Inhibition Of O-glycosylation In Human Intestinal Epithelial Cells Reduces Bacteria Adherence And Mucin2Expression

Background and ObjectiveThe intestinal tract is unique among mucosal tissues, because it is heavily colonized bymicroorganisms, mostly bacteria, starting from birth. Mucus as a critical mediator ofhost–bacteria interactions. Recent work has established that the mucin-rich mucus layer actsas an essential barrier between the luminal microbiota and the underlying immune cells. Theintestinal mucus is primarily composed of Mucin-2(MUC2), a prominent member of themucin family. Mice deficient in Muc2, the principal mucin forming the intestinal mucuslayers, completely lack a colonic mucus layer and fail to restrict bacterial attachment to themucosal tissues, which likely leads to the severe spontaneous colitis and colorectal cancer thatthese mice develop. Mucins represent a major group of high-molecular-weight O-linkedglycoproteins, which are widely expressed in the body and play many essential roles inphysiologic and pathologic settings. O-glycans have four main core structures, referred to asCore1-, Core2-, Core3-and Core4-derived O-glycans. Core1-derived O-glycans which isthe major O-glycans found on mouse Muc2, is largely existed in most organization and celltypes. Loss of C1GALT1specifically within the developing intestinal epithelium resulted inspontaneous colitis. However, the question remains exactly unclear how O-glycans promotemucus barrier integrity and homeostasis with the gut microbiota. In this study, an inhibitor ofO-glycosylation (benzyl-α-GalNAc) was used to treat HT-29and differentiated HT-29cells(HT-29-Gal) to block the synthesis of their O-glycans, and further observing the adhesionbehavior changes of enterohemorrhagic Escherichia coli serotvpe O157:H7(EHEC O157:H7)and enteropathogenic Escherichia coli (EPEC) in intestinal epithelial cells. We established thestably transfected HT-29cells with shRNA-C1GALT1/EGFP plasmid, to provide thenecessary model of intestinal epithelial cells with Core1synthase inhibition to observe thebacterial adhesion change. By studying the role of mucin-type O-glycans in regulating interactions with the microbiota, it will help us to understand the mechanism of EPEC orEHEC induced intestinal infection.Methods1Colonic carcinoma cell line HT-29was induced to undergo the differentiation, culturedin Galactose media.2HT-29or differentiated HT-29cells (HT-29-Gal) were treated with benzyl-α-GalNAc,an inhibitor of O-glycosylation.3MUC2protein and its mRNA in HT-29and HT-29-Gal cells treated withbenzyl-α-GalNAc or not were detected using western-blotting and real-time PCR.4HT-29-Gal, HT-29, benzyl-α-GalNAc treated HT-29-Gal (named as HT-29-Gal-OBN)and benzyl-α-GalNAc treated HT-29cells (named as HT-29-OBN) were incubated withEHEC O157:H7or EPEC, the attached bacteria were quantified by determining the CFUfollowing the plating of serial dilutions of the bacteria or Immunofluorescence staining toevaluate the effect of benzyl-α-GalNAc on bacteria adherence.5To construct the interference plasmid of C1GALT1, then transfected into HT-29cellswhich Core1synthase is abundantly expressed and the stably transfected HT-29cells wereobtained with G418.Results1MUC2expression levels were significangly lower in HT-29-Gal-OBN andHT-29-OBN cells at both protein and mRNA levels respectively, compared with untreatedcells.2The bacterial adherence assay found that the adherence of EHEC O157:H7or EPEC toHT-29-Gal or HT-29cells after treatment with benzyl-α-GalNAc was significantly decreasedcompared with the untreated HT-29-Gal or HT-29cells, respectively.3The stably transfected HT-29cells which C1GALT1were knocked down wereestablished for further experiments.Conclusions Inhibition of O-glycosylation in intestinal epithelial cells reduce theirMUC2expression and bacteria adherence.