The Interaction of Helicobacter pylori with the Adherent Mucus Gel Layer Secreted by Polarized HT29-MTX-E12 Cells

Abstract

Helicobacter pylori colonises the gastric mucosa of humans. The majority of organisms live in mucus. These organisms are an important reservoir for infection of the underlying epithelium. Cell culture models for H. pylori infection do not normally possess a mucus layer. The interaction of H. pylori with TFF1, a member of the trefoil factor family found in gastric mucin, is mediated by lipopolysaccharide. To test the hypothesis that the interaction of H. pylori with TFF1 promotes mucus colonization we characterised the interaction of H. pylori with a mucus secreting cell line, HT29-MTX-E12. An isogenic mutant of H. pylori with truncated core oligosaccharides was produced and binding to TFF1 and ability to colonise HT29-MTX-E12 cells determined. The adherent mucus layer of HT29-MTX-E12 cells contained the gastric mucin MUC5AC and trefoil factors, TFF1 and TFF3. H. pylori was found within the mucus layer in discrete clusters and in close association with TFF1. It also interacted with the membrane bound mucin MUC1 and replicated when co-cultured with the cells. An isogenic mutant of H. pylori with a truncated LPS core did not interact with TFF1, and colonization of HT29-MTX-E12 cells was reduced compared to the wild-type strain (p<0.05). Preincubation of cells with wild type LPS but not with truncated LPS resulted in reduced colonization by H. pylori. These results demonstrate that the interaction of TFF1 with H. pylori is important for colonization of gastric mucus and the core oligosaccharide of H. pylori LPS is critical for this interaction to occur. HT29-MTX-E12 cells are a useful system with which to study the interaction of bacteria with mucosal surfaces and the effect of such interactions on mediating colonization.

Divergent Mechanisms of Interaction of Helicobacter pylori and Campylobacter jejuni with Mucus and Mucins

“…We have shown previously that HT29-MTX-E12 cells can be infected by both H. pylori and C. jejuni (20,21). In this study, C. jejuni infection was enhanced in, but not dependent on, the presence of mucus, whereas H. pylori required the presence of the adherent mucus layer for effective infection.…”

“…Gastric mucins have been shown to promote the proliferation of H. pylori and to alter gene expression by the organism (58). Recently, we have shown that the interaction of H. pylori with the trefoil peptide TFF1, present in the adherent mucus layer of HT29-MTX E12 cells, promotes infection (21). This finding highlights the role of nonmucin components of mucus in mediating the colonization of mucosal surfaces and warrants their further investigation.…”

“…The observation that H. pylori bound poorly to HT29-MTX- E12 mucin was surprising given that the adherent mucus layer of this cell line contains the gastric mucin MUC5AC (21). H. pylori does not colonize the colon or adhere in vivo to colonic cells.…”

“…The LS174T (29) and HT29 (30) human colonic carcinoma cell lines, methotrexate (MTX)-adapted HT29 cells (HT29-MTX) (31), and a mucus-secreting subclone, the HT29-MTX-E12 cell line (32), were used. The HT29-MTX-E12 cell line is a subclone of HT29-MTX cells that expresses the gastric mucin MUC5AC (21), selected on the basis of tight-junction formation and the production of an adherent mucus layer (32). HT29 cells were maintained in McCoy's medium (Lonza) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) nonessential amino acids (Sigma), 2 mM L-GlutaMAX (Invitrogen), 100 U/ml penicillin (Sigma), 100 g/ml streptomycin (Sigma), and 125 g/ml amphotericin B (Sigma).…”

“…These include cell lines that produce adherent mucus layers (20,21) and novel mucin microarrays that contain natural mucins from different animal species (22). This study aimed to examine the interactions of both H. pylori and C. jejuni with mucus and mucins by harnessing these novel techniques.…”