A role for the tfs3 ICE-encoded type IV secretion system in pro-inflammatory signalling by the Helicobacter pylori Ser/Thr kinase, CtkA

Abstract

Two distinct type IV secretion systems (T4SSs) can be identified in certain Helicobacter pylori strains, encoded on mobile genetic elements termed tfs3 and tfs4. Although their function remains unknown, both have been implicated in clinical outcomes of H. pylori infection. Here we provide evidence that the Tfs3 T4SS is required for activity of the pro-inflammatory Ser/Thr kinase protein, CtkA, in a gastric epithelial cell infection model. Previously, purified recombinant CtkA protein has been shown to upregulate NF-kappaB signalling and induce TNF-alpha and IL-8 cytokine secretion from cultured macrophages suggesting that it may potentiate the H. pylori-mediated inflammatory response. In this study, we show that CtkA expressed from its native host, H. pylori has a similar capacity for stimulation of a pro-inflammatory response from gastric epithelial cells. CtkA interaction was found to be dependent upon a complement of tfs3 T4SS genes, but independent of the T4SSs encoded by either tfs4 or the cag pathogenicity island. Moreover, the availability of CtkA for host cell interaction was shown to be conditional upon the carboxyl-terminus of CtkA, encoding a putative conserved secretion signal common to other variably encoded Tfs3 proteins. Collectively, our observations indicate a role for the Tfs3 T4SS in CtkA-mediated pro-inflammatory signalling by H. pylori and identify CtkA as a likely Tfs3 T4SS secretion substrate.

Fig 1. Genetic context of ctkA in H. pylori tfs3 ICE gene clusters.

(A) The tfs3 ICE of H. pylori strain PeCan18b. Genes are annotated according to the ‘pz’ gene designation (pz1-41) initially used to describe the tfs3 gene cluster of strain PeCan18b. Corresponding genes in selected reference genomes are provided in S3 Table. vir-homologous genes (virB2-11 [T4SS assembly], virD4, virD2 [relaxase], virC1 [ancillary relaxosome protein]), encoding key components associated with the putative Tfs3 T4SS are coloured red and labelled accordingly. (B) The variable context of ctkA within comparable ends of the tfs3 ICE gene cluster is shown for representative strains with reference to a strain lacking ctkA (PeCan18). Additional ctkA+ strain genomes in GenBank with an equivalent organisation of genes to those shown are indicated in parentheses after the named reference strains and are as follows; CPY1313 (CPY6261, UM038, UM066, UM085, UM370, UM411, FD577, FD703, A-27, L7, 83, 22, J99), OK310 (UM303R/S, BM013A/B, FD506, ML3, 59, F32, UM096), PZ5080 (NQ4076, NQ4099, NQ4200) and SJM180 (Puno135), Puno120, PZ5026 (H-1, H-16, H-18, H-36, P-2, P-26, Aklavik117, Shi112, PeCan4), H. cetorum (strains MIT 00–7128 and MIT 99–5656). NCBI GenBank Accession numbers for strain sequences shown are; PeCan18 (AF487344 and CP00347), CPY1313 (AKNK01), OK310 (AP012601), PZ5080 (ASYV01), SJM180 (CP002073), Puno120 (CP002980), PZ5026 (ASYT01) and Helicobacter cetorum MIT 00–7128 (CP003479).

Fig 2. Sequence alignments showing conservation of C-terminal sequence between tfs3 ICE-encoded proteins.

Sequence alignment demonstrates conservation in the last 25 C-terminal amino acid residues of CtkA, PZ39 and Fic proteins encoded in tfs3 gene clusters from a representative selection of H. pylori strains.

Fig 3. CtkA expressed from H. pylori stimulates pro-inflammatory cytokine secretion from gastric epithelial cells.

Wild-type H. pylori, cagE-deficient strains and isogenic derivatives constitutively expressing GSK-CtkA were assessed for their ability to induce IL-8 secretion from MKN28 cells in co-culture. Supernatants were sampled 48 h post infection and assayed for IL-8 secretion. In comparison with the parent cagE mutants alone, all tfs3(+) strains expressing GSK-CtkA (AB31, 10A, AB5) showed a trend towards induction of elevated levels of IL-8 when expressing GSK-CtkA, whereas a tfs3(-) strain (64) did not. The difference in levels of induced IL-8 due to GSK-CtkA expression was significant when expressed from strain AB5. Levels of IL-8 attributable to GSK-CtkA were modest relative to cagPAI-mediated responses from all wild-type strains. Graph shows means and SDs from three independent experiments, each performed in triplicate.

Fig 4. Signalling pathways involved in CtkA-mediated stimulation of IL-8 secretion.

MKN28 cell monolayers remained untreated (-), or were treated with chemical inhibitors for NF-κB (6 amino-4-(4-phenoxyphenylethylamino) quinazoline), JNK (SP600125) and MEK1 (U0126) for 1 h before and during infection with H. pylori strain AB5ΔcagE::gsk-ctkA. IL-8 levels were subsequently assessed by ELISA. Basal levels of IL-8 detected in uninfected supernatants are also indicated for reference (‘Cells’). Graph shows means and SDs from three independent experiments, each performed in triplicate. p values indicate significant differences in the presence of inhibitor compared with infection alone.

Fig 5. Pro-inflammatory signalling in response to CtkA requires the Tfs3 type IV secretion system and the C-terminus of CtkA.

MKN28 cells were co-cultured with H. pylori AB5ΔcagE derivative strains (MOI = 20) for 48 or 72 h prior to determination of IL-8 concentrations in supernatants by ELISA. Both inactivation of (A) tfs3 virB9 and (B) deletion of the last 23 C-terminal amino acid residues of CtkA in strain AB5ΔcagE::gsk-ctkA(1–906) resulted in abrogation of IL-8 secretion. (C) Similar effects were observed following co-culture of the complement of AB5 strains with gastric AGS cells although IL-8 responses for all strains were markedly lower. Graph shows means and SDs from three independent experiments, each performed in triplicate. p values indicate significant differences in IL-8 levels compared to the AB5ΔcagE::gsk-ctkA strain. (D) Western immunoblot analysis of 100X concentrated supernatants from H. pylori strains AB5ΔcagE, AB5ΔcagE:gsk-ctkA, AB5ΔvirB9ΔcagE:gsk-ctkA and AB5ΔcagE:gsk-ctkA_1-906 (Lanes 1–4 respectively) grown in RPMI 1640. Blots were probed with anti-GSK, anti-GAPDH and anti-CagA specific antiserum. Detection of GAPDH and CagA in supernatants indicates non-secretory release of intracellular protein in all strain backgrounds.