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.

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability

“…In these experimental systems, ammonia is present at concentrations sufficiently high to influence VacA-induced vacuolation (45). In addition, ammonia-producing H. pylori enzymes, such as urease, ␥-glutamyl transpeptidase, asparaginase, and glutaminase, are also present (46,60). Therefore, previous studies using H. pylori strains or BCF were not able to assess the effect of VacA on host cells in the absence of ammonia.…”

“…Consequently, in experimental studies in which cells are treated with purified VacA, the cell culture medium is often supplemented with NH 4 Cl. The presence of weak bases in cell culture medium may mimic the conditions in the stomach during H. pylori infection, as H. pylori generates ammonia in vivo through the actions of urease and other enzymes, such as ␥-glutamyl transpeptidase, asparaginase, and glutaminase (44)(45)(46). In this study, we investigated the mechanism(s) by which NH 4 Cl influences the magnitude of VacA-induced cell death.…”

A simple and cost‐efficient adherent culture platform for human gastric primary cells, as an in vitro model for Helicobacter pylori infection

“…The development of primary gastric culture systems is beneficial for the study of a wide array of research questions, the most popular of which is the interaction of H. pylori with normal cells that line the stomach lumen. Considering that our cell culture system (GPCs) was fully established in vitro, we have been able to develop an extended model (up to 96 hours) for H. pylori infection, whereas previously reported cell culture systems were mostly of short term (≤24 hours). In our model, we demonstrated a clear tropism of H. pylori cells for gastric epithelial islands (and not the feeder cells), which underwent the expected vacuolation and elongation following H. pylori infection.…”

Helicobacter pyloriouter inflammatory protein A (OipA) suppresses apoptosis of AGS gastric cells in vitro

“…pylori virulence factors, host factors, and environmental factors (El‐Omar et al, ). Among bacterial virulence factors, the virulence factor vacA was shown to be involved in cell vacuolation in vitro (Ling, Khoo, Hwang, Yeoh, & Ho, ) and reported to be implicated in several clinical conditions (Sahara et al, ; Zhang et al, ). The vacuolating events are linked to changes in the vacA structures (Atherton et al, ).…”

“…The risk of developing of chronic gastritis and progressing to severe atrophic gastritis as a result of H. pylori infection differs significantly from person to person due to differences in several factors, including H. pylori virulence factors, host factors, and environmental factors (El-Omar et al, 2003). Among bacterial virulence factors, the virulence factor vacA was shown to be involved in cell vacuolation in vitro (Ling, Khoo, Hwang, Yeoh, & Ho, 2015) and reported to be implicated in several clinical conditions (Sahara et al, 2012;Zhang et al, 2016). The vacuolating events are linked to changes in the vacA structures (Atherton et al, 1995).…”