Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis
Fig 5
H. pylori soluble non-proteinaceous metabolite of the HldE-dependent heptose phosphate reaction activates cells when experimentally transfected.
A), B), C) HEK293T luciferase reporter cell transfection with treated lysate ETL preparations of H. pylori strains reveal HldE-dependent activation in a transient transfection setting using NF-κB luciferase plasmid (pNFκB-luc). A) transiently-transfected HEK293T luciferase reporter cells were coincubated for 3 h with live H. pylori of either wild type strain N6, its isogenic heptose core pathway mutants or the cagPAI function-negative mutant HP0527, as indicated, or parental strain 88–3887, its isogenic cagPAI deletion mutant (dcagPAI), or cagA mutant, respectively. B) transiently transfected HEK293T luciferase reporter cells were coincubated with ETL preparations of H. pylori strains, heptose pathway mutants, or cagPAI mutants added to the cell medium in the absence of transfection agent for 3 h (see silver-stained gel of ETL preparations in Fig 1); C) in the third assay set-up, transiently transfected HEK293T cells were super-transfected with ETL preparations of H. pylori strains and their isogenic mutants. In B) and C, ultrapure H. pylori N6 wt LPS was also coincubated or transfected as an additional control. Cells in A), B), C) were incubated prior to the luciferase measurement for 3 h. All assay conditions were measured in triplicates. Two-tailed, non-paired Student’s t-test indicates significant differences of *p<0.05, **p<0.01, ***p<0.001, ns = not significant. D) time-dependent activation of HEK-Blue Null1 SEAP reporter cells (Invivogen) after transfection with treated lysate preparations (ETL) from H. pylori N6 and its isogenic core heptose biosynthesis mutants. Cell activation by release of secreted alkaline phosphatase into the medium was monitored over a time course of 25 hours post transfection using HEK-Blue real time detection medium (Methods). All conditions were assayed in triplicates. Statistical significance of differences in D) was calculated by Student’s t-test for comparisons between mock and all other conditions (blue symbols) and HPN6 wt and all other conditions (red symbols); ns = non-significant; **p<0.01; ***p<0.001; ****p<0.0001.