Fig. 6: Binding effect of GV-971 on Rib adhesion domain and its inhibitory effect on L.m. adhesion, lactate-SAA pathway, and Th1 activation.
a Protein structure of the Rib domain from L.m., which we named Rib_gene1738. The black bracket demonstrates the protein structure of one representative Rib repeated domain, of which the repeated GIANLDKL residues were present (also see Supplementary Fig. S6a). b Detailed interaction of 2 to 6 sugar units of GV-971 (solid sticks) with repeated GIANLDKL residues of the Rib_gene1738 (color ribbon) protein from Ribhigh-L.m. The binding sites are shown. Hydrogen bonds and salt bridges are depicted as yellow and purple dashed lines, respectively. Different oligomers of GV-971 are shown as sticks. The repeated GIANLDKL residues are displayed in green, orange, and blue ribbons. c Type of connection between GV-971 and GIANLDKL residues of the Rib_gene1738 protein (color ribbon) from Ribhigh-L.m. d, e Molecular dynamics simulations of 5 sugar units of GV-971 interacting with repeated GIANLDKL residues of the Rib_gene1738 protein (color ribbon) from Ribhigh-L.m. using AMBER at total 100 nanoseconds (ns) simulation. RMSD values through 100 ns (d) and interacting forces including hydrogen bonds and salt bridges are depicted as yellow and purple dashed lines, respectively (e). f SPR sensor grams of the Rib_gene2117-GV-971 interaction. Concentrations of GV-971 containing 2–10 sugar units (from bottom to top) are 0.5, 1, 2, 5, and 10 mM, respectively. KD value is 10−5 M. g The effect of GV-971 of two different doses (200 μg/mL and 400 μg/mL, final concentration) on the adhesion of Ribhigh-L.m. to HT29 cells at 2-h incubation time. The numbers of bacteria per HT29 cells were calculated in 4 to 5 separate areas for each well and replicated 3 times. Data are represented as mean ± SD. ***P < 0.001 by Student’s t-test, n = 15. h The effect of GV-971 of two different doses (200 μg/mL and 400 μg/mL, final concentration) on GPR81-NF-κB signaling and SAA levels of HT29 cells in adhesion assays in g. s, short exposure; l, long exposure. i The effect of 100 mg/kg GV-971 on the relative abundance of the 41-week-age Tg mice gut microbiota species L.m. Data are represented as mean ± SEM. *P < 0.05 by Student’s t-test, n = 5–7. Tg: n = 7, 3 M + 4 F; Tg+GV-971: n = 5, 3 M + 2 F. M, male mice; F, female mice. j The relative abundance of fecal L.m. in the WT recipient mouse that received 40-week-age Tg mouse feces treated or not treated by 100 mg/kg GV-971. Data are represented as mean ± SEM. **P < 0.01 by Student’s t-test, n = 7–8. k Effect of 100 mg/kg GV-971 on fecal lactate levels of the 41-week-age Tg mice feces detected by targeted metabolomics. Data are represented as mean ± SD. *P < 0.05 by Student’s t-test, n = 8–10. l The relative abundance of fecal lactate in the WT recipient mouse that received 40-week-age Tg mouse feces treated or not treated by 200 mg/kg GV-971. The levels of lactate were detected by targeted metabolomics. Data are represented as mean ± SEM, n = 5–8. m Effect of one-month treatment of 100 mg/kg GV-971 on Ileum SAA levels of 9-month-old Tg mice. Data are represented as mean ± SEM, n = 7. Tg: n = 7, 7 M; Tg+GV-971: n = 7, 5 M + 2 F. M, male mice; F, female mice. n The relative abundance of ileum SAA in the WT recipient mouse that received 40-week-age Tg mouse feces treated or not treated by 200 mg/kg GV-971. Data are represented as mean ± SEM. *P < 0.05 by Student’s t test, n = 7. o Effect of 100 mg/kg GV-971 on blood SAA levels of the 41-week-age Tg mice. The levels of SAA were determined by ELISA. Data are represented as mean ± SEM, n = 7–8. Tg: n = 8, 4 M + 4 F; Tg + GV-971: n = 7, 4 M + 3 F. M, male mice; F, female mice. p Effect of 100 mg/kg GV-971 on blood Th1 cells of the 41-week-age Tg mice. Data are represented as mean ± SEM. ***P < 0.001 by Student’s t-test, n = 7. Tg: n = 7, 7 M; Tg+GV-971: n = 7, 5 M + 2 F. M, male mice; F, female mice. All recipient mice in FMT assays are male mice.
