The Role And Mechanism Of A Novel Postbiotic Named HM0539 From Lactobacillus Rhamnosus GG Prevents Intestinal Infection By Enterohaemorrhagic E.coli O157:H7

BACKGROUND AND PURPOSEE.coli O157:H7(EHEC O157:H7)is a highly contagious foodborne pathogen that induce severe gastrointestinal diseases and pose a potentially serious threat to human health.At present,there is no effective treatment for EHEC O157:H7 infection.Antibiotic treatment has not improved the course of enteritis caused by EHEC O157:H7,and even increased the risk of concurrent HUS.Therefore,it is urgent to change strategies and develop prevention-based prevention and control methods to reduce the burden of disease and economy caused by EHEC O157:H7 infection.LGG and its metabolites are the most researched probiotics.Our previous study demonstrated that LCS promoted development of neonatal intestinal defense and protects the neonatal rats against oral E.coli K1 infection;Using high performance liquid chromatography-tandem mass spectrometry technology,we identified a potential biologically active protein,named HM0539,with a beneficial effect on intestinal barrier function.The purpose of this study is to determine the protective effect and mechanism of HM0539 on EHEC O157:H7-induced gastrointestinal infection.METHODS1.HT-29 cells were cultured in a 24-well plate,placed in a 37? 5%carbon dioxide incubator for 10 days;then according to different concentrations of HM0539(10 ng/mL and 100 ng/mL)groups and different treatments of HM0539(pretreatment and co-treatment)groups,adding 1×107 CFU EHEC 0157:H7 treatment for 4h for executing adhesion and invasion experiment;EHEC 0157:H7 relative adhesion/invasion rate =(number of colonies in each group of treatment group/average number of colonies in control group)×100%.2.HT-29 cells were cultured,grouped and treated as above;the Mucin expression of each group of cells was detected by PAS protein concentration quantitative assay.IFC and Wb technology were used to compare the expression of MUC2,ZO-1 and Occludin protein in each group of cells.3.The mice were randomly divided into 4 groups(before challenge,the NC group and the EHEC O157:H7 group were gavaged with blank pectin for 7 days;the HM0539 group and the HM0539+EHEC O157:H7 group were gavaged with HM0539 pectin for 7 days),10 mice/group.The mice were fed with streptomycin water for 3 days before the challenge and fasted for 12 hours.The mice in the EHEC O157:H7 group and HM0539+EHEC O157:H7 group were irrigated with 100?L×1011 CFU/mL EHEC O157:H7 bacterial solution,and mitomycin(2.5 mg/kg),gavaging the mice again with the same amount of bacteria after 12h interval.The NC group and HM0539 group were challenged with 100 ?L of sterile PBS.We observed the infection status of the mice for a week,recorded the weight and survival,and detected the intestinal injury by HE staining,PAS staining and IHC.4.HT-29 cells and Caco-2 cells were cultured,grouped and treated as above.The expression of IL-1? in the cell culture supernatant of each group was compared by ELISA.Wb was used to compare the activation of TLR4/NF-?B pathway in each group of cells;the mice were grouped and treated as above,extracting the total protein of intestinal tissue,comparing the expression of IL-1? in the intestinal tissue of each group of mice by ELISA,and comparing the activation of TLR4/NF-?B pathway in the intestinal tissue of each group of mice by Wb.5.Data analysis and graphs production were executed by software SPSS20.0 and GraphPad Prisn5.0.The t test was used for comparison between the two groups,and one-way ANOVA was used for comparison between multiple groups.The measurement data were expressed in the form of mean±SD(Mean+SD),and all experiments were repeated 3 times,P<0.05 was considered statistically significant.RESMLTS1.For the effect of HM0539 on EHEC O157:H7 adhesion and invasion to HT-29 cells in vitro.We found that HM0539 had potential to inhibit EHEC O157:H7 adhesion and invasion of HT-29 cells in a concentration-dependent manner(P<0.05).On the other hand,we also found that the pretreatment effect of HM0539 was better than that of the co-incubation of HM0539 and EHEC O157:H7(P<0.05).2.In vivo study showed that HM0539 reduced mortality and inhibited weight loss in EHEC O157:H7-challenged mice(P<0.05).HE assays showed that HM0539 could attenuate EHEC O157:H7-induced villous atrophy of the jejunum and crypt abscess.3.PAS staining of HT-29 and mouse intestine revealed that EHEC O157:H7 reduced mucin expression while pretreatment with HM0539 inhibited the degradation of Mucin by EHEC O157:H7(P<0.05).4.Intestinal barrier tests showed that HM0539 could inhibit EHEC O157:H7 from destroying tight junction proteins(P<0.05),thereby protecting the integrity of the intestinal barrier5.EHEC O157:H7 group had higher IL-1? levels and more TLR4/NF-?B signalling activation than control group both in vitro and in vivo,while pretreatment with HM0539 could attenuate these parameters(P<0.05).CONCLUSION1.The protective effect of prophylactic use of HM0539 against EHEC O157:H7-induced gastrointestinal infection could be better than therapeutic administration.2.HM0539 could inhibit EHEC O157:H7-indcued Mucin degradation and tight junction damage,thereby protecting the integrity of the intestinal barrier3.The protective mechanism of HM0539 on EHEC O157:H7-induced inflammatory response might via inhibit TLR4/NF-?B activation and IL-1? secretion.