Instrumental Role of Helicobacter pylori γ-Glutamyl Transpeptidase in VacA-Dependent Vacuolation in Gastric Epithelial Cells

Abstract

Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.

Fig 1. Live-cell micrographs of H. pylori-infected AGS cells.

AGS cells were infected with H. pylori WT or Δggt at MOI of 1:100 over a 24 hour period and the process of infection was recorded using Biostation IMQ. Uninfected cells served as control. Micrographs shown here are at 2h, 12h, 16h and 24h time-points. Perinuclear vacuoles are indicated with red arrows. Large vacuoles are indicated with yellow arrows. Insets show a close-up of vacuole position and size in AGS cells. (See S2 Fig and S1–S3 Videos respectively for the full time course).

Fig 2. GGT contributes to vacuolation induced by H. pylori.

Phase-contrast micrographs of (A) AGS cells and (B) primary gastric epithelial cells before and after neutral red uptake assay following co-culturing at MOI of 1:100 with H. pylori WT or Δggt for 24 hours. Uninfected cells (i), WT-treated cells (ii) or Δggt-treated cells (iii) were observed using phase contrast microscopy. The cells were subjected to neutral red dye assay (iv, v and vi, respectively) and observed under the microscope. Original magnification, ×400. Extent of vacuolation is indicated by the amount of neutral red retained within the cells. (C) AGS cells were co-cultured with WT H. pylori (■) or Δggt (□) at MOI 100 for various time-points up to 24 hours. (▲) indicates uninfected cells. The cells were then subjected to neutral red dye uptake assay. (D) Primary gastric epithelial cells were co-cultured with H. pylori WT or Δggt for 24 hours and subjected to neutral red uptake assay. Experiments were performed in triplicates and values represent the means ± SD from 3 independent experiments. *P<0.05.

Fig 3. H. pylori GGT potentiates VacA-dependent vacuolation induction in gastric epithelial cells through glutamine hydrolysis.

(A) AGS cells were co-cultured with H. pylori WT, Δggt, ΔvacA, ΔvacA/ggt (□) and/or rGGT (■) for 24 hours. Untreated cells (UN) served as control. (B) AGS cells were co-cultured with WT, Δggt or ΔvacA at MOI 1:100 for 24 hours in the presence (■) or absence (□) of 2mM glutamine. (C) AGS cells were co-cultured with WT, Δggt or ΔvacA at MOI 1:100 for 24 hours in the absence or presence of 1-3mM ammonium chloride. The cells were then subjected to neutral red dye uptake assay. (D) AGS cells were co-cultured with H. pylori WT or Δggt for 24 hours in the presence (■) of 2mM glutamine or in glutamine-free media (□). Concentration of ammonia in the spent culture media was measured using an ammonia assay kit. Experiments were performed in triplicates and values represent the means ± SD from 3 independent experiments. *P<0.05.

Fig 4. Role of GGT and urease in VacA-mediated vacuolation.

AGS cells were co-cultured with WT, Δggt, ΔvacA, ΔureAB or ΔureAB/ggt at MOI 1:100 for 24 hours. Vacuolating activity was measured using neutral red dye uptake assay. Experiments were performed in triplicates and values represent the means ± SD from 3 independent experiments. *P<0.05.

Fig 5. Neutralizing ability of GGT MAbs on rGGT and H. pylori 88–3887 GGT activity.

(A) rGGT (5mU) or (B) a population of 5 × 10⁶ H. pylori WT was pre-incubated with various amounts of MAb 1G1 (■), 4F11 (□), 3F4 (▲) or 3C10 (Δ) (0 to 30μg of IgG protein) for 1 hour at 37°C on microtitre plates. Mouse IgG_2a (●) and IgG_2b (○) isotype controls were used in parallel at the same concentrations. Inhibition of GGT activity by MAbs was measured using GGT assay and values represent GGT activity inhibition (expressed as a percentage) compared to the control where no MAb was added. Experiments were performed in triplicates and values represent the means ± SD from 3 independent experiments.

Fig 6. Inhibition of GGT activity decreases H. pylori-induced vacuolation.

(A) H. pylori WT was pre-incubated with GGT MAbs (10μg) for 1 hour before they were used to infect AGS cells for another 24 hours. Mouse IgG_2a and IgG_2b isotype controls were used in parallel at the same concentration. (B) AGS cells were co-cultured with H. pylori WT or Δggt in the absence or presence of SBC (2-10mM). Uninfected cells (UN) served as control. The cells were then subjected to neutral red dye uptake assay. Experiments were performed in triplicates and values represent the means ± SD from 3 independent experiments. *P<0.05.