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Title:Understanding the molecular mechanism on cell surface receptor interaction of helicobacter pylori vacuolation cytotoxin
Author(s):Oh, Seung Jin
Director of Research:Blanke, Steven R.
Doctoral Committee Chair(s):Blanke, Steven R.
Doctoral Committee Member(s):Wilson, Brenda A.; Morrissey, James H.; Fratti, Rutilio A.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Vacuolating cytotoxin (VacA)
Helicobacter pylori
Bacterial toxin
Toxin-receptor interaction
Toxin-membrane interaction
Abstract:Chronic infection with the human specific gastric pathogen Helicobacter pylori (H. pylori) is a risk factor for the development of gastric ulcer disease and gastric adenocarcinoma. The vacuolating cytotoxin (VacA) is a secreted toxin of H. pylori, initially discovered as the proteinacious factor within H. pylori culture filtrates that intoxicates epithelial cells and induces vacuole biogenesis. Throughout 25 years of VacA research, several interesting and important properties of VacA have become apparent; the gene encoding VacA (vacA) is characterized by a high degree of genetic variation, and experimental evidence strongly supports the idea that VacA promotes colonization and persistence of H. pylori, and the pathophysiology associated with H. pylori infection. At cellular level, VacA functions as an endomembrane channel subsequent to internalization into a host cell and targets the mitochondria to modulate host cell physiology. An abundant cell surface membrane sphingolipid, sphingomyelin (SM), functions as a cell surface receptor, mediating cellular activity of VacA. However, major gaps in knowledge include the molecular detail on the interaction between VacA and its cell surface receptor, and the contribution of the VacA-receptor interaction to the high degree of genetic variation on vacA. In this dissertation, I report that three VacA residues, arginine 552 (Arg-552), tryptophan 603 (Trp-603), and arginine 647 (Arg-647), are the active receptor-interacting residues important for VacA-mediated cellular activity and SM-interaction. A mutant form of VacA with alanine substitution on the three VacA residues, VacA (R552A/W603A/R647A), showed dramatic attenuation of vacuole biogenesis on human epithelial-derived cells, suggesting the importance of the three VacA residues for cellular activity. VacA (R552A/W603A/R647A) binding to SM-coated plates was attenuated relative to wildtype VacA, demonstrated the importance of the three VacA residues for SM interaction. Binding of VacA (R552A/W603A/R647A) to the plasma membrane of the epithelial-derived cells was reduced relative to wildtype VacA, suggesting the importance of the three VacA residues for cell surface binding. A larger fraction of total membrane bound wildtype VacA than VacA (R552A/W603A/R647A) bound to a specific component or components on the surface of cultured cells, suggesting the importance of the three VacA residues for receptor-mediated plasma membrane binding of VacA on the cells. Also, a smaller fraction of total membrane bound VacA (R552A/W603A/R647A) than wildtype VacA partitioned to detergent resistant membranes (DRMs), suggesting that the three VacA residues are important for VacA association into specialized microdomains on the cell surface called lipid rafts. In order to understand the contribution of the active receptor-interacting residues in the high degree of genetic variation on vacA, I examined two variant forms of VacA from two H. pylori strains; a toxic form of VacA from H. pylori strongly associated with gastric disease and a less toxic form of VacA from H. pylori less associated with the gastric disease. By generating a chimeric form of VacA, I observed that the difference on the central region with the most heterogeneity mediates VacA cellular activity. Mapping the central region by smaller subdomain substitution between the toxic form and the less toxic form of VacA revealed that chimeric forms of VacA lacking the active receptor-interacting residues showed attenuation in cellular activity of the toxin. It was demonstrated that the difference on the region that contains the active receptor-interacting residues contribute to the difference in the cytotoxicity between toxic variant and less toxic variant form of VacA. In Chapter 4, I described the importance of the oligomerization state of VacA for its cellular activity. In Chapter 5, I discuss the current model on how VacA interacts and becomes anchored onto the membrane by a mechanism involving the active receptor-interacting residues. My finding of the active receptor-interacting residues sheds a new insight in designing inhibitors to block the VacA-receptor interaction, which can supplement current H. pylori eradication strategy of antibiotic treatment.
Issue Date:2017-12-01
Rights Information:Copyright 2017 Seung J. Oh
Date Available in IDEALS:2018-03-13
Date Deposited:2017-12

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