Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Xianke Meng; Geert-Jan Boons; Marc Wösten; Tom Wennekes

doi:10.1002/anie.202107181

Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

Original language	English
Pages (from-to)	24811-24816
Number of pages	6
Journal	Angewandte Chemie-International Edition
Volume	60
Issue number	47
Early online date	14 Sept 2021
DOIs	https://doi.org/10.1002/anie.202107181
Publication status	Published - 15 Nov 2021

Bibliographical note

Funding Information:
We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC–MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with analysis. We thank Xuefeng Cao and Linda Heijmen‐van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.). in silico

Funding Information:
We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC?MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with in silico analysis. We thank Xuefeng Cao and Linda Heijmen-van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.).

Publisher Copyright:
© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH

Keywords

bacterial flagella
carbohydrates
glycoproteins
glycosyltransferase
metabolic labeling

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Angew Chem Int Ed - 2021 - Meng - Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of CampylobacterFinal published version, 1.2 MBLicence: CC BY

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@article{e7b3f854949e41948e8b5e5f43c7b0ff,

title = "Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase",

abstract = "Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.",

keywords = "bacterial flagella, carbohydrates, glycoproteins, glycosyltransferase, metabolic labeling",

author = "Xianke Meng and Geert-Jan Boons and Marc W{\"o}sten and Tom Wennekes",

note = "Funding Information: We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC–MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with analysis. We thank Xuefeng Cao and Linda Heijmen‐van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.). in silico Funding Information: We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC?MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with in silico analysis. We thank Xuefeng Cao and Linda Heijmen-van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.). Publisher Copyright: {\textcopyright} 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH",

year = "2021",

month = nov,

day = "15",

doi = "10.1002/anie.202107181",

language = "English",

volume = "60",

pages = "24811--24816",

journal = "Angewandte Chemie-International Edition",

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publisher = "John Wiley and Sons Ltd",

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Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase. / Meng, Xianke; Boons, Geert-Jan ; Wösten, Marc et al.
In: Angewandte Chemie-International Edition, Vol. 60, No. 47, 15.11.2021, p. 24811-24816.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

AU - Meng, Xianke

AU - Boons, Geert-Jan

AU - Wösten, Marc

AU - Wennekes, Tom

N1 - Funding Information: We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC–MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with analysis. We thank Xuefeng Cao and Linda Heijmen‐van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.). in silico Funding Information: We thank Richard Wubbolts (Utrecht University) for technical support with microscopy. We thank Gerlof Bosman and Minglong Liu (both of Utrecht University) for assistance with UPLC?MS measurements. We thank Yifei Lang and Vito Thijssen (both of Utrecht University) for assistance with in silico analysis. We thank Xuefeng Cao and Linda Heijmen-van Dijk (both of Utrecht University) for technical support in the biolab. This research was supported by grants from The Netherlands Organisation for Scientific Research (NWO, VIDI grant 723.014.005, to T.W.) and China Scholarship Council 201606180014 (to X.M.). Publisher Copyright: © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH

PY - 2021/11/15

Y1 - 2021/11/15

N2 - Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

AB - Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

KW - bacterial flagella

KW - carbohydrates

KW - glycoproteins

KW - glycosyltransferase

KW - metabolic labeling

UR - https://www.scopus.com/pages/publications/85118227560

U2 - 10.1002/anie.202107181

DO - 10.1002/anie.202107181

M3 - Article

C2 - 34519150

SN - 1433-7851

VL - 60

SP - 24811

EP - 24816

JO - Angewandte Chemie-International Edition

JF - Angewandte Chemie-International Edition

IS - 47

ER -

Metabolic Labeling of Legionaminic acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase

Abstract

Bibliographical note

Keywords

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