In this report, we demonstrate that the major virulence genes of H. pylori, cagA and vacA, are strongly induced in bacteria associated with the gastric epithelial cell line AGS. Fur that acts as a global BMN 673 regulator in H. pylori  has a role in the AGS cell contact-dependent upregulation of cagA and vacA. The H. pylori strain 26695 was grown in GC agar medium (BD Difco, Franklin Lakes, NJ, USA) supplemented with 6.8% defibrinated horse blood (Remel, Lenexa, KS, USA), 8% horse serum (GIBCO, Grand Island, NY, USA), L-cysteine hydrochloride monohydrate (MP Biomedicals, Santa Ana, CA, USA) and IsoVitaleX (BD BBL, Sparks,
MD, USA) at 37 °C under microaerobic conditions (7.5% CO2, 5% O2
in air) and subcultured every 2 days. Helicobacter pylori cultures growing in GC agar plates were enumerated by CFU assay every 24 hours for up to 96 hours. As has been reported previously , the cultures attained the mid-logarithmic phase of growth at 48 hours; find more hence, H. pylori cultures grown on GC agar plates for 48 hours were used in this study. The bacteria were maintained as frozen stocks at −80 °C in brain heart infusion medium supplemented with 20% glycerol. AGS cells were grown to about 75% confluency in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and washed thrice with PBS, and RPMI with 10% FBS was added. Helicobacter pylori cultures grown in GC agar plates for 48 hours and suspended in RPMI were added
to the AGS monolayer at multiplicity of infection (MOI) 50 and incubated at 37 °C under microaerobic conditions for the desired period of time. At the end of the incubation period, unadhered bacteria in the supernatant were removed, the H. pylori-infected AGS cell line was washed three to four times with PBS, and the adhered bacteria were released by lysing the AGS cells with 1% Triton X-100 in PBS for about 5 minutes followed by vigorous pipetting. The bacterial CFU was determined by serial dilution and plating on BHI agar plates. It was separately confirmed that treatment with 1% Triton X-100 had no effect Bay 11-7085 on the viability or gene expression in H. pylori. Adherence of H. pylori to HeLa and Hep-2 cells was performed similarly. Parallel experiments were performed with H. pylori under identical conditions but without cell line. In some experiments, the iron chelator 2,2′-dipyridyl (dpp) was added at a concentration of 200 μmol/L during adherence assays. All experiments were repeated at least thrice, and the data are expressed as means ± standard deviation (SD). The statistical significance of the data was analyzed using the Students t-test, and p values <.05 were considered significant. RNA was extracted from unadhered and adhered H. pylori using TRIzol reagent (Gibco BRL) following the manufacturer’s instructions.