strains. Number of EntF DNA copies per gram of feces measured four days after treatment with placebo (300 μL BHI medium) (limit of detection: 105 copies/g) (left). EntF* concentration in colon content. No EntF* was detected in the placebo group (the red line indicates the limit of detection) (middle). EntF* concentration in serum content. No EntF* was detected in the placebo group (the red line indicates the limit of detection) (right)