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

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

doi:10.3389/fcimb.2021.705468

Abstract

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.

Highlights

Whereas an increase in the availability of resources such as clean water and professional healthcare has significantly decreased the mortality from E. coli infections in low and middle-income countries (LMICs), the number of non-fatal infections remains high and so does the cost of these infections to societies in lowresource settings due to childhood stunting and delays in cognitive development as well as increased risk of dying from other infectious diseases (Anderson et al, 2019; Khalil et al, 2021)
We show that antibodies from patients exposed to Enterotoxigenic Escherichia coli (ETEC) infections predominantly recognize glycosylated over non-glycosylated YghJ which points to increased immunogenicity of glycosylated YghJ compared to the non-glycosylated antigen
ETEC secretes enzymatically active YghJ into the growth medium (Luo et al, 2014), to ensure that only fully processed and modified YghJ was analyzed by BEMAP, we purified the exported protein from the culture supernatant

Summary

Introduction

Whereas an increase in the availability of resources such as clean water and professional healthcare has significantly decreased the mortality from E. coli infections in low and middle-income countries (LMICs), the number of non-fatal infections remains high and so does the cost of these infections to societies in lowresource settings due to childhood stunting and delays in cognitive development as well as increased risk of dying from other infectious diseases (Anderson et al, 2019; Khalil et al, 2021). Enterotoxigenic Escherichia coli (ETEC) globally continues to cause severe diarrhea and death in high risk patient groups, including older individuals (Poolman and Anderson, 2018) and is the most common cause of diarrhea in travelers to endemic regions (Olson et al, 2019). E. coli remains a World Health Organization (WHO) priority pathogen and vaccine target given its high burden and the increasing emergence of Extended-spectrum b-lactamase producing Enterobacteriaeceae including ESBL-ETEC (Shrivastava et al, 2018; Tacconelli et al, 2018). The Wellcome Trust and Boston Consulting Group recommend that vaccine development for enteric E. coli including ETEC be accelerated due to the increasing antimicrobial resistance (AMR) threat (Wellcome Trust, 2019) and this recommendation was repeated in the WHO Action Framework: Leveraging Vaccines to Reduce Antibiotic Use and Prevent Antimicrobial Resistance (World Health Organization, 2020). Whereas ETEC is a global challenge, both the commercial and academic E. coli vaccine pipeline remains inadequate (Barry et al, 2019; Theuretzbacher et al, 2019; Bekeredjian-Ding et al, 2020; Giersing, 2020)

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