Session: Session 5

Characterization of nanofractionation of intestinal microbiota using metaproteomics in patients with Crohn's disease

Céline HENRY¹, Maxime LONG^1,2, Romain SAUSSET², Catherine JUSTE², Marianne DE-PAEPE²

¹Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, PAPPSO, Jouy-en-Josas, France
²Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France

Introduction : Recent studies have indicated differences in the composition of the intestinal virome associated with chronic inflammatory bowel diseases (IBD), including Crohn's disease. The role of membrane vesicles secreted by both bacteria and human cells in the context of intestinal inflammation has also been recognized. The nanometric fraction of the microbiota, specifically extracellular vesicles (Evs) and viruses, is believed to play a crucial role in intestinal inflammation. However, limited research has been conducted on this subject, partly due to technical challenges associated with the small size and diverse nature of viruses and Evs.

We have developed a technique for separating Evs and viruses from the intestinal microbiota based on their density and enhanced techniques for quantifying viruses and Evs in stools.

Methods : EVs were characterized in the intestinal microbiota of both healthy subjects and Crohn's disease patients. Metaproteomics has been conducted on a timsTOF Pro (Bruker) coupled to nanoElute chromatography. Iterative mode was used with databases IGC 10.4, Homo sapiens Swiss-Prot-TrEMBL and contaminants. The grouping of proteins into protein subgroups was done using i2MassChroQ (http ;//pappso.inrae.fr). To compare protein number and abundance between clinical groups, we implemented Wilcoxon tests with Benjamini–Hochberg stepwise adjustment for multiple pairwise comparisons between clinical groups.

Results : We identified more than 22 000 subgroups of proteins corresponding to 100 000 peptides. Among them, 900 are human proteins and most of them are part of exosome, confirming the quality of the Evs preparation. Human proteins are also more abundant in Crohn's disease patient.

Conclusion : In summary, the study employs advanced techniques to investigate the role of EVs and viruses in Crohn's disease, shedding light on potential mechanisms underlying microbiome disturbances in the hope of informing future therapeutic approaches.