The Peptidoglycan From Lactobacillus DM9811: Isolation Extraction And Immunological Activity

Lactobacillus is one of important probiotics. The biological properties of peptidoglycan (PNG) include biological antagonism, nutritional effects, immune regulation, anti-tumor and anti-aging, etc. Lactobacillus plays immune role via the interaction between its specific cellular components and the host immune cell.PNG also called mucopeptide, is a major cell wall component of Lactobacillus and functions its physical and chemical properties. The basic structure of PNG consists of a disaccharide unit of N-acetyl-D-glucosamine and N-acetylmuramic acid (GlcNAc-MurNAc) byβ-1, 4 linkages. Each GlcNAc-MurNAc has a side chain of tetrapeptide, which is are interlinked by short pentapeptide of (Gly)5. PNG is a continuous covalent-linked sac-liked macromolecular structure that is found on the outside of the cytoplasmic membrane of almost all bacteria. For the past few years, the biological activity of PNG has been concerned.TLRs (Toll like receptors, TLRs) have recently emerged as a key component of innate immune system which can detect microbial infection and trigger antimicrobial host defense responses. TRLs regulate the acquired immune though up-regulating Antigen-presenting cell surface costimulatory molecules and antigen-presenting cells to secrete cytokines which could induce the differentiation of T and B lymphocytes to the effects of T and B lymphocyte. TLRs can recognize pathogen associated molecular patterns (MAMPs).PNG is one of the most abundant MAMPs in Lactobacillus. It maybe recognized by TLRs and activated innate immunity system.ObjectiveIn this paper, Lactobacillus DM9811 separated by Dalian Medical University, Department of Microbiology Lab were used as the research object. Extraction of PNG from Lactobacillus DM9811 and the effect and mechanism of Lactobacillus PNG on modulating mammal immune response were investigated.Methods1) Isolation and extraction PNG from Lactobacillus DM9811 The bacteria Lactobacillus DM9811 were harvested after culture of 48h and boiled in 10% Trichloracetic acid (TAC) and 4% SDS in PBS for 20mins and for 30mins, consequently. After digested by trypsin for 48hs, the crude PNG extract precipitates were obtained by centrifugation. The partial purified PNG components were obtained further by lysozyme digestion followed by Sephacrly S-300HR gel filtration.2) Characterization of PNG. Phenol- sulfuric acid was used to detect sugar content. Lysozyme was used to detect the hydrolysis of PNG while absorbances of PNG solution were examined. Paradimethylaminobenzaldehyde was used to detect N-acetyl-D-glucosamine.3) Immunological effect of PNG Mouse Macrophages Ana-1 cells were treated with or without various concentration of PNG for 24hs. The protein amounts of IL-10, IL-12 and TNF-αwere examined by ELISA. Flow Cytometry was used to detect Macrophages Ana-1 cells proliferation and the expression of TLR2 and TLR4 on Macrophages Ana-1 cells.Results1) PNG with apparent homogeneous molecular weight were isolated from the bacteria Lactobacillus DM9811.2) Compared with the control group, upon treatment of PNG, IL-10, IL-12 and TNF-αwere secreted from mouse Macrophages Ana-1 cells. The proliferation of macrophages Ana-1 significantly increased the expression level of TLR2 and TLR4 on Ana-1 cells rose in the PNG group compared with the control group.Conclusion:PNG from Lactobacillus DM9811 can activate macrophages Ana-1 cells in vitro.