TY - JOUR
T1 - Glycosyltransferase Jhp0106 (PseE) contributes to flagellin maturation in Helicobacter pylori
AU - Yang, Kai Yuan
AU - Kao, Cheng Yen
AU - Su, Marcia Shu Wei
AU - Wang, Shuying
AU - Chen, Yueh Lin
AU - Hu, Shiau Ting
AU - Chen, Jenn Wei
AU - Teng, Ching Hao
AU - Tsai, Pei Jane
AU - Wu, Jiunn Jong
N1 - Funding Information:
We would like to thank Drs. Ryan Holroyd and Xiaoxi Brook Lin for their helpful comments and editing on this manuscript. We also thank Wei-Jiun Tsai for his great assistance with the PyMOL software and Chia-Hsuan Su for graphic design. We appreciate the technical support from Yui-Ying Yu and Shu-Li Ho at the Electron Microscopy Facility in the National Yang-Ming University. This study was supported by the Ministry of Science and Technology, Taiwan (the MOST grants 107-2320-B-010-012 and 108-2320-B-010-002).
Funding Information:
We would like to thank Drs. Ryan Holroyd and Xiaoxi Brook Lin for their helpful comments and editing on this manuscript. We also thank Wei‐Jiun Tsai for his great assistance with the PyMOL software and Chia‐Hsuan Su for graphic design. We appreciate the technical support from Yui‐Ying Yu and Shu‐Li Ho at the Electron Microscopy Facility in the National Yang‐Ming University. This study was supported by the Ministry of Science and Technology, Taiwan (the MOST grants 107‐2320‐B‐010‐012 and 108‐2320‐B‐010‐002).
Publisher Copyright:
© 2021 The Authors. Helicobacter published by John Wiley & Sons Ltd
PY - 2021/4
Y1 - 2021/4
N2 - Background: Flagella-mediated motility is both a crucial virulence determinant of Helicobacter pylori and a factor associated with gastrointestinal diseases. Flagellar formation requires flagellins to be glycosylated with pseudaminic acid (Pse), a process that has been extensively studied. However, the transfer of Pse to flagellins remains poorly understood. Therefore, the aim of this study is to characterize a putative glycosyltransferase jhp0106 in flagellar formation. Materials and Methods: Western blotting and chemical deglycosylation were performed to examine FlaA glycosylation. Protein structural analyses were executed to identify the active site residues of Jhp0106, while the Jhp0106-FlaA interaction was examined using a bacterial two-hybrid assay. Lastly, site-directed mutants with mutated active site residues in the jhp0106 gene were generated and investigated using a motility assay, Western blotting, cDNA-qPCR analysis, and electron microscopic examination. Results: Loss of flagellar formation in the Δjhp0106 mutant was confirmed to be associated with non-glycosylated FlaA. Furthermore, three active site residues of Jhp0106 (S350, F376, and E415) were identified within a potential substrate-binding region. The interaction between FlaA and Jhp0106, Jhp0106::S350A, Jhp0106::F376A, or Jhp0106::E415A was determined to be significant. As well, the substitution of S350A, F376A, or E415A in the site-directed Δjhp0106 mutants resulted in impaired motility, deficient FlaA glycosylation, and lacking flagella. However, these phenotypic changes were regardless of flaA expression, implying an indefinite proteolytic degradation of FlaA occurred. Conclusions: This study demonstrated that Jhp0106 (PseE) binds to FlaA mediating FlaA glycosylation and flagellar formation. Our discovery of PseE has revealed a new glycosyltransferase family responsible for flagellin glycosylation in pathogens.
AB - Background: Flagella-mediated motility is both a crucial virulence determinant of Helicobacter pylori and a factor associated with gastrointestinal diseases. Flagellar formation requires flagellins to be glycosylated with pseudaminic acid (Pse), a process that has been extensively studied. However, the transfer of Pse to flagellins remains poorly understood. Therefore, the aim of this study is to characterize a putative glycosyltransferase jhp0106 in flagellar formation. Materials and Methods: Western blotting and chemical deglycosylation were performed to examine FlaA glycosylation. Protein structural analyses were executed to identify the active site residues of Jhp0106, while the Jhp0106-FlaA interaction was examined using a bacterial two-hybrid assay. Lastly, site-directed mutants with mutated active site residues in the jhp0106 gene were generated and investigated using a motility assay, Western blotting, cDNA-qPCR analysis, and electron microscopic examination. Results: Loss of flagellar formation in the Δjhp0106 mutant was confirmed to be associated with non-glycosylated FlaA. Furthermore, three active site residues of Jhp0106 (S350, F376, and E415) were identified within a potential substrate-binding region. The interaction between FlaA and Jhp0106, Jhp0106::S350A, Jhp0106::F376A, or Jhp0106::E415A was determined to be significant. As well, the substitution of S350A, F376A, or E415A in the site-directed Δjhp0106 mutants resulted in impaired motility, deficient FlaA glycosylation, and lacking flagella. However, these phenotypic changes were regardless of flaA expression, implying an indefinite proteolytic degradation of FlaA occurred. Conclusions: This study demonstrated that Jhp0106 (PseE) binds to FlaA mediating FlaA glycosylation and flagellar formation. Our discovery of PseE has revealed a new glycosyltransferase family responsible for flagellin glycosylation in pathogens.
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U2 - 10.1111/hel.12787
DO - 10.1111/hel.12787
M3 - Article
C2 - 33586844
AN - SCOPUS:85101434030
SN - 1083-4389
VL - 26
JO - Helicobacter
JF - Helicobacter
IS - 2
M1 - e12787
ER -