Characterization of the extracellular mucus layer of HT29-MTX-E12 cells

<p>. Frozen sections of 21 day old HT29-MTX-E12 cells were stained with (A) PAS and haematoxylin or with specific antibodies against (B) MUC5AC, and trefoil peptides, (C) TFF1, and (D) TFF3. An adherent extracellular mucus layer could be visualised using PAS and haematoxylin staining. MU5AC, TFF1 and TFF3 were detected in this extracellular mucus.</p

Oligonucleotide primers used for reverse transcriptase PCR.

<p>Oligonucleotide primers used for reverse transcriptase PCR.</p

Colonization of HT29-MTX-E12 cells by <i>H. pylori</i>

<p>. (A and B) Immunofluorescent microscopy was used to visualise <i>H. pylori</i> colonizing HT29-MTX-E12 cells. <i>H. pylori</i> strain PU4 was used to infect HT29-MTX-E12 cells for 24 h after which time frozen sections were stained with specific antibodies against <i>H. pylori</i> (red) and MUC5AC or MUC1 (green). DAPI (blue) staining was used to visualise cell nuclei. Organisms were found in discrete foci or clusters throughout the mucus layer and close to the epithelial cells. (C) Colonization of 21 day old HT29-MTX-E12cells with <i>H. pylori</i> strain PU4 over a 24 h period of infection. There were significantly more organisms associated with the cells after 24 h of infection compared with 2 h or 4 h of infection, (* p≤0.05). (D) Replication of <i>H. pylori</i> strain PU4 co-cultured with 21 day old HT29-MTX-E12 cells over a 24 h period. Cells were infected with <i>H. pylori</i> for 2 h after which time the cells were washed thoroughly to remove any non adherent bacteria. Cells were incubated for a further 22 h and the total number of bacteria associated with the cells was determined. The number of bacteria associated with the cells was significantly increased at the 24 h time point compared to the 2 h time point, indicating that the organism reproduced while associated with the cells (*p≤0.05). Error bars indicate mean ± Standard Deviation of three separate experiments.</p

TFF1 in HT29-MTX-E12 cells interacts with <i>H. pylori</i> LPS.

<p>A <i>H. pylori</i> total cell lysate (Lane 1)and purified LPS (Lane 2) were electrophoresed on SDS PAGE and (A) stained with Coomassie blue which stains proteins or alcian blue which stains carbohydrate moieties or (B and C) transferred onto PVDF membrane and probed with a total cell lysate made from uninfected HT29-MTX-E12 cells. The filter was then probed with either an antibody to TFF1 (B) or an antibody to TFF3 (C) and a horseradish peroxidase conjugated secondary antibody. Strong TFF1 binding and weak TFF3 binding to low molecular weight RF LPS of <i>H. pylori</i> was detected using the ECL system from Amersham. Identical overlay blots (controls) were probed with just the secondary antibody. This confirmed that only the low molecular weight (6 kDa) band was specific for TFF1 and TFF3 binding.</p

Tight junction formation in HT29-MTX-E12 cells over a 21 day period of differentiation.

<p>(A) Transepithelial electrical resistance values (Ω/cm²) were measured at different timepoints throughout a 21 day period of differentiation. (B) ZO-1 staining of HT29-MTX-E12 cells at day 21. Error bars indicate mean ± Standard Deviation of three separate experiments.</p

The interaction of <i>H. pylori</i> with TFF1 in HT29-MTX-E12 cells.

<p>HT29-MTX-E12 cells grown for 21 days on transwell filters were infected with <i>H. pylori</i> for 24 h. Infected cells were stained with antibodies against <i>H. pylori</i> (red) and against TFF1 (green). DAPI staining (blue) was used to visualise cell nuclei.</p

<i>H. pylori</i> mutants producing truncated LPS do not interact with TFF1 and exhibit reduced colonization of HT29-MTX-E12 cells.

<p>(A) LPS from <i>H. pylori</i> strains PA4 and PA4ΔHP1191 was run on SDS PAGE and stained with alcian blue or blotted onto PVDF membrane and probed with either anti Lewis x antibody or with dimeric TFF1. (B) <i>H. pylori</i> strains PA4ΔHP1191 and P12ΔHP1191 exhibited significantly reduced colonization of HT29-MTX-E12 cells compared with WT parental strains (*p<0.05). (C) Incubation of cells with purified LPS from wild type strain PA4 but not with LPS from strain PA4ΔHP1191 reduced subsequent colonization of the cells with strain PA4 (*p<0.05).</p

Cellular localization of MUC1, MUC2, MUC5AC, TFF1, TFF2 and TFF3 in HT29-MTX-E12 cells.

<p>Paraffin embedded HT29-MTX-E12 cells grown on transwell filters for 21 days were stained with specific antibodies for MUC1, MUC2, MUC5AC, TFF1, TFF2 and TFF3.</p

Expression and Characterization of a Novel Recombinant Version of the Secreted Human Mucin MUC5AC in Airway Cell Lines

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins’ size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease

The effect of TFF1 and copper on <i>H. pylori</i> colonization of AGS-AC1 cells.

<p>AGS-AC1 cells, induced and uninduced, were infected with wild type strain (P12), or mutant strain P12ΔHP1191. The infections were carried out in the presence of Cu 100 µM or BCS 500 µM. Adherence was evaluated in comparison to control untreated AGS-AC1 cells. <b>a:</b> Effects of TFF1 and copper on <i>H. pylori</i> P12 cell adhesion. <b>b:</b> Effects of copper on <i>H. pylori</i> P12 cell adhesion. <b>c:</b> Effects of TFF1 and/or BCS on <i>H. pylori</i> P12 cell adhesion. <b>d:</b> Effects of TFF1 and/or copper on <i>H. pylori</i> P12ΔHP1191 cell adhesion.</p