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. 2022 Feb 1;23(3):1705.
doi: 10.3390/ijms23031705.

Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii

Sandrine Auger  1 , Camille Kropp  1 , Esther Borras-Nogues  1 , Wasaporn Chanput  1 , Gwenaelle Andre-Leroux  2 , Oscar Gitton-Quent  1 , Leandro Benevides  1 , Natalia Breyner  1 , Vasco Azevedo  3 , Philippe Langella  1 , Jean-Marc Chatel  1
Affiliations

Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii

Sandrine Auger et al. Int J Mol Sci. .

Abstract

The commensal bacterium Faecalibacterium prausnitzii has unique anti-inflammatory properties, at least some of which have been attributed to its production of MAM, the Microbial Anti-inflammatory Molecule. Previous phylogenetic studies of F. prausnitzii strains have revealed the existence of various phylogroups. In this work, we address the question of whether MAMs from different phylogroups display distinct anti-inflammatory properties. We first performed wide-scale identification, classification, and phylogenetic analysis of MAM-like proteins encoded in different genomes of F. prausnitzii. When combined with a gene context analysis, this approach distinguished at least 10 distinct clusters of MAMs, providing evidence for functional diversity within this protein. We then selected 11 MAMs from various clusters and evaluated their anti-inflammatory capacities in vitro. A wide range of anti-inflammatory activity was detected. MAM from the M21/2 strain had the highest inhibitory effect (96% inhibition), while MAM from reference strain A2-165 demonstrated only 56% inhibition, and MAM from strain CNCM4541 was almost inactive. These results were confirmed in vivo in murine models of acute and chronic colitis. This study provides insights into the family of MAM proteins and generates clues regarding the choice of F. prausnitzii strains as probiotics for use in targeting chronic inflammatory diseases.

Keywords: Faecalibacterium prausnitzii; microbial anti-inflammatory molecule; probiotic.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Genetic organization of the nucleotide region encoding the MAM protein in F. prausnitzii. Arrows indicate the direction of gene transcription. (A) Organization in the A2-165 strain. The putative functions of the genes are indicated. (B) Comparison of the organization in several representative strains of F. prausnitzii. Genes conserved in different genomes are indicated by the same color label.
Figure 2
Figure 2
Sequence alignment of promoter regions from mam genes. We examined 200 nucleotides upstream of the annotated translational start site, indicated by +1. The sequences of the ribosome binding site are indicated by a green frame. The sequences of the TATA box are indicated by purple frames.
Figure 3
Figure 3
Cluster map of identified full-length MAM proteins. The dataset of 108 MAM proteins was clustered using CLANS, which classified all MAM proteins into families. CLANS runs BLAST on a given set of sequences and clusters them in 3D according to their all-against-all pairwise similarity. A 2D representation was obtained by seeding sequences randomly in the arbitrary distance space. For each clan, some known member strains are indicated in parentheses in the legend. Connections with a P-value higher than 1E-22 are rendered in blue, those with a P-value between 1E-22 and 1E-33 are rendered in gray, and those with a P-value lower than 1E-33 are rendered in black.
Figure 4
Figure 4
Unrooted maximum likelihood phylogenetic tree of MAM proteins. The MAM phylogeny was reconstructed using MEGA-X (Materials and Methods). Bootstrap support values are shown.
Figure 5
Figure 5
In vitro anti-inflammatory properties of MAM proteins. Anti-inflammatory properties of the MAM proteins were evaluated using an NF-kB reporter luciferase assay. The HEK 293 cell line was transfected with an NF-kB luciferase plasmid, a pCarma1 plasmid, and a probiH1 plasmid containing MAM cDNA from different strains of F. prausnitzii. Results are expressed as % of NF-kB luciferase activity, with 100% representing the value obtained after transfection with NF-kB luciferase, Carma1, and proBiH1 EMPTY plasmids. Each plasmid was tested in triplicates (technical replicate) and experiments were performed three times (biological replicate). Results are expressed as mean of the three biological replicates.
Figure 6
Figure 6
Protective effect of MAM A2-165 or MAM M21/2 in a DNBS-induced model of acute colitis. Recombinant bacteria containing vectors with MAM from strain A2-165, MAM from strain M21/2, or proBiH1-EMPTY were administered orally to mice (n = 8/group) for 7 days prior to colitis induction by intrarectal injection of DNBS (150 mg/kg). Mice were then sacrificed 3 days after DNBS injection. (A) Daily monitoring of mouse weight; (B) Macroscopic score; (C) Immune response in MLN. Data represent mean ± SD. * p < 0.05.
Figure 7
Figure 7
Protective effect of MAM A2-165 or MAM M21/2 in a DNBS-induced model of chronic colitis Recombinant strains were also tested in a model of chronic inflammation. Mice were administered a lower dose of DNBS (100 mg/kg) two times, with a 3-week interval between doses. (A) Experimental design; (B) Daily monitoring of mouse weight; (C) Macroscopic score; (D) Immune response in MLN. Data represent mean ± SD. * p < 0.05.
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