Whole Genome Sequence Analysis Of Lactobacillus Reuteri And Its Regulation In Intestinal Mucosal Immunity Of Piglets

Lactobacillus reuteri15007, initially known as L. fermentum15007and isolated from the colons of healthy weaning piglets in our lab, has been extensively investigated for its probiotic activities including strong adhesion which could make improvements in the gut health of piglets. Four experiments were conducted to investigate the genetic basis of its probiotic characteristics and the effects of L. reuteri15007on intestinal mucosal immunity in piglets. In Exp.1, genome sequencing and bioinformatics analysis of L. reuteri15007was conducted by a whole-genome shotgun strategy and bioinformatics analysis. The genome contained a1,947,706bp chromosome with1,891protein-coding genes and six plasmids with a total number of163protein-coding genes. L. reuteri15007has a complete primary metabolic pathway, and is capable of encoding the main enzymes of the glycolysis pathway, some of the enzymes of the pentose phosphate pathway, and some enzymes of fatty acid, amino acid and nucleic acid metabolism. The L. reuteri15007genome sequence revealed some of the genes that are known to be involved in the resistance to low pH, bile salt, oxidative stress and adhesion to intestinal epithelial cells. L. reuteri15007genome encoded two gene clusters for exopolysaccharide biosynthesis. Comparative genome revealed a large inversion which was visible compared with other L. reuteri strains. Furthermore, L. reuteri15007genome showed strain-specific features, such as330unique genes, a uniquely clustered regularly interspaced short palindromic repeat （CRISPR）, and a specific exopolysaccharide cluster. Exp.2studied the adhesion property of L. reuteri15007and verified its adhesion genes. L. reuteri15007has strong hydrophobic characteristics and self-aggregation ability, and the adhesion ability was associated with the surface composition of protein and lipid. Six genes were selected and recombinants were built successfully. The LRI₃25and LRI₆05were expressed successfully by obtaining the purified proteins. However, LRI₃52（extracellular protein） and LRI₆05（peptidase M23） protein were shown not adhere to porcine small intestinal epithelial cell in the experiments. Exp.3was conducted to investigate the effect of L. reuteri15007on the expression of defense peptide in porcine intestinal epithelial cells in vitro and in vivo. After in vitro exposure to108CFU/mL of L. reuteri15007for6h, the defense peptide （pBD-2, pBD-3, pBD-114, pBD-129and PG1-5） expression was significantly improved in a porcine small intestinal epithelial cell line （IPEC-J2）. In vivo, L. reuteri15007supplementation significantly increased the expression of porcine β-defensin2（pBD-2） in the jejunum, and pBD-2, pBD-3, pBD-114and pBD-129in the colon （P<0.05）. L. reuteri15007supplementation significantly increased the concentration of butyric acid in colonic digesta. In addition, the colonic peroxisome proliferator-activated receptor-y （PPAR-y） and G protein-coupled receptor41（GPR41） mRNA expression of L. reuteri15007group was significantly higher than that of the control group （P<0.05）. The expression of pattern-recognition receptors （PRRs） were not affected by L. reuteri15007. The above-mentioned findings suggest that L. reuteri15007could stimulate defense peptide expression in neonatal piglets by increasing the concentration of short chain fatty acids in colonic digesta. In Exp.4, IPEC-J2cell lines were taken to investigate whether L. reuteri15007induced inflammation as the defense peptide was stimulated by L. reuteri15007in porcine intestinal epithelial cells. L. reuteri15007significantly increased Toll-like receptor2（TLR2）, TLR6and TLR9expression, and also stimulated the expression of interleukin-10and transforming growth factor-β3（P<0.05）, while interleukin-6and tumor necrosis factor-α （TNF-α） expression were not changed. In addition, L. reuteri15007exerted some exclusion ability against E. coli K88in IPEC-J2cells, and could decrease the over-expression of tumor necrosis factor-a and interleukin-6induced by lipopolysaccharide or E. coli K88, and could also increase interleukin-10expression （P<0.05）. We also found E. coli K88could stimulate defense peptide （pBD-2, pBD-114, pBD-129and PG1-5） expression. However no synergistic or antagonistic effect was found in stimulating defense peptide expression when combinations of L. reuteri15007and E. coli K88were used. In conclusion, the present study provides new perspectives for researching the probiotic mechanism of L. reuteri15007. The results supported and extended the previous studies for the probiotic characteristics of L. reuteri15007, more importantly, valuable information about L. reuteri15007improvement in maintaining advantages and eliminating the disadvantage for probiotic was also provided. L. reuteri15007could improve mucosal immunity and gut health of neonatal piglets by increasing the concentration of short chain fatty acid in colonic digesta and stimulating the expression of defense peptide in the colon. L. reuteri15007could stimulate the expression of defense peptide without inducing inflammation. In addition, L. reuteri15007suppressed the inflammatory responses induced by E. coli K88via inhibiting adhesion of E. coli K88and modulating the cytokines.