Identification of a muropeptide precursor transporter from gut microbiota and its role in preventing intestinal inflammation

Abstract

The gut microbiota is a considerable source of biologically active compounds that can promote intestinal homeostasis and improve immune responses. Here, we used large expression libraries of cloned metagenomic DNA to identify compounds able to sustain an anti-inflammatory reaction on host cells. Starting with a screen for NF-κB activation, we have identified overlapping clones harbouring a heterodimeric ATP-binding cassette (ABC)-transporter from a Firmicutes. Extensive purification of the clone's supernatant demonstrates that the ABC-transporter allows for the efficient extracellular accumulation of three muropeptide precursor, with anti-inflammatory properties. They induce IL-10 secretion from human monocyte-derived dendritic cells and proved effective in reducing AIEC LF82 epithelial damage and IL-8 secretion in human intestinal resections. In addition, treatment with supernatants containing the muropeptide precursor reduces body weight loss and improves histological parameters in Dextran Sulfate Sodium (DSS)-treated mice. Until now, the source of peptidoglycan fragments was shown to come from the natural turnover of the peptidoglycan layer by endogenous peptidoglycan hydrolases. This is a report showing an ABC-transporter as a natural source of secreted muropeptide precursor and as an indirect player in epithelial barrier strengthening. The mechanism described here might represent an important component of the host immune homeostasis.

Keywords: ABC-transporter; inflammation; microbiota; muropeptide precursor.

Fig. 1.

(A) Reads sequences of 169 human gut metagenomic samples mapped to the F4 sequence with a focus on the portion of the fosmid insert which includes the split region between the two MAGs (GUT_GENOME259254 and GUT_GENOME139568). The red arrow indicates the separation point between the two MAGs. (B) Distance tree view of the top 100 match using blast pairwise alignment with the ABC heterodimer as query.

Fig. 2.

The activity of F4 clone is mediated by the ABC heterodimeric transporter by a MyD88-independent pathway. NF-κB activity in (A–C) HEK Null- or (D and E) THP-1-NF-κB/SEAP reporter cells induced by (A) supernatant of F4 clone and of the transposed F4D5 clone; (B) supernatants from E. coli Epi300 transformed with single (p-ORF3 and p-ORF4) or both ORFs of the ABC heterodimer (pBAD-ABC in the Figure); (C) supernatants from F4D5:ABC (complemented clone), pBAD-ABC (ABC-transporter cloned in pBAD30 vector), and F4D5-pBAD (transposed clone transformed with empty pBAD30 vector). (D) NF-κB activity induced on THP-1 and (E) THP-1 MyD88^−/− NF-κB/SEAP reporter cells by LPS (10 ng/mL), TNF (10 ng/mL), Flagellin (100 ng/mL), F4, F4D5, and Epi300 supernatants. Supernatants were all used at 10% v/v. Data (A–C) are pooled from biological triplicates, and error bars represent SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). Data (D and E) are shown as mean ± SD of triplicate measurements of a representative of two independent experiments.

Fig. 3.

F4D5:ABC-derived active fractions signal through NOD1 and NOD2 receptors and induce IL-10 secretion from moDC. NF-κB activity (A and B) induced by F13 and F24 fractions in (A) HEK-NOD1 NF-κB-SEAP and (B) HEK-NOD2 NF-κB-SEAP reporter cells. Cytokine secretion (C) IL-10 and (D) IL-8 by human moDC following stimulation with F13 and F24 fractions. TriDAP and MDP were used at 0.01 to 0.1 to 1 µg/mL. TNF (10 ng/mL) and PHA (3 µg/mL) were used as positive controls. F13 and F24 fractions were from purification of F4D5:ABC (blue bars) and F4D5-pBAD (blue-striped bars) supernatants. Results are shown as mean ± SD of triplicate measurements of a representative of two independent experiments.

Fig. 4.

The muropeptide precursor M-TriDAP-MP induces IL-10 secretion from human moDC. (A) IL-10 and (B) TNF secretion by human moDC stimulated for 18 h with MDP (gray bars), M-TriDAP-MP (orange bars), M-TriDAP (green bars), and TriDAP (blue bars) at 1 to 10 to 100 mM. Dataare pooled from biological triplicates. Error bars represent SEM (*P < 0.05; ns, not significant) as calculated by the paired t test.

Fig. 5.

Muropeptide precursor present in F4D5:ABC extract protect intestinal epithelium from E. coli LF82-induced inflammation. Human ileal resections (mounted on Ussing chambers) were treated with culture media (control, white bars), 1 × 10⁹ CFU E. coli LF82 (orange bars), F4D5-pBAD (blue-striped bars), and F4D5:ABC (blue bars) supernatants submitted to partial purification (ultrafiltration using 10-kDa cutoff filters followed by two cycles of acetone precipitation and a C18 solid phase extraction). (A) Transepithelial resistance (TEER), (B) IL-8 quantification by AlphaLisa, and (C) histology. One supernatant from F4D5-pBAD-treated resection lost during manipulation. Experiments are biological quadruplicates, and error bars represent SEM (*P < 0.05) as calculated by the unpaired t test.

Fig. 6.

Anti-inflammatory properties of F4D5:ABC supernatant in the DSS model of colitis in C57bl/6 mice. (A) Design of the study: Mice were gavaged daily with 200 µL of either vehicle, F4D5-pBAD supernatant, F4D5:ABC supernatant, or Pentasa granules, starting 2 d before the beginning of DSS treatment in the drinking water. All mice were killed at day 12 postbeginning of DSS treatment. (B) Loss of body weight was monitored all along the study. (C) Ratio weight/size (mg/cm) of the colon of mice at killing. (D) Histological score at killing. Data represent the mean of 10 mice/group. Errors bars represent the SEM (*P < 0.05 and **P < 0.01).

Fig. 7.

Homolog genes to the F4-derived ABC-transporter fail to stimulate NF-κB activity in HEK-Null-NF-κB cells and IL-10 secretion from human moDC. (A) NF-κB activity on HEK-Null reporter cells and (B) IL-10 secretion from human moDC induced by supernatants of E. coli Epi300 expressing ABC-Am and ABC-Hb heterodimeric transporters. Supernatant of pBAD-ABC was used as positive control. Supernatants were derived from cultures performed under identical conditions and recovered at similar OD600. Supernatants were used at 10% v/v. Experiments are biological duplicates, and error bars represent SD as calculated by the unpaired t test.

Fig. 8.

F4D5:ABC transporter modelling results. (A) Structure homology comparison between the F4D5:ABC transporter prediction template and 6quz.1 template using the SWISS-Model system. (B) 3D prediction overlaps between F4D5:ABC (red) and 6quz.1 (blue). (C) Output data including coordinates, target-template alignment, modelling log, and quality estimation information. (D) SWISS-Model template Modelling: in pink the chain A and in blue the chain B. (E) AlphaFold2 Modelling (AF2) with in red the chain A and in blue the chain B. (F) Matching sequences using Matchmaker sequence analysis tools from ChimeraX 1.3. The zoomed D-lock regions of the 2 structures are shown. The arrows indicate the D-lock regions.