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.

Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex

“…Cag T4SS complexes were purified from either a wild-type H. pylori strain or a ∆cag3 mutant strain, each engineered to produce an epitope-tagged form of CagF, as described previously ( Chung et al, 2019 ; Frick-Cheng et al, 2016 ). The resulting preparations were analyzed by SDS-PAGE and colloidal Coomassie blue staining, and the composition of the preparations was determined by LC-MS/MS analysis ( Frick-Cheng et al, 2016 ; Lin et al, 2020 ).…”

“…Coomassie blue staining, and the composition of the preparations was determined by LC-MS/MS analysis (Frick-Cheng et al, 2016;Lin et al, 2020).…”

Type IV secretion systems: Advances in structure, function, and activation

“…through DNA translocation (Cover et al, 2020;Lin et al, 2020;Varga et al, 2016). T4SS Cag -mediated translocation of HBP was implicated in activation of the NFk B pathway, and HBP was thus designated as a new pathogen-associated molecular pattern or PAMP (Zimmermann et al, 2017).…”

“…Translocation of CagA induces various changes in eukaryotic target cells, including but not limited to cytoskeletal alterations and changes in cell polarity and adhesion, disruption of cell–cell junctions and increased cell motility and cell migration (Backert and Tegtmeyer, 2017; Hatakeyama, 2014). Independently of CagA translocation and, curiously, VirD4‐like Cagβ, the T4SS Cag stimulates interleukin‐8 (IL‐8) induction (Fischer et al., 2001), and also activates the Toll‐like receptor 9 (TLR9) through DNA translocation (Cover et al., 2020; Lin et al., 2020; Varga et al., 2016). T4SS Cag ‐mediated translocation of HBP was implicated in activation of the NF‐ k B pathway, and HBP was thus designated as a new pathogen‐associated molecular pattern or PAMP (Zimmermann et al., 2017).…”

Temporal Control of the Helicobacter pylori Cag Type IV Secretion System in a Mongolian Gerbil Model of Gastric Carcinogenesis

“…CagA translocation assay. CagA translocation into AGS gastric epithelial cells was analyzed using previously described methods (84)(85)(86)(87)(88)(89)(90). Briefly, H. pylori strains were cocultured with AGS cells at a MOI of 100:1 for 4 to 6 h at 37°C.…”