| Heparosan, a member of the glycosaminoglycan (GAG) family, is the polysaccharide found in the capsule of certain bacteria as well as the biosynthetic precursor of heparin or heparan sulfate (HS) in animals. Heparin and HS are a class of the most important polysaccharide substances of the organisms, involve in a variety of biological processes, such as embryonic development, cell growth and differentiation, blood coagulation, lipid metabolism, inflammation, angiogenesis, cell signaling and microbial infection. Heparin is widely used as anticoagulant and antithrombotic in clinical, apart from that, heparin also exhibits lipidemic regulating, anti-inflammatory and anticancer properties. Recently, the anti-infection effects of heparin got a lot of attentions. It has been found that exogenous heparin can inhibit the infection of variety of infectious pathogenic microorganisms to eukaryotic cells, including viruses, bacteria and parasites. The inhibition effects of heparin to the adhesion and intrusion of variety of pathogenic microorganisms make it promising to be developed into broad-spectrum anti-infectives. As the strong anticoagulant effect of heparin, the application of exogenous heparin has the risk of bleeding, and the dose and operation should be strictly controlled, that restrict its wide applications. Heparosan has the similar sugar backbone with heparin or HS, except the polymer is nonsulfated, and there is no epimerization of glucuronic acid to iduronic acid, without anticoagulant activity, it can be directly extracted from bacteria and modified by sulfation or epimerization to generate a variety of derivatives with similar sturcture. It was found that heparosan and its derivatives had multiple heparin-linking activities, such as anticoagulant, anti-inflammatory, anti-viral and anti-cancer, but no related study was reported about the effect of heparosan on bacterial infection. In the present study, we evaluated the effect of heparosan on the adhesion and colonization of some common intestinal bacteria, and investigated the effects of heparosan on the overall regulation of intestinal flora as well as its preventive effects on the pathogen infection in vivo, providing basis for its further development as anti-infective.1Extraction and isolation of heparosanObjective To prepare heparosan through the cultivation of Pasteurella multocida type D P-934. Methods The conditions of the carbon sources, metal ions, inculum size and liquid volume were optimized through investigation of heparosan yield to determine the final fermentation conditions. After the determination of the main purification processes, the key factors were investigated to optimize the isolation of heparosan. Heparosan isolated was characterized and determined according to the relevant references. Results After optimization, the final fermentation conditions were as follows:bovine brain heart infusion20g/L, peptone10g/L, glucose20g/L, NaCl5.0g/L, MgSO40.2g/L, MnCl20.5g/L, pH7.2,50mL medium in a250mL Erlenmeyer flask, and inoculation quantity5%(v/v), fermentation for48h. The yield of heparosan was increased from the0.13g/L in brain heart infusion to0.41g/L. The isolation and purification process was simple, with the addition of cetylpyridinium chloride, heparosan in the supernatant was precipitated, then the precipitate was dissolved in NaCl solution, finally heparosan was isolated with the precipitation of ethanol. High purity heparosan was got through the control of the concentration of NaCl in the alcohol precipitation process. The heparosan extracted was characterized through heparinase Ⅲ degradation and1H-NMR analysis, and the preparation technology furnished a heparosan with above98%purity. Conclusion The yield of heparosan was improved through the optimize of culture conditions. The extraction and isolation processes were simple and feasible, result a high purity heparosan that applicable for further tests.2Effects of heparosan on the adhesion and colonization of tested intestinal bacteria in vitro. Objective To investigate the effects of heparosan on the adhesion and colonization of tested intestinal bacteria. Methods Intestinal enterocytes and mucus layer models were established to evaluate the effects of heparosan on the adhesion of tested intestinal pathogens, entero-pathogentic Escherichia coli (EPEC), Staphylococcus aureus, Pasteurella multocida and Salminella, as well as the common probiotics Lactobacillus rhamnosus GG (LGG) to intestinal enterocytes and mucus. In addition, the effects of heparosan on the colonization of LGG and S. aureus were evaluated by the evaluation of effects on their biofilm formation. The effects of other common GAGs (heparin, chondroitin sulfate and hyaluronic acid) as well as the positively charged chitosan were comparatively studied. Results The inhibition of heparosan on the adhesion of tested pathogens were shown to increase in a dose-dependent fashion. A concentration of50μg/ml heparosan showed efficient ability of anti-adhesion of EPEC,S. aureus and P. muliocida to HT-29enterocytes and mucus models individually, but it had no influence on the adhesion of LGG. When the density of LGG was low, the combination of heparosan showed better results from competition and resistance binding assay to EPEC. There were no obvious differences between the inhibition effects of heparosan and heparin. The inhibition of heparosan to the adhesion of pathogens may be related to its similar structure with heparin or HS. Heparosan showed no significant effects on the biofilm formation of LGG and S. aureus, while heparin promoted the biofilm formation of LGG with above40%increase. This increase was partially related to the strong negative charges of heparin. Conclusion Heparosan can inhibit the adhesion of tested pathogens to intestinal enterocytes and mucus, and relative advantages the competition of probiotics. Heparosan has the potential to be developed into anti-infectives.3The overall regulation effects of heparosan on intestinal flora and its preventive effects on pathogen infection in vivo.Objective To evaluate the overall regulation effects of heparosan on rats intestinal flora and its preventive effects on pathogen infection in vivo. Methods Rats were randomly divided into three groups and given0.9%NaCl,0.5mg/mL and1mg/mL heparosan, respectively. The dose of intragastric administration was1mL per100g body weight everyday, and lasting for2weeks. The fecal samples of the7d and14d were taken and analyzed with denaturing gradient gel electrophoresis, the different bands of heparosan tests groups compared with that of saline group were selected and sequenced to analysis the changes in the composition of intestinal flora, for the evaluation of the effects of heparosan on the intestinal dominant strains.2mL EPEC suspension of109CFU/mL were given to rats by intragastric administration for3consecutive days to induce entertis for the evaluation of the preventive effects of heparosan on pathogen infection. Results Heparosan showed significant regulation effects on the rat intestinal flora, the proportion of lactobacillus probiotics groups were significantly increased. With the administration of EPEC, the rats of saline group showed apparently intestinal inflammation, while the heparosan treated groups were not. Conclusion Heparosan advantages the dominate of lactobacillus probiotics, which is helpful in the maintenance of a stable and healthy intestinal environment, reducing the incidence of disease. Heparosan can effectively prevent the infection of EPEC. |
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