Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation
Protein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In Helicobacter pylori, glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In additio...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2025-12-01
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| Series: | Gut Microbes |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19490976.2025.2455513 |
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| author | Hong Li Xiaoqiong Tang Tiandi Yang Tingting Liao Aleksandra W. Debowski Tiankuo Yang Yalin Shen Hans-Olof Nilsson Stuart M. Haslam Barbara Mulloy Anne Dell Keith A. Stubbs Wolfgang Fischer Rainer Haas Hong Tang Barry J. Marshall Mohammed Benghezal |
| author_facet | Hong Li Xiaoqiong Tang Tiandi Yang Tingting Liao Aleksandra W. Debowski Tiankuo Yang Yalin Shen Hans-Olof Nilsson Stuart M. Haslam Barbara Mulloy Anne Dell Keith A. Stubbs Wolfgang Fischer Rainer Haas Hong Tang Barry J. Marshall Mohammed Benghezal |
| author_sort | Hong Li |
| collection | DOAJ |
| description | Protein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In Helicobacter pylori, glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In addition to flagellins, several other proteins including RecA, AlpA/B, and BabA/B in H. pylori have also been reported to be glycosylated and to be dependent on the lipopolysaccharide (LPS) biosynthetic pathway. However, these proteins have not been purified for sugar-specific staining or structural analysis to confirm the existence of carbohydrate motifs. Here, using a combined approach of genetics, protein purification, and sugar-specific staining, we demonstrate that RecA is not a glycoprotein. Moreover, using LPS-protein reconstitution experiments, we demonstrate that the presence of O-antigen containing full-length LPS interferes with the electrophoretic mobility of H. pylori RecA and many other proteins including AlpA/B on SDS-PAGE. Finally, we demonstrate that full-length LPS extracted from E. coli affects electrophoretic migration of H. pylori proteins, while full-length LPS extracted from H. pylori similarly influences the electrophoretic migration of E. coli proteins. The impact is more subtle with E. coli LPS compared to H. pylori LPS, indicating that the magnitude of effect of LPS effects on protein mobility is dependent on bacterial source of the LPS. These findings suggest that the effects of full-length LPS on protein electrophoresis may represent a more general phenomenon. As LPS is a unique component of virtually all Gram-negative bacteria, our data suggest that when observing protein electrophoretic mobility shifts between wild-type and LPS mutant strains or between subcellular fractionation samples, the influence of LPS on protein electrophoretic migration should be considered first, rather than interpreting it as potential protein glycosylation that is dependent upon LPS biosynthetic pathway. |
| format | Article |
| id | doaj-art-528310e85ed8480e8d72b0ea14a90dfe |
| institution | Kabale University |
| issn | 1949-0976 1949-0984 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Gut Microbes |
| spelling | doaj-art-528310e85ed8480e8d72b0ea14a90dfe2025-01-21T06:07:56ZengTaylor & Francis GroupGut Microbes1949-09761949-09842025-12-0117110.1080/19490976.2025.2455513Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylationHong Li0Xiaoqiong Tang1Tiandi Yang2Tingting Liao3Aleksandra W. Debowski4Tiankuo Yang5Yalin Shen6Hans-Olof Nilsson7Stuart M. Haslam8Barbara Mulloy9Anne Dell10Keith A. Stubbs11Wolfgang Fischer12Rainer Haas13Hong Tang14Barry J. Marshall15Mohammed Benghezal16Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaCenter of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaDepartment of Life Sciences, Imperial College London, London, UKHelicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Nedlands, AustraliaHelicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Nedlands, AustraliaCenter of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaCenter of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaHelicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Nedlands, AustraliaDepartment of Life Sciences, Imperial College London, London, UKDepartment of Life Sciences, Imperial College London, London, UKDepartment of Life Sciences, Imperial College London, London, UKSchool of Molecular Sciences, University of Western Australia, Crawley, AustraliaMax von Pettenkofer Institute of Hygiene and Medical Microbiology, Faculty of Medicine, and German Center for Infection Research (DZIF), LMU Munich, Munich, GermanyMax von Pettenkofer Institute of Hygiene and Medical Microbiology, Faculty of Medicine, and German Center for Infection Research (DZIF), LMU Munich, Munich, GermanyCenter of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaHelicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Biomedical Sciences, University of Western Australia, Nedlands, AustraliaCenter of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, ChinaProtein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In Helicobacter pylori, glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In addition to flagellins, several other proteins including RecA, AlpA/B, and BabA/B in H. pylori have also been reported to be glycosylated and to be dependent on the lipopolysaccharide (LPS) biosynthetic pathway. However, these proteins have not been purified for sugar-specific staining or structural analysis to confirm the existence of carbohydrate motifs. Here, using a combined approach of genetics, protein purification, and sugar-specific staining, we demonstrate that RecA is not a glycoprotein. Moreover, using LPS-protein reconstitution experiments, we demonstrate that the presence of O-antigen containing full-length LPS interferes with the electrophoretic mobility of H. pylori RecA and many other proteins including AlpA/B on SDS-PAGE. Finally, we demonstrate that full-length LPS extracted from E. coli affects electrophoretic migration of H. pylori proteins, while full-length LPS extracted from H. pylori similarly influences the electrophoretic migration of E. coli proteins. The impact is more subtle with E. coli LPS compared to H. pylori LPS, indicating that the magnitude of effect of LPS effects on protein mobility is dependent on bacterial source of the LPS. These findings suggest that the effects of full-length LPS on protein electrophoresis may represent a more general phenomenon. As LPS is a unique component of virtually all Gram-negative bacteria, our data suggest that when observing protein electrophoretic mobility shifts between wild-type and LPS mutant strains or between subcellular fractionation samples, the influence of LPS on protein electrophoretic migration should be considered first, rather than interpreting it as potential protein glycosylation that is dependent upon LPS biosynthetic pathway.https://www.tandfonline.com/doi/10.1080/19490976.2025.2455513Helicobacter pylorimolecular weight shiftSDS-PAGEprotein glycosylationlipopolysaccharide |
| spellingShingle | Hong Li Xiaoqiong Tang Tiandi Yang Tingting Liao Aleksandra W. Debowski Tiankuo Yang Yalin Shen Hans-Olof Nilsson Stuart M. Haslam Barbara Mulloy Anne Dell Keith A. Stubbs Wolfgang Fischer Rainer Haas Hong Tang Barry J. Marshall Mohammed Benghezal Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation Gut Microbes Helicobacter pylori molecular weight shift SDS-PAGE protein glycosylation lipopolysaccharide |
| title | Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation |
| title_full | Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation |
| title_fullStr | Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation |
| title_full_unstemmed | Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation |
| title_short | Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in Helicobacter pylori protein glycosylation |
| title_sort | reinvestigation into the role of lipopolysaccharide glycosyltransferases in helicobacter pylori protein glycosylation |
| topic | Helicobacter pylori molecular weight shift SDS-PAGE protein glycosylation lipopolysaccharide |
| url | https://www.tandfonline.com/doi/10.1080/19490976.2025.2455513 |
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