IDENTIFICATION OF A FUNCTIONAL DOMAIN OF A PROBIOTIC FUNCTIONAL FACTOR FOR THE LONG-LASTING EFFECT ON PREVENTION OF COLITIS

Background & Aims: Probiotic-derived secretory proteins, such as p40, serve as functional factors for benefiting host health. However, there is a paucity of structure-function analysis of these factors making it difficult to define the mechanism(s) of action. We previously demonstrated the sustained effects of p40 on preventing intestinal inflammation. This study aimed to define the p40 functional domain that contributes to p40-regulated epigenetic programs in intestinal epithelial cells in early life that sustains TGFβ production and prevention of colitis in adulthood.

Methods: p40 sequence was analyzed by molecular evolutionary genetics analysis. 3D structure of p40 was predicted by using YASARA, Alpha-fold, and Modeller. p40 1-27 aa is a signal peptide, thus, two recombinant p40 peptides, 28-120aa (N120) and 28-180aa (N180) (Figure 1), were generated using an E. coli expression platform and validated by gel staining and Western blot. Human 3D colonoids and a mouse colonic epithelial cell line (YAMC) were treated with full-length p40 (p40F), p40N180 and p40N120. Pups received p40F and p40N120 from postnatal days 2–21. Regulatory T cell (Treg) differentiation (CD4⁺Foxp3⁺ cells) in 8-week-old Foxp3-GFP Balb/c mice were tested by flow cytometry and the status of colitis induced by 3% DSS treatment for 4 days in 8-week-old C57BL/6 mice was assessed. Gene expression was examined by RT-PCR, methylation of histone 3 on lysine 4 (H3K4me)1/3 and localizing ZO1, a tight junction protein by immunostaining , TGFb-regulated SMAD activation and H3K4me1/3 by Western blot.

Results: The p40 N-terminus is an uncharacterized domain conserved in bacteria whereas the C-terminus shows high homology to a CHAP domain. In-silico analysis revealed that the N-terminus is composed of a coiled-coil domain (a1-7, residues 28–300) and a b-sheet structure in the C-terminus (Figure 1). Compared to p40F, p40N180 and p40N120 both up-regulated expression of Setd1b (encoding a methyltransferase) and H3K4me1/3, and enhanced Tgfb expression and SMAD2 phosphorylation in human colonoids and YAMC cells. Neonatal supplementation with p40F and p40N120 increased H3K4me1 cells (p<0.05) and Tgfb expression (p<0.05) in the colon and Treg expansion in the colonic lamina propria (p<0.05) of adult Foxp3-GFP mice. DSS-induced inflammation (score:10.40±0.68) in adult mice was mitigated by neonatal supplementation with p40F (6.6±0.75, p<0.05) and p40N120 (2.2±1.0, p<0.05). In addition, p40F and p40N120 significantly reduced DSS-induced expression of proinflammatory cytokines (TNF and IL6) and prevented DSS-disrupted epithelial barrier.

Conclusions: These data indicate that the 28-120aa peptide of p40 contains the functional domain for sustained prevention of colitis. Thus, intervention with functional peptides of probiotic functional factors may serve as a novel strategy for maintaining human health.