Sterile cotton swabs

For infection H. pylori wt and H. pylori ΔhtrA were grown for 2 days at 37 °C in anaerobic chambers containing a CampyGen gas mix (Oxoid, Wesel/Germany) (Wiedemann et al., 2012 (link)). H. pylori was harvested and resuspended in phosphate buffered saline (PBS, pH 7.4) using sterile cotton swabs (Carl Roth, Karlsruhe/Germany). The bacterial concentration was measured in a spectrophotometer as optical density (OD) at 600 nm (Eppendorf, Hamburg/Germany). Apical marker expression such as microvilli and tight junction formation were routinely checked as described (Tegtmeyer et al., 2017b (link)). Infections were performed from the apical side at a multiplicity of infection (MOI) of 100 as described (Kim et al., 2013 (link)), unless indicated otherwise. All infection assays were repeated four times.

Human MKN-28 cells (JCRB, #0253) were originally isolated from gastric adenocarcinoma. The Caco-2 cells (ATCC HTB-37) were obtained from a human colon adenocarcinoma. Both cell lines have been extensively used over the last twenty years as models for studying the gastrointestinal barrier. Cells were cultured in 6-well plates with RPMI1640 or DMEM medium, respectively, containing 4 mM glutamine (Invitrogen, Karlsruhe/Germany), and 10% FCS (Invitrogen, Karlsruhe/Germany) [68 (link)]. H. pylori strains 26695, P12 and their mutants were grown on horse serum GC agar plates supplemented with nystatin (1 μg/mL), vancomycin (10 μg/mL) and trimethoprim (5 μg/mL), and if necessary with 4 μg/chloramphenicol per mL. Growth was performed for 2 days at 37 °C in anaerobic chambers containing a CampyGen gas mix (Oxoid, Wesel/Germany) at 37 °C [69 (link)]. H. pylori was harvested and resuspended in phosphate buffered saline (PBS, pH 7.4) using sterile cotton swabs (Carl Roth, Karlsruhe/Germany). The bacterial concentration was measured in a spectrophotometer as optical density (OD) at 600 nm (Eppendorf, Hamburg/Germany). Infections were carried out at a multiplicity of infection (MOI) of 50 [70 (link)]. All infection assays were done in triplicates.

Bacteria (H. pylori wild-type strains and the isogenic ∆cagY Cuz20 mutant) were harvested from agar plates using sterile cotton swabs (Carl Roth, Karlsruhe/Germany) and resuspended in phosphate buffered saline, pH 7.4 (PBS) at concentrations of identical OD₆₀₀. The exact numbers of colony-forming units (CFU) was established by plating serial dilutions on agar plates [55 (link)]. Cells were washed with once with PBS and culture medium devoid of antibiotics or antimycotics and H. pylori was added at an MOI of 100 (approximately 3.5 × 10⁷ CFU/well) in all experiments. The cells were then incubated for 6 h as reported previously [56 (link)]. An uninfected mock control was included where PBS lacking bacteria was added.
For binding assays, after 6 h of incubation the cells were washed three times with 1 mL of pre-warmed culture medium without antibiotics to eliminate non-attached bacteria, after which the cells were lysed by incubation with 1 mL 0.1% saponin in PBS at 37 °C for 15 min [57 (link)]. After collection serial dilutions of the cell lysates were cultivated on GC agar plates for quantification of cell-bound H. pylori CFU.