Analysis of Helicobacter pylori binding site on HEp‐2 cells and three cell lines from human gastric carcinoma

Abstract

Helicobacter pylori (H. pylori) is a pathogen responsible for chronic gastritis and peptic ulcer diseases. It colonises the gastric mucus layer and adheres to the gastric epithelial cell surface. As this adherence is the first step of infection, it is important to study the adherence mechanism. The aim of this study was to analyse the specific binding assay of H. pylori to HEp-2 cells and three gastric phenotype cell lines, AGS, MKN-45 and AZ-521. H. pylori NCTC 11637 grown on agar plates was harvested and used in experiments. H. pylori was inoculated to pre-cultured cell monolayers. Adhered bacteria were labelled with an anti-H. pylori antibody and an FITC-conjugated secondary antibody and quantified by using a fluorescent plate reader. Microbial adherence to HEp-2 cells increased with incubation time and incubated concentration of H. pylori. No further increase was obtained with four or more hours of incubation or with a concentration of 4 x 10(7) bacteria/well or more. Scatchard analysis revealed a linear plot and the Bmax value was 88.3. Similar adherence patterns were obtained when AGS, AZ-521 and MKN-45 cells were used for adherence assays, but they had a lower binding affinity than HEp-2 cells and AZ-521. MKN-45 cells had less receptors than HEp-2 and AGS cells. In conclusion, H. pylori adhered to the cell surface could be quantified by this assay method. H. pylori adhesion to cell surfaces has a single population of binding site and one type of binding site on HEp-2, AGS, AZ-521 and MKN-45 cells.

Host Cell Contact Induces Fur‐dependent Expression of Virulence Factors CagA and VacA in Helicobacter pylori

“…It has been reported that in addition to human gastric cells, H. pylori can also efficiently adhere to other cell lines [37,38]. Indeed, the adherence of H. pylori to HeLa and Hep-2 cell lines was similar to the AGS cell line (Table S2).…”

Lafutidine inhibits Helicobacter pylori‐induced interleukin‐8 production in human gastric epithelial cells

“…Adherence was assayed as previously reported 14 . Briefly, cells grown on a 24‐well plate were stimulated with H. pylori (2 × 10 7 CFU/mL) at 37°C in serum‐free RPMI 1640 for 3 h. To determine the effect of drugs, serum‐free RPMI 1640 with and without drugs was added to the cells 1 h before the addition of H. pylori .…”

“…Adherence was assayed as previously reported. 14 Briefly, cells grown on a 24-well plate were stimulated with H. pylori (2 ¥ 10 7 CFU/mL) at 37∞C in serumfree RPMI 1640 for 3 h. To determine the effect of drugs, serum-free RPMI 1640 with and without drugs was added to the cells 1 h before the addition of H. pylori. The medium and unbound H. pylori were discarded and the cells with bound H. pylori were washed with assay medium and fixed with 10% formaldehyde.…”

Identification of two new Helicobacter pylori surface proteins involved in attachment to epithelial cell lines

“…Antibodies to bacterial surface adhesins may also prove valuable in the selective blocking of specific adhesins, and mutational inactivation of discrete adhesin genes may provide additional information. However, these approaches are unlikely to give distinct results for micro-organisms such as Helicobacter pylori that express multiple strong adhesins on their surface, and where additional adhesive proteins may become involved subsequent to the initial attachment (Nishihara et al, 1999;Palovuori et al, 2000;Su et al, 1998).…”