PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome

Abstract

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p><jats:italic>Campylobacter</jats:italic> 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 <jats:italic>Escherichia coli</jats:italic> strain capable of generating multiple glycoconjugate vaccine candidates against <jats:italic>Campylobacter jejuni</jats:italic>.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We generated a glycoengineering <jats:italic>E. coli</jats:italic> strain containing the conserved <jats:italic>C. jejuni</jats:italic> 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 <jats:italic>C. jejuni</jats:italic>, <jats:italic>Pseudomonas aeruginosa</jats:italic> (both Gram-negative), and <jats:italic>Clostridium perfringens</jats:italic> (Gram-positive) as acceptors. Using this <jats:italic>pgl</jats:italic> integrant <jats:italic>E. coli</jats:italic> strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from <jats:italic>C. jejuni</jats:italic> (double<jats:italic>-</jats:italic>hit vaccines)<jats:italic>,</jats:italic> 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 <jats:italic>C. jejuni</jats:italic> 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 <jats:italic>E. coli pgl</jats:italic> integrant strain.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>We 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.</jats:p> </jats:sec>