Journal: BMC Biology
Article Title: The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction
doi: 10.1186/s12915-022-01317-z
Figure Lengend Snippet: 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 ( P app ) of PapRIV, EntF*, and EDF-analog in Caco-2 cells. Bars represent mean P app 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: LC 1 -MS 1 , reversed-phase ultra-high-performance liquid chromatography (RP-UPLC) using triple quadrupole (TQ) in MRM mode; LC 1 -MS 2 , high-resolution quadrupole time-of-flight; LC 1 -MS 3 , high-resolution quadrupole-orbitrap; LC 2 -MS 1 , 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: 10 5 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)
Article Snippet: At day 0, the placebo group was treated with the cell medium (BHI), the control group received a mixture of 3 EntF-negative E. faecium strains (NCIMB 10415; W54 and LMG S-28935 each 10 8 CFU per strain) suspended in 300 μL of BHI, while the test group received a mixture of 3 EntF-producing E. faecium strains (LMG 23236; T-110 and ATCC 8459 each 10 8 CFU/strain) suspended in 300 μL of BHI.
Techniques: In Vitro, In Vivo, MANN-WHITNEY, Permeability, Sampling, Liquid Chromatography with Mass Spectroscopy, High Performance Liquid Chromatography, Targeted Proteomics, Hydrophilic Interaction Liquid Chromatography, Mass Spectrometry, Concentration Assay