Unique TLR9 Activation by Helicobacter pylori Depends on the cag T4SS, But Not on VirD2 Relaxases or VirD4 Coupling Proteins

Abstract

The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.

Fig. 1

Genetic composition of three T4SSs in H. pylori strain P12. Conserved structural components VirB2-VirB4 and VirB7-VirB11 of the T4SSs, as well as DNA coupling proteins TraG/VirD4, DNA relaxases Rlx/VirD2 and recombinase XerD are highlighted. The TFS3 and TFS4 VirB6 proteins are located within the blanked out regions, in the cagPAI VirB6 is represented by CagW. The P12 locus tag numbers are indicated next to the T4SS. The fourth H. pylori T4SS (the ComB system) was not included in this alignment, because it facilitates the import of free DNA, which was not subject of this study

Fig. 2

Stimulation of TLR9 activation by T4SS-positive type-I H. pylori strains. A Infection of AGS cells with various H. pylori wt strains. In contrast to type-II strains, type-I H. pylori isolates express a functional T4SS that enables injection and subsequent phosphorylation of CagA in the cytoplasm. CagA phosphorylation and expression was monitored by Western blot analysis of corresponding protein lysates using antibodies against PY99 and CagA. The asterisk in panel A indicates an unidentified phosphorylated 125 kDa protein of the host cell. GADPH was used as loading control. B Quantification of TLR9 activation by the SEAP reporter assay. C IL-8 secretion measured by ELISA. Quantitative data are presented as means ± SD. Safr7: SouthAfrica7

Fig. 3

TLR9 activation by H. pylori requires the cagPAI, but not CagA, ADP-heptose or peptidoglycan effector molecules. A Infection of AGS cells with the parental wt strain P12 and isogenic mutants. Western blots of corresponding protein lysates were analyzed with the antibodies shown in the panel. α-PY99: phosphorylated CagA; α-CagA: total CagA; α-GAPDH: loading control. The asterisk in panel A indicates an unidentified phosphorylated 125 kDa protein of the host cell. B Quantification of TLR9 activation by the SEAP reporter assay. C IL-8 secretion measured by ELISA. Quantitative data are presented as means ± SD

Fig. 4

TLR9 activation by H. pylori does not involve VirD2 relaxases, VirD4 coupling proteins or XerD recombinase. A Infection of AGS cells with the parental wt strain P12 and isogenic mutants. Western blots of corresponding protein lysates were analyzed with the antibodies shown in the panel. α-PY99: phosphorylated CagA; α-CagA: total CagA; α-GAPDH: loading control. The asterisk in panel A indicates an unidentified phosphorylated 125 kDa protein of the host cell. B Quantification of TLR9 activation by the SEAP reporter assay. C IL-8 secretion measured by ELISA. Quantitative data are shown as means ± SD