Insights into the proteome of next-generation probiotic Faecalibacterium duncaniae A2-165 through label-free proteomics approach

Faecalibacterium duncaniae A2-165 is a rod-shaped, non-motile, and Extremely Sensitive to Oxygen microorganism, belonging to one of the most abundant genera in the human gut microbiome. A decreased abundance of Faecalibacterium species is correlated with Inflammatory Bowel Diseases (IBDs), highlighting this genus as a marker of gut health and a promising Next-Generation Probiotic. While the anti-inflammatory effects of F. duncaniae A2-165 are well known, current studies lack a global protein-level understanding of this species’ metabolism. To address this issue, this study investigated the proteome of F. duncaniae A2-165 during the stationary phase using a label-free LC-MS/MS proteomics approach. We quantified 1,280 proteins in total, corresponding to 44.7% of the in silico predicted proteome, with a clear distinction between insoluble and soluble protein abundances. The subcellular localization predictions for the quantified proteins identified 802 cytoplasmic proteins, 265 membrane proteins, six extracellular proteins, eight cell wall proteins, and 199 proteins with unknown localization. Functional analysis of the differentially abundant proteins between insoluble and soluble fractions showed a predominance of transporter proteins in the insoluble fraction. At the same time, the metabolism of amino acids, carbohydrates, and nucleotides was predominant in the soluble fraction. Further analysis of enriched pathways for each fraction showed that energy metabolism, carbon cycle, and amino acid metabolism were enriched in the soluble fraction. In contrast, ABC transporters, quorum sensing, and oxidative phosphorylation were enriched in the insoluble fraction. We identified proteins associated with anti-inflammatory effects, notably the key butyrate-production protein ButCoAT, the MAM protein and its ABC transporter, and shikimate pathway enzymes. This study characterized, for the first time, the F. duncaniae A2-165 proteome in the stationary phase, profiling the subproteomes of soluble and insoluble fractions and identifying key proteins involved in F. duncaniae A2-165 metabolism at the protein level. The results presented here could provide new insights into the study of F. duncaniae A2-165.