SlpB Protein Enhances the Probiotic Potential of L. lactis NCDO 2118 in Colitis Mice Model

Abstract

Bacteria used in the production of fermented food products have been investigated for their potential role as modulators of inflammation in gastrointestinal tract disorders such as inflammatory bowel diseases (IBD) that cause irreversible changes in the structure and function of gut tissues. Ulcerative colitis (UC) is the most prevalent IBD in the population of Western countries, and it is marked by symptoms such as weight loss, rectal bleeding, diarrhea, shortening of the colon, and destruction of the epithelial layer. The strain Propionibacterium freudenreichii CIRM-BIA 129 recently revealed promising immunomodulatory properties that greatly rely on surface-layer proteins (Slp), notably SlpB. We, thus, cloned the sequence encoding the SlpB protein into the pXIES-SEC expression and secretion vector, and expressed the propionibacterial protein in the lactic acid bacterium Lactococcus lactis NCDO 2118. The probiotic potential of L. lactis NCDO 2118 harboring pXIES-SEC:slpB (L. lactis-SlpB) was evaluated in a UC-mice model induced by Dextran Sulfate Sodium (DSS). During colitis induction, mice receiving L. lactis-SlpB exhibited reduced severity of colitis, with lower weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score, with significant differences, compared with the DSS group and the group treated with L. lactis NCDO 2118 wild-type strain. Moreover, L. lactis-SlpB administration increased the expression of genes encoding tight junction proteins zo-1, cln-1, cln-5, ocln, and muc-2 in the colon, increased IL-10 and TGF-β, and decreased IL-17, TNF-α, and IL-12 cytokines in the colon. Therefore, this work demonstrates that SlpB recombinant protein is able to increase the probiotic potential of the L. lactis strain to alleviate DSS-induced colitis in mice. This opens perspectives for the development of new approaches to enhance the probiotic potential of strains by the addition of SlpB protein.

Keywords: Lactococcus lactis; SlpB; colitis; inflammatory bowel disease; propionibacterium.

FIGURE 1

Lactococcus lactis-Surface-layer protein (Slp)B is able to control weight loss in Dextran Sulfate Sodium (DSS)-induced colitis. Time-course of body weight during the seven experimental days (A) and weight loss (B) are shown. The two-way ANOVA (A), one-way ANOVA (B), and Tukey’s post-hoc tests were used for the multiple comparisons (The data represent the mean ± SD of 12 mice per group). Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 2

L lactis-SlpB alleviates clinical symptoms in DSS-colitis mice model and reduces colon mucosal damage. Disease activity index over the seven experimental days (A), at the last day (B), Colon length analysis (C), Micrograph images of the histopathological analysis of the colon tissue (D) and analysis of the histopathological score (E) are shown. The slides were stained in hematoxylin and eosin (H&E) and analyzed under ×20 magnification. The two-way ANOVA (A), one-way ANOVA (B), and Tukey’s post-hoc tests were used for the multiple comparisons (The data represent the mean ± SD of 12 mice per group). Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 3

L. lactis-SlpB mitigates histological signs of DSS-colitis. Model images of micrographs for analysis of goblet cells in colon tissue (A) and the result of goblet cell quantification by field (B) and depth of colon intestinal crypts (C) are shown. The slides were stained in Periodic Acid-Schiff (PAS), goblet cells have intense purple-pink tones, and analyzed under ×40 magnification. The data represent the mean ± SD of 12 mice per group. One-way ANOVA and Tukey’s post-hoc tests were used for multiple comparisons. Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 4

L. lactis Wild-type and L. lactis-SlpB strains prevent DSS-induced increase of myeloperoxidase (MPO) and eosinophilic peroxidase (EPO) activity. Quantification of the myeloperoxidase [MPO, (A)] and eosinophilic [EPO, (B)] enzymes in the colon tissue is shown. One-way ANOVA and Tukey’s post-hoc tests were used for multiple comparisons. The data represent the mean ± SD of six mice per group. Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 5

L. lactis-SlpB increases the expression of genes involved in epithelial barrier protection. Quantification of the expression of the genes Mucin 2 (muc2) (A), Zonula Occludes 1 (zo-1) (B), Zonula Occludes 2 (zo-2) (C), Claudin-1 (cln-1) (D), Claudin-5 (cln-5) (E), and Occludin (ocln) (F), in the mice colon, is shown. One-way ANOVA and Tukey’s post-hoc tests were used for multiple comparisons. The data represent the mean ± SD of six mice per group. Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 6

L. lactis-SlpB strain modulates expression of pro and anti-inflammatory genes implicated in ulcerative colitis. Quantification of the expression of the genes inducible nitric oxide synthase (inos) (A), peroxisome proliferator-activated receptor-gamma (pparg) (B), as well as interleukin-17 (il-17) (C) and interleukin-10 (il-10) (D) pro and anti-inflammatory cytokines, is shown. The data represent the mean ± SD of six mice per group. One-way ANOVA and Tukey’s post-hoc tests were used for multiple comparisons (n = 6). Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

FIGURE 7

L. lactis-SlpB strain modulates cytokines production in the mice colon. Colonic cytokines concentrations levels of IL-17 (A), IL-12 (B), TNF-α, (C), IL-10 (D), TGF-β (E), IL-1β (F) were quantified by enzyme-linked immunosorbent assay (ELISA). The data represent the mean ± SD of six mice per group. One-way ANOVA and Tukey’s post-hoc tests were used for multiple comparisons. Asterisks represent statistically significant differences as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.