Construction Of Mouse β-defensin 14-expressing Lactiplantibacillus Plantarum And Its Effect On Colitis

Inflammatory bowel diseases(IBDs)such as ulcerative colitis and Crohn’s disease,are a group of chronic disorders affecting the intestinal tract with a global prevalence of more than0.3%.IBDs are thought to be the result of inappropriate immune activation in response to the gut microbiota induced by genetic and environmental factors.The therapeutic drugs for IBDs are mainly biological agents that inhibit gut inflammation.These drugs cannot solve the underlying causes of IBDs patients,such as gut microbiota imbalance and intestinal barrier dysfunction.Moreover,serious adverse reactions are likely to occur with long-term use of these drugs.Therefore,it is necessary to develop novel therapeutic strategies for IBDs that target intestinal homeostasis.β-defensins are cationic antimicrobial peptides secreted mainly by epithelial cells and immune cells.They play an important role in resisting microbial infection and regulating host immunity.Mouse β-defensin 14(mBD14)is an ortholog of human β-defensin 3.In addition to the classic antibacterial activity,its anti-inflammatory effect has been proved in vitro.However,the instability of mBD14 in the process of transmission in vivo limits its application in intestinal inflammation.The living therapeutic of delivering antimicrobial peptides through oral administration of engineered probiotics has been widely concerned.The engineered probiotics will continuously secrete antimicrobial peptides in the intestinal tract and achieve therapeutic effects.This study aimed to construct an engineered probiotic that can produce mBD14.In addition,the effect of the engineered probiotic on colitis in mice was revealed.The main results and conclusions are as follows:(1)In order to construct a mBD14-expressing engineering probiotic,the p NZ8148 plasmid and the fusion gene fragment encoding mBD14 were connected by genetic engineering technology,and then the plasmid was transformed into Lactiplantibacillus plantarum to obtain mBD14-expressing Lactiplantibacillus plantarum.A band corresponding to 382 bp was confirmed from single colonies.The content of mBD14 in the secreted protein was significantly higher than that in the intracellular protein.Besides,the mBD14 level in mouse colon content was increased significantly with mBD14-expressing Lactiplantibacillus plantarum administration.These results suggest that mBD14-expressing Lactiplantibacillus plantarum could secrete mBD14 in the colon of mice.(2)In order to confirm the role of mBD14-expressing Lactiplantibacillus plantarum in colitis,we established a mouse model of colitis with dextran sodium sulfate,and mice were treated with mBD14-expressing Lactiplantibacillus plantarum.We demonstrated the protective effect of mBD14-expressing Lactiplantibacillus plantarum against colitis,as evidence by the reduction in weight loss,disease activity index scores,colon shortening and histological changes in mice.(3)The effect of mBD14-expressing Lactiplantibacillus plantarum on the typical bacteria in colitis was revealed.The results depicted that mBD14-expressing Lactiplantibacillus plantarum significantly increased the relative expression of Akkermansia muciniphila and Faecalibacterium prausnitzii in colitis mice,and also prevented the abnormal enrichment of pathogenic Escherichia coli,Fusobacterium nucleatum and Staphylococcus aureus,thus contributing to the maintenance of gut microbiota homeostasis.(4)The effects of mBD14-expressing Lactiplantibacillus plantarum on the intestinal barrier and inflammatory response in mice with colitis were investigated.The results indicated that mBD14-expressing Lactiplantibacillus plantarum enhanced the expression of tight junction proteins and reduced intestinal permeability,thereby protecting the intestinal barrier function.The detection of pro-inflammatory cytokines and NOD-like receptor pyrin domain containing 3 and its downstream inflammatory pathways in colon showed that mBD14-expressing Lactiplantibacillus plantarum could inhibit the intestinal inflammatory response in colitis mice.The study is the first time to successfully construct mBD14-expressing Lactiplantibacillus plantarum and confirm its protective effect on colitis,providing a theoretical basis for antimicrobial peptide-engineered probiotics to treat or prevent IBDs.