Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines

Abstract

BackgroundActinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection.ResultsIn this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer.ConclusionsA vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1751-2) contains supplementary material, which is available to authorized users.

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

“…2 and Supplementary Table 4). Previous works indicate that in A. pleuropnemoniae , the glucose installed by ApNGT is modified by the polymerizing Apα1-6 glucosyltransferase to form N -linked dextran 29 and that this structure could be a useful vaccine antigen 16,35 . Recent work also showed that the β1-4 galactosyltransferase LgtB from Neisseria meningitis (NmLgtB) can modify an ApNGT-installed glucose in E. coli , forming N -linked lactose (Galβ1-4Glc-Asn) 28 .…”

Synthetic Glycobiology: Parts, Systems, and Applications

“… 168 Other studies have developed short, optimized GlycTag sequences for NGT 80 and have shown that the modification of a target protein with these GlycTags (such as GGNWTT) can successfully direct efficient NGT glycosylation of diverse recombinant proteins in vivo and in vitro . 80 , 160 , 173 Later studies have found that the single glucose residue installed by NGT can be elaborated to a dextran polymer 160 (which could be useful for vaccines against pathogenic bacteria that use NGTs to adhere to human cells), polysialic acids 131 (which may prolong the serum-half-life of small therapeutic proteins), N -acetyllactosamine (LacNAc), 174 , 175 and other fucosylated and sialylated forms of lactose 174 , 175 by overexpression of elaborating GTs within the cell. 131 This sequential elaboration technique may also allow an NGT-based system to circumvent the limits on glycan structure found in OST systems.…”

“…Recently, several N - and O -linked glycosylation systems that function in the bacterial cytoplasm have been discovered. These systems often glycosylate extracellular adhesion and autotransporter proteins that facilitate adherence of pathogenic bacteria to human cells. − N -glycosyltransferases (NGTs) are one such class of enzymes that have been recently characterized ,,− and have elicited great interest from the glycoengineering community for their ability to initiate N -linked glycosylation in the bacterial cytoplasm when heterologously expressed in E. coli . ,,,,,,− NGTs bear structural homology to eukaryotic OGTs, but were first identified as part of an extracellular adhesion operon in Haemophilus influenzae , founding a new functional class of GTs that install monosaccharides onto asparagine residues in the cytoplasm using UDP-Glc or UDP-Gal as soluble sugar donors . In some species, the single glucose residues installed by NGTs are extended into a dextran polymer by a glucose polymerase (α 1,6 GlcT) .…”

“…NGTs are particularly promising glycoengineering tools because they are the only known cytoplasmic enzyme class capable of installing glycans onto asparagine residues at eukaryotic-like N-X-S/T sequons. ,,, For example, ApNGT has been functionally expressed in E. coli where it was found to glycosylate several autotransporter proteins, some native E. coli proteins, and recombinant human erythropoietin (EPO) . Other studies have developed short, optimized GlycTag sequences for NGT and have shown that the modification of a target protein with these GlycTags (such as GGNWTT) can successfully direct efficient NGT glycosylation of diverse recombinant proteins in vivo and in vitro . ,, Later studies have found that the single glucose residue installed by NGT can be elaborated to a dextran polymer (which could be useful for vaccines against pathogenic bacteria that use NGTs to adhere to human cells), polysialic acids (which may prolong the serum-half-life of small therapeutic proteins), N -acetyllactosamine (LacNAc), , and other fucosylated and sialylated forms of lactose , by overexpression of elaborating GTs within the cell . This sequential elaboration technique may also allow an NGT-based system to circumvent the limits on glycan structure found in OST systems.…”

Recent advances in the production of recombinant glycoconjugate vaccines

“…56–58 Much of the research with this enzyme has focused on biochemical characterisation and its potential for vaccine assembly is currently being explored. 59…”