Influence of commensal bacteria on the proteolytic and antigenic profiles of INFOGEST-like digested wheat gliadin

Celiac disease (CeD) is a chronic autoimmune enteropathy developed by genetically predisposed individuals when exposed to gluten. Gluten gliadins, along with gut microbiota, may influence CeD onset and progression through mechanisms that remain unclear.

Gliadin-degrading bacterial isolates obtained from CeD patients, and their 1st-degree relatives' stool and blood were identified (Bacillus tropicus, Enterococcus faecalis, Micrococcus sp., Cronobacter sakazakii, Pseudomonas aeruginosa, and Serratia marcescens) and used in an INFOGEST-like protocol to simulate gliadin digestion after 4 h (digested gliadin, d-gliadin). The d-gliadin digesta were analyzed by fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), fluorescence spectroscopy, and polyclonal and monoclonal (R5 and G12) enzyme-linked immunosorbent assays (ELISA).

In the absence of the bacterial isolates, gliadin is poorly digested and self-assembles within 1 day into intermediate and large protein oligomers/aggregates, enriched in β-sheet structure (FTIR amide I band between 1,600 and 1,700 cm^-1) and able to bind thioflavin T and Congo red. Conversely, in the presence of the bacterial isolates, gliadin is further digested, leading to an increase in protein fragments. After 4 h, the P. aeruginosa, C. sakazakii, and B. tropicus d-gliadin digesta presented a mixture of d-gliadin peptides and aggregates that showed higher antigenicity (associated with the exposure of the 5-amino acid QQPFP and 6-amino acid QPQLPY epitopes, present in the 25-mer and 33-mer, respectively) than control digestions (without bacteria), while E. faecalis led to lower antigenicity. In turn, within 24 h of incubation, all bacterial isolates led to the formation of undigested material with lower antigenicity, either due to fewer 33-mers and 25-mers in solution, or to fragment aggregation into amorphous material, not exposing antigenic sequences. Hence, intestinal flora may enhance or diminish the antigenicity of gliadin, thereby modulating the immunogenic response to gliadin/gluten.