Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease

Abstract

Background: Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined.

Methods: Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice.

Results: The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice.

Conclusions: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Keywords: CELL BIOLOGY; CROHN'S DISEASE; IBD; INFLAMMATION; INTESTINAL BACTERIA.

Figure 1

IL-8 response of human Caco2 cells to stimulation in DMEM medium with IL-1β, F. prausnitzii supernatant (SN) or LyBHI medium, and F. prausnitzii supernatant fractions F1 (20% of acetonitrile), F2 (40% acetonitrile) and F3 (80% acetonitrile). The values are expressed as the mean ± SEM in pg IL-8/mg protein. * p < 0.05 (compared to DMEM control with IL-1β in three experiments).

Figure 2

(A) MALDI TOF MS spectra (zoom scan for the m/z range 1630–1933) generated from F2′ (A) and F2 fractions (B) showing two ions at m/z 1733.93, 1833.92, only in (B) MS spectrum. (C) FTICR CID spectrum of the [M + 2H]²⁺ m/z 1073.61 precursor ion (corresponding to the ion of interest [M + H]⁺ m/z 2146.94). De novo sequencing generated a probable partial amino acid sequence from singly charged ions. The accuracy of mass determination made it possible to attribute this series unambiguously and to differentiate between the isobaric amino acids K and Q.

Figure 3

Sequence alignments between MAM protein and 7 other homologous proteins of F. prausnitzii. Sequences were identified using a BLAST search and aligned using ClustalW2 program. The protein identifiers correspond to the following F. prausnitzii strains: C7H4X2, A2-165; R6QJG8 and R6Q1X1, sp. CAG:82; D4KBR2, SL3/3; A8SAI8, M21/2; E2ZMJ5, KLE1255; D4K191 and D4K193, L2-6. The region corresponding to identified peptides Pep1-5 is shown by an arrow.

Figure 4

Homology model of MAM protein based on a GGDEF domain template. (A) Sequence alignment of MAM and GGDEF protein from M. capsulatus (Q60BX6); (B) X-ray structure of template protein (PDB entry 3ICL) showing the secondary structure elements; (C) three-dimensional model of MAM calculated with Modeller. The rms deviation on Cα positions of aligned residues is 1.1 Å. The region 49-68 corresponding to the identified peptides Pep1-5 is coloured in yellow.

Figure 5

Decrease in activation of the NF-κB pathway after transfection of MAM protein in different epithelial cells: in HEK293T in a dose-dependent manner (A), in TLR4/MD2/CD14 stably transfected HEK293T stimulated by LPS 100 ng. mL⁻¹ (B) and in intestinal cells HT29 (C). SN50 (50 μM) was used as positive control of NF-κB pathway inhibition in HEK293T. No activity of transfected MAM protein was observed on the STAT3 pathway activated by colivelin 0.1 nM (D). * p < 0.05 (compared to activation control).

Figure 6

Decrease in activation of the NF-κB pathway after co-transfection of MAM protein and IκκB in HEK293T ( * p< 0.05)

Figure 7

Subcellular location of MAM protein in HeLa cells (A, B, C, D) and co-localisation of MAM protein with Iκκβ in HEK293T cells (F, G, H, I). E and J labels correspond to staining controls in untreated cells.

Figure 8

Western blot with anti-flag antibody for MAM protein detection in small intestine enterocytes (1) and large intestine enterocytes (2) of mice fed with pILEmpty L. lactis and in small intestine enterocytes (3) and large intestine enterocytes (4) of mice fed with pILMAM L. lactis

Figure 9

Effects of intragastric administration of L. lactis bacterial suspension (pILMAM or Empty equivalent) on TNBS-induced colitis in C57BL/6 mice considering Wallace score (A), weight after induction of colitis (B) and quantification using ELISA of IL-17A (C) and INF-γ (D) in colons obtained 48 h after DNBS colitis induction (in pg/mL of total proteins). The values are expressed as the mean ± SEM (*p < 0.05 compared to L. lactis pILEmpty controls).

Figure 10

PCR detection of MAM gene on RNA extract from feces of dixenic mice colonized with Faecalibacterium prausnitzii and Escherichia coli (1), on pILMAM plasmid (2, positive control) and on RNA extract of same sample (1) traited by RNAse before RT-PCR (3, negative control).