An engineering probiotic producing defensin-5 ameliorating dextran sodium sulfate-induced mice colitis via Inhibiting NF-kB pathway.

BACKGROUND: Human defensin-5 (HD-5) is a key antimicrobial peptide which plays an important role in host immune defense, while the short half-life greatly limits its clinical application. The purpose of this study was to investigate the effects of an engineering probiotic producing HD-5 on intestinal barrier and explore its underlying mechanism METHODS: We constructed the pN8148-SHD-5 vector, and transfected this plasmid into Lactococcus lactis (L. lactis) to create the recombinant NZ9000SHD-5 strain, which continuously produces mature HD-5. NZ9000SHD-5 was administrated appropriately in a dextran sodium sulfate (DSS)-induced colitis model. Alterations in the wounded intestine were analyzed by hematoxylin-eosin staining. The changes of intestinal permeability were detected by FITC-dextran permeability test, the tight junction (TJ) proteins ZO-1 and occludin and cytokines were analyzed by western blotting or enzyme linked immunosorbent assay. In Caco-2 cell monolayers, the permeability were analyzed by transepithelial electrical resistance, and the TJ proteins were detected by western blotting and immunofluorescence. In addition, NF-κB signaling pathway was investigated to further analyze the molecular mechanism of NZ9000SHD-5 treatment on inducing intestinal protection in vitro. RESULTS: We found oral administration with NZ9000SHD-5 significantly reduced colonic glandular structure destruction and inflammatory cell infiltration, downregulated expression of several inflammation-related molecules and preserved epithelial barrier integrity. The same protective effects were observed in in vitro experiments, and pretreatment of macrophages with NZ9000SHD-5 culture supernatants prior to LPS application significantly reduced the expression of phosphorylated nuclear transcription factor-kappa B (NF-κB) p65 and its inhibitor IκBα. CONCLUSIONS: These results indicate the NZ9000SHD-5 can alleviate DSS-induced mucosal damage by suppressing NF-κB signaling pathway, and NZ9000SHD-5 may be a novel therapeutic means for ulcerative colitis.

Rapid detection of ermB gene in Clostridium difficile by loop-mediated isothermal amplification.

Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. Here, we describe a sensitive and rapid molecular method to detect ermB in C. difficile to contribute to the wider epidemiological study. Five sets of loop-mediated isothermal amplification (LAMP) primers were designed and optimized for rapid detection of ermB. The specificity and sensitivity of the primers for ermB were detected, and the ermB LAMP assay was compared to conventional PCR with 80 clinical isolates of C. difficile. Real-time monitoring of turbidity and chromogenic reaction were used to determine negative and positive results. A total of 26 pathogenic bacterial strains of different species were found to be negative for ermB, which indicated the high specificity of the primers. ermB was detected in 78.8 % (63/80) of the clinical isolates by both LAMP and conventional PCR. The detection limit of LAMP was 36.1  pg DNA µl- 1 and its sensitivity was 10-fold greater than that of conventional PCR. This study is the first report regarding the development and application of the LAMP assay for detection of the ermB gene in C. difficile strains. The developed LAMP method is sensitive, specific and provides a user-friendly visual approach for the rapid detection of ermB-bearing C. difficile.

Syndecan-1 Acts in Synergy with Tight Junction Through Stat3 Signaling to Maintain Intestinal Mucosal Barrier and Prevent Bacterial Translocation.

BACKGROUND: Intestinal epithelial tight junction (TJ) is the principal determinant of mucosal permeability, defects of which have been correlated to inflammatory bowel disease. In this study, we investigated whether syndecan-1 (Sdc1), the predominant cell surface heparan sulfate proteoglycan, affects TJ proteins to protect intestinal barrier function. METHODS: The role of Sdc1 in barrier function was examined in cultured colonic epithelial cells and dextran sodium sulfate-induced colitis mouse model. Barrier function was determined by transepithelial electrical resistance, bacterial translocation, and FITC-dextran flux. Canonical TJ proteins ZO-1 and occludin were measured by Western blot and immunofluoresence. Role of the Stat3 pathway was detected by Western blot and chromatin immunoprecipitation. RESULTS: Overexpressed Sdc1 in Caco-2 cells attenuated transepithelial electrical resistance reduction, prevented bacterial translocation, and repressed FITC-dextran flux, whereas Sdc1 knockdown in HT29 cells resulted in a greater loss of barrier function. Supplementation of exogenous Sdc1 in colitis mice ameliorated the activity of colitis and barrier defect. Mechanistically, Sdc1 significantly modulated expressions of ZO-1 and occludin by activating Stat3, which directly bound to the promoter regions of ZO-1 and occludin. Furthermore, ZO-1 and occludin were found to bind to each other, and their repression could induce Sdc1 upregulation. CONCLUSIONS: Sdc1 plays an important role in protecting the intestinal barrier function and preventing bacterial translocation, in synergy with TJ through Stat3 signaling in an Sdc1/Stat3/ZO-1 and occludin feedback loop. Sdc1 participates in the mechanism that is related to intestinal barrier function and colitis and represents a therapeutic target for novel anti-inflammatory bowel disease strategies.