Linking inherent O-Linked Protein Glycosylation of YghJ to Increased Antigen Potential

Mette Thorsing, Thøger Jensen Krogh, Lars Vitved, Arkadiusz Nawrocki, Rikke Jakobsen, Martin R. Larsen, Subhra Chakraborty, A. Louis Bourgeois, Ann Zahle Andersen, Anders Boysen*

*Corresponding author for this work

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Enterotoxigenic Escherichia coli (ETEC) is a WHO priority pathogen and vaccine target which causes infections in low-income and middle-income countries, travelers visiting endemic regions. The global urgent demand for an effective preventive intervention has become more pressing as ETEC strains have become increasingly multiple antibiotic resistant. However, the vaccine development pipeline has been slow to address this urgent need. To date, vaccine development has focused mainly on canonical antigens such as colonization factors and expressed toxins but due to genomic plasticity of this enteric pathogen, it has proven difficult to develop effective vaccines. In this study, we investigated the highly conserved non-canonical vaccine candidate YghJ/SsLE. Using the mass spectrometry-based method BEMAP, we demonstrate that YghJ is hyperglycosylated in ETEC and identify 54 O-linked Set/Thr residues within the 1519 amino acid primary sequence. The glycosylation sites are evenly distributed throughout the sequence and do not appear to affect the folding of the overall protein structure. Although the glycosylation sites only constitute a minor subpopulation of the available epitopes, we observed a notable difference in the immunogenicity of the glycosylated YghJ and the non-glycosylated protein variant. We can demonstrate by ELISA that serum from patients enrolled in an ETEC H10407 controlled infection study are significantly more reactive with glycosylated YghJ compared to the non-glycosylated variant. This study provides an important link between O-linked glycosylation and the relative immunogenicity of bacterial proteins and further highlights the importance of this observation in considering ETEC proteins for inclusion in future broad coverage subunit vaccine candidates.

Original languageEnglish
Article number705468
JournalFrontiers in Cellular and Infection Microbiology
Number of pages10
Publication statusPublished - 19. Aug 2021

Bibliographical note

Publisher Copyright:
© Copyright © 2021 Thorsing, Krogh, Vitved, Nawrocki, Jakobsen, Larsen, Chakraborty, Bourgeois, Andersen and Boysen.


  • Enterotoxigenic Escherichia coli
  • immunogenicity
  • mass spectrometry
  • protein glycosylation
  • SslE
  • sub-unit vaccines
  • vaccine development
  • YghJ


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