Abstract
BackgroundCampylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni.ResultsWe generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain.ConclusionsWe determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.
Highlights
Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens
As the live attenuated vaccines (LAVs) created in our previous study [13] were intended to be used in poultry which have a higher body temperature than humans or other farm animals [19], we investigated the effect of temperature on protein expression and glycosylation
E. coli SDB1 [18] is a W3110-derivative strain lacking the O-antigen with the additional deletion of waaL and wecA genes encoding the O-antigen ligase and the initiating glycosyltransferase involved in O-antigen and enterobacterial common antigen (ECA) biosynthesis, respectively
Summary
Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. In 2011 Fisher et al, demonstrated that the addition of a sequence consensus ‘glycotag’ at either the N-or C-termini of a protein was sufficient for glycosylation [9] This discovery meant that double-hit approach vaccines (where the protein and glycan are from the same pathogen) are a possibility, as any protein can become an acceptor. The use of new carrier proteins contributes towards avoiding possible interference between different vaccines [11], carrierinduced immune suppression [12], as well as increasing vaccine specificity and coverage
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