{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/99489"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/99489","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Understanding the molecular mechanism on cell surface receptor interaction of helicobacter pylori vacuolation cytotoxin","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-12-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-12-01","abstract_has_math":false,"creators":["Oh, Seung Jin"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Blanke, Steven R.","Wilson, Brenda A.","Morrissey, James H.","Fratti, Rutilio A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-03-13T17:32:22Z","date_published":"2018-03-13T17:32:22Z","updated_at":"2026-07-22T22:24:37Z","subjects":["Bacterial toxin","Vacuolating cytotoxin (VacA)","Helicobacter pylori","Sphingomyelin","Toxin-receptor interaction","Toxin-membrane interaction"],"languages":["en"],"rights":["Copyright 2017 Seung J. Oh"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/99489","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Blanke, Steven R.","Wilson, Brenda A.","Morrissey, James H.","Fratti, Rutilio A."]},{"key":"dc:creator","label":"Author","values":["Oh, Seung Jin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-03-13T17:32:22Z","2020-03-14T09:15:22Z","2017-12-01","2017-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Bacterial toxin","Vacuolating cytotoxin (VacA)","Helicobacter pylori","Sphingomyelin","Toxin-receptor interaction","Toxin-membrane interaction"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2017 Seung J. Oh"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/99489"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-12-01","The student, Seung Oh, accepted the attached license on 2017-11-27 at 15:21.","The student, Seung Oh, submitted this Dissertation for approval on 2017-11-27 at 15:28.","This Dissertation was approved for publication on 2017-12-01 at 10:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #11761 on 2018-03-13 at 10:33:34","Made available in DSpace on 2018-03-13T17:32:22Z (GMT). No. of bitstreams: 2 OH-DISSERTATION-2017.pdf: 48560294 bytes, checksum: 4e542835a5b80ebd169b1145bd63a033 (MD5) LICENSE.txt: 4205 bytes, checksum: 88d44b982d1d01ed2478bab2637135e9 (MD5) Previous issue date: 2017-12-01","Embargo set by: Seth Robbins for item 105457 Lift date: 2020-03-13T17:32:30Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 105457 Lift date: 2020-03-13T17:36:05Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 105457 on 2020-03-14T09:15:22Z.","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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Understanding the molecular mechanism on cell surface receptor interaction of helicobacter pylori vacuolation cytotoxin"]}]}],"canonical_facts":{"dc:contributor":["Blanke, Steven R.","Wilson, Brenda A.","Morrissey, James H.","Fratti, Rutilio A."],"dc:creator":["Oh, Seung Jin"],"dc:date":["2018-03-13T17:32:22Z","2020-03-14T09:15:22Z","2017-12-01","2017-12"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2019-12-01","The student, Seung Oh, accepted the attached license on 2017-11-27 at 15:21.","The student, Seung Oh, submitted this Dissertation for approval on 2017-11-27 at 15:28.","This Dissertation was approved for publication on 2017-12-01 at 10:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #11761 on 2018-03-13 at 10:33:34","Made available in DSpace on 2018-03-13T17:32:22Z (GMT). No. of bitstreams: 2 OH-DISSERTATION-2017.pdf: 48560294 bytes, checksum: 4e542835a5b80ebd169b1145bd63a033 (MD5) LICENSE.txt: 4205 bytes, checksum: 88d44b982d1d01ed2478bab2637135e9 (MD5) Previous issue date: 2017-12-01","Embargo set by: Seth Robbins for item 105457 Lift date: 2020-03-13T17:32:30Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 105457 Lift date: 2020-03-13T17:36:05Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 105457 on 2020-03-14T09:15:22Z.","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."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/99489"],"dc:language":["en"],"dc:rights":["Copyright 2017 Seung J. Oh"],"dc:subject":["Bacterial toxin","Vacuolating cytotoxin (VacA)","Helicobacter pylori","Sphingomyelin","Toxin-receptor interaction","Toxin-membrane interaction"],"dc:title":["Understanding the molecular mechanism on cell surface receptor interaction of helicobacter pylori vacuolation cytotoxin"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:37Z"}