title: 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.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Samantha Shi Min
authors.initials: SS
authors.lastname: Ling
authors.affiliation: Monoclonal Antibody Unit, Institute of Molecular and Cell Biology, Singapore, Singapore.
authors.forename: Lawrence Han Boon
authors.initials: LH
authors.lastname: Khoo
authors.affiliation: Monoclonal Antibody Unit, Institute of Molecular and Cell Biology, Singapore, Singapore.
authors.forename: Le-Ann
authors.initials: LA
authors.lastname: Hwang
authors.affiliation: Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Khay Guan
authors.initials: KG
authors.lastname: Yeoh
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Bow
authors.initials: B
authors.lastname: Ho
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001426:Bacterial Proteins / Q000502:physiology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D005753:Gastric Mucosa / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D016481:Helicobacter Infections / Q000382:microbiology* / Q000473:pathology; D016480:Helicobacter pylori / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D061251:Primary Cell Culture; D013272:Stomach Diseases / Q000382:microbiology*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005723:gamma-Glutamyltransferase / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C074689:VacA protein, Helicobacter pylori; D005723:gamma-Glutamyltransferase