Abstract
BackgroundHow host cell glycosylation affects EPEC or EHEC O157:H7 invasion is unclear. This study investigated whether and how O-glycans were involved in EPEC or EHEC O157:H7 invasion into HT-29 cells.ResultsLectin histochemical staining confirmed stronger staining with PNA, which labeled Galβ1, 3 GalNAc (core 1 structure) in HT-29-Gal-OBN and C2GnT2-sh2/HT-29 cells, compared with control cells. EPEC or EHEC O157:H7 invasion into HT-29 and its derived cells was based on the intracellular presence of GFP-labeled bacteria. The differentiation of HT-29 cells led to a reduction in EPEC internalization compared with HT-29 cells (p < 0.01). EPEC or EHEC O157:H7 invasion into HT-29-OBN and HT-29-Gal-OBN cells increased compared with HT-29 and HT-29-Gal cells (p < 0.05 and p < 0.01). Core 2 O-glycan-deficient HT-29 cells underwent a significant increase in EPEC (p < 0.01) or EHEC O157:H7 (p < 0.05) invasion compared with control cells.MethodsBacterial invasion into cultured cells was determined by a gentamicin protection assay and a GFP-labeled bacteria invasion assay. O-glycans biosynthesis was inhibited by benzyl-α-GalNAc, and core 2 O-glycan-deficient HT-29 cells were induced by C2GnT2 interference.ConclusionThese data indicated that EPEC or EHEC O157:H7 invasion into HT-29 cells was related to their O-glycosylation status. This study provided the first evidence of carbohydrate-dependent EPEC or EHEC O157:H7 invasion into host cells.
Highlights
IntroductionThis study investigated whether and how O-glycans were involved in Enteropathogenic E. coli (EPEC) or EHEC O157:H7 invasion into HT-29 cells
How host cell glycosylation affects Enteropathogenic E. coli (EPEC) or EHEC O157:H7 invasion is unclear
We identified the O-glycosylation status of HT-29-OBN cells and mucin-type core 2 O-glycan deficient HT-29 cells, using a series of lectin recognition: Maackia amurensis (MAA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Ulex europaeus (UEA-I), Griffonia simplicifolia (GSAII), Canavalia ensiformis (ConA), and Sambucus nigra (SNA), and we further demonstrated that core 2 mucin-type O-glycan inhibited EPEC or EHEC O157:H7 invasion into HT-29 cells
Summary
This study investigated whether and how O-glycans were involved in EPEC or EHEC O157:H7 invasion into HT-29 cells. The mucin layer functions as a barrier to gastrointestinal tract (GI) bacterial infection, effectively hampering bacteria from adhering to and invading into cells [1, 2]. Mucin-type core 2 O-glycan is biosynthesized by the enzyme core 2 β1, 6-N-acetylglucosaminyltransferase 2 (C2GnT2) (Fig. 1), which is mainly expressed in the colon [3, 4]. O-glycans might be related to the selection of the commensal flora in the distal colon, and it could act as an attachment site for different bacteria [6]. We have reported that core 2 O-glycan deficiency in HT-29 cells and enhanced MUC3 expression in HT-29-Gal cells resulted in decreased EPEC or EHEC O157:H7 adherence [7, 8]
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