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

• Published in: PloS one Vol. 10; no. 6; p. e0131460
• Main Authors: Ling, Samantha Shi Min, Khoo, Lawrence Han Boon, Hwang, Le-Ann, Yeoh, Khay Guan, Ho, Bow
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 25-06-2015; Public Library of Science (PLoS)
• Subjects: Ammonia; Ammonium; Ammonium chloride; Antibodies, Monoclonal - pharmacology; Bacterial Proteins - physiology; Cells, Cultured; Cytotoxicity; Deoxyribonucleic acid; DNA; Enzymes; Epithelial cells; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - microbiology; Epithelial Cells - pathology; g-Glutamyltransferase; gamma-Glutamyltransferase - immunology; gamma-Glutamyltransferase - pharmacology; gamma-Glutamyltransferase - physiology; Gastric Mucosa - drug effects; Gastric Mucosa - metabolism; Gastric Mucosa - microbiology; Gastric Mucosa - pathology; Glutamine; Helicobacter Infections - microbiology; Helicobacter Infections - pathology; Helicobacter pylori; Helicobacter pylori - enzymology; Helicobacter pylori - physiology; Humans; Infections; Laboratory animals; Medicine; Microscopy; Monoclonal antibodies; Primary Cell Culture; Proteins; Recombinant; Serine; Severe acute respiratory syndrome; Stomach Diseases - microbiology; Substrates; Ulcers; Vacuoles; Vacuoles - drug effects; Vacuoles - metabolism; Vacuoles - pathology; Virulence; Virulence factors; γ-Glutamyltransferase; Singapore
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0131460

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.