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

Abstract

Background 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. Results We 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. Conclusions 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.

Modification of avian pathogenic Escherichia coli χ7122 lipopolysaccharide increases accessibility to glycoconjugate antigens

“…The absence of significant reactivity of the antibody against 6xHis until the cells are permeabilised may be a consequence of the C-terminal location of the tag and topology of the proteins in the membrane. As demonstrated in previous studies, we observed that glycosylation of NetB was temperature dependent (P < 0.05) [15]. However, a temperature-dependent effect was not observed with FlpA glycosylation.…”

“…As demonstrated in previous studies, we observed that glycosylation of NetB was temperature dependent (P < 0.05) [ 15 ]. However, a temperature-dependent effect was not observed with FlpA glycosylation.…”

“…The effect of temperature was also assessed, with each culture being grown at 28ºC, 37ºC and 42ºC. The temperatures represent the optimal glycosylation temperature, optimal E. coli growth temperature and avian body temperature, respectively [ 15 ]. Bacterial cells were examined for the presence of the 6xHis-tag, glycan and both 6xHis-tag and glycan.…”

“…However, although vaccination of chickens with this strain was able to reduce APEC colonization of the lungs upon experimental challenge, no reduction in C. jejuni colonization of the caeca was detected after challenge, indicating that further optimisation of the vaccine strain was required. Importantly, surface display of the antigen was limited in the study, particularly at the body temperature of chickens [ 14 , 15 ].…”

A combinatorial DNA assembly approach to biosynthesis ofN-linked glycans inE. coli

“…Expression of heterologous genes from the chromosome rather than multicopy plasmids is a well-recognised strategy for alleviating the toxic effects of metabolically burdensome enzymes, such as PglB, which resides in the cytoplasmic membrane ( Bentley et al 1990 ; Bassalo et al 2016 ; Englaender et al 2017 ). However, in a recent study, Terra et al integrated the entire unmodified pgl pathway onto the chromosome of the chassis strain SDB1 ( wecA - and waaL -; Terra et al 2022 ) and observed greater glycoconjugate yield in 3 out of 4 acceptor proteins tested when an additional inducible copy of pglB was also provided on a plasmid. Optimal glycosylation was achieved when both the glycan biosynthesis genes and pglB were encoded on a plasmid in their native configuration.…”