ABSTRACT: MAM (Microbial-Anti-Inflammatory Molecule) is a 14,5 kDa protein that is one of the best-known effector molecules with anti-inflammatory properties in Faecalibacterium duncaniae, a critical species in the human gut microbiota. Despite its importance, MAM function and molecular features remain poorly understood. Therefore, in this study, we sought to elucidate MAM's physiological importance. We investigated MAM localization using mass-spectrometry, immunogold labeling, and peptide secretion dynamics during bacterial growth. Bioinformatic analysis and microscopy further supported our understanding of MAM protein domain organization, interactions, and putative macromolecular assembly. Our results identified MAM as the most abundant protein in the cell envelope, and the second most abundant one in the overall proteome of F. duncaniae, with confirmed localization at the bacterial surface through immunogold labeling. Bioinformatics analysis highlights that MAM could comprise an N-terminal 21 residue leader peptide whose sequence contains all the motifs to be recognized and cleaved by a peptidase, followed by a 114 residue cargo peptide. Appropriately, in silico modeling suggests that the MAM leader peptide nicely accommodates the peptidase-domain-containing ABC transporter (PCAT) that is adjacent to MAM in the genome of F. duncaniae. After N-terminal excision, the cargo protein could be transported to the cell envelope via this PCAT, where it could assemble into a hexameric, pore-like structure, as revealed by AlphaFold3 modeling. Electron microscopy images of In situ F.duncaniae cells revealed a highly ordered lattice with repetitive units of hexamers. Moreover, an enriched fraction of MAM protein was obtained with in vitro LiCl extraction, exhibiting the same organizational pattern as the predicted hexameric organization. These findings provide the first comprehensive characterization and molecular export mechanisms of MAM as a key protein component of the F. duncaniae cell envelope, suggesting roles in cell structure, permeability, and communication with the host environment. It reveals a novel, lattice-like organization on the F. duncaniae cell envelope that may play a critical role in maintaining bacteria structure. This work introduces a novel discussion about the unique organization of the F.duncaniae cell envelope, having MAM as a key component for the bacteria, supporting the understanding of the unique biology of F. duncaniae and its potential as a next-generation probiotic or Live Biotherapeutics
Céline Henry 
