Bacterial Energetic Requirements for Helicobacter pylori Cag Type IV Secretion System-Dependent Alterations in Gastric Epithelial Cells

Abstract

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.

Keywords: ATPases; Helicobacter pylori; gastric cancer; secretion systems.

FIG 1

Individual Cag T4SS ATPases are not required for assembly of the Cag T4SS core complex. H. pylori mutant strains lacking genes encoding individual ATPases (Δcagα, Δcagβ, and ΔcagE) were modified by introducing a gene encoding HA-CagF into the ureAB chromosomal locus, resulting in strains with the genotypes indicated in the figure (corresponding to FC1.1 [Δcagα/HA-CagF], FC2.1 [Δcagβ/HA-CagF], and FC3.1 [cagE::kan/HA-CagF] in Table 1). T4SS core complexes were immunopurified from these strains, and the preparations were analyzed and visualized by negative-stain electron microscopy (magnification, ×28,000). (A) Core complexes purified from a wild-type (WT) strain engineered to produce HA-CagF (WT/HA-CagF) (positive control). (B to D) Core complexes isolated from mutant strains engineered to produce HA-CagF (Δcagα/HA-CagF, Δcagβ/HA-CagF, and cagE::kan/HA-CagF). Scale bars, 25 nm.

FIG 2

Individual Cag T4SS ATPases are essential for CagA translocation into AGS gastric epithelial cells. Wild-type (WT) strain 26695, a Δcag PAI mutant strain, and the indicated unmarked deletion mutant strains (Δcagα, Δcagβ, and ΔcagE) were cocultured with AGS cells. Genetically manipulated strains containing restored wild-type ATPase sequences (named ASL12.1 [restored WT cagα], ASL14.1 [restored WT cagβ], and ASL16.1 [restored WT cagE]; Table 1) were tested as controls. Extracts from H. pylori-gastric epithelial cell cocultures were immunoblotted with an anti-CagA antibody to detect CagA and an anti-phosphotyrosine antibody (anti-PY99) to detect phosphorylated CagA.

FIG 3

Cagα and CagE, but not Cagβ, are required for three Cag T4SS-dependent alterations in host cells. Wild-type (WT) strain 26695, a Δcag PAI mutant strain, and the indicated unmarked deletion mutant strains (Δcagα, Δcagβ, and ΔcagE) were cocultured with AGS cells, AGS-NF-κB reporter cells, or HEK293-hTLR9 reporter cells. Genetically manipulated strains containing restored wild-type ATPase sequences (named ASL12.1, ASL14.1, and ASL16.1; Table 1) were tested as controls. IL-8 production, NF-κB activation, or TLR9 activation were quantified as described in Materials and Methods. (A, B) Cagα and CagE are required for IL-8 induction and NF-κB activation in AGS gastric epithelial cells. (C) Cagα and CagE are required for H. pylori-induced TLR9 activation in HEK293-hTLR9 reporter cells. The data represent results of three independent experiments with multiple technical replicates. Values represent means ± standard error of the mean (SEM). Statistical significance among groups was determined by Kruskal-Wallis test with Dunnett’s multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 compared to WT.

FIG 4

Functional Walker motifs in individual Cag T4SS ATPases are required for CagA translocation into AGS gastric epithelial cells. Strains containing mutations in Walker motifs of Cagα, Cagβ, or CagE (cagα WB, cagβ WB, and cagE WB mutants, corresponding to ASL11.1, ASL13.1, or ASL 15.1, respectively, in Table 1) were generated as described in Materials and Methods. Wild-type (WT) strain 26695, a Δcag PAI mutant strain, and the indicated mutant strains were cocultured with AGS cells. Genetically manipulated strains containing restored wild-type ATPase sequences (named ASL12.1, ASL14.1, and ASL16.1; Table 1) were tested as controls. Extracts from H. pylori-gastric epithelial cell cocultures were immunoblotted with an anti-CagA antibody to detect CagA and an anti-phosphotyrosine antibody (anti-PY99) to detect phosphorylated CagA.

FIG 5

Functional Walker motifs in Cagα and Cagβ are required for three Cag T4SS-dependent phenotypes. Wild-type strain 26695, a Δcag PAI mutant, and strains containing mutations in Walker motifs of Cagα, Cagβ, or CagE (ASL11.1, ASL13.1, or ASL 15.1; Table 1 and Fig. S1) were cocultured with AGS cells, AGS-NF-κB reporter cells, or HEK293-hTLR9 reporter cells. IL-8 production, NF-κB activation, or TLR9 activation were quantified as described in Materials and Methods. (A, B, and C) Functional Walker motifs in Cagα and CagE are essential for H. pylori-induced IL-8 induction, NF-κB activation, and TLR9 activation. The data represent results of three independent experiments with multiple technical replicates. Values represent mean ± standard error of the mean (SEM). Statistical significance among groups was determined by Kruskal-Wallis test with Dunnett’s multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 compared to WT.