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

Published version
Peer-reviewed

Type

Article

Change log

Authors

Mauri, Marta 
Sannasiddappa, Thippeswamy H 
Smith, Alexander A 
Stevens, Mark P 

Abstract

jats:titleAbstract</jats:title>jats:sec jats:titleBackground</jats:title> jats:pjats:italicCampylobacter</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:italicEscherichia coli</jats:italic> strain capable of generating multiple glycoconjugate vaccine candidates against jats:italicCampylobacter jejuni</jats:italic>.</jats:p> </jats:sec>jats:sec jats:titleResults</jats:title> jats:pWe generated a glycoengineering jats:italicE. coli</jats:italic> strain containing the conserved jats:italicC. 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:italicC. jejuni</jats:italic>, jats:italicPseudomonas aeruginosa</jats:italic> (both Gram-negative), and jats:italicClostridium perfringens</jats:italic> (Gram-positive) as acceptors. Using this jats:italicpgl</jats:italic> integrant jats:italicE. coli</jats:italic> strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from jats:italicC. jejuni</jats:italic> (doublejats: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:italicC. 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:italicE. coli pgl</jats:italic> integrant strain.</jats:p> </jats:sec>jats:sec jats:titleConclusions</jats:title> jats:pWe 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>

Description

Keywords

Journal Title

Microbial Cell Factories

Conference Name

Journal ISSN

1475-2859

Volume Title

21

Publisher

Springer Science and Business Media LLC
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/N001591/1)