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

Abstract

Background: Actinobacillus 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.

Results: In 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.

Conclusions: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.

Keywords: Actinobacillus pleuropnuemoniae; Glycoengineering; N-linked glycosylation.

Fig. 1

Demonstration of glycosylation of the peptide AtaC_1866–2428 by a single chromosomal copy or multiple plasmid copies of the ngt and agt operon. Glycosylated AtaC was resolved by SDS-PAGE and detected by coomassie staining (left panel) or immunoblot analysis (right panel), probed with anti-6xHis (green) and anti-dextran (red) antibody

Fig. 2

Expression and solubility of ApxIA peptide fragments. a- Schematic representation of the domain architecture of ApxIA. b- The ‘hydrophobic’ domain (D1) and Ca²⁺-binding domain (D3) of ApxIA were recombinantly expressed in E. coli, lysed and soluble and insoluble fractions were resolved by SDS-PAGE. c- Expression and solubility of ApxIA fragments immunoblot detected using an anti-6xHis (green) antibody. Lane 1: Ladder; lane 2: E. coli expressing ApxIAD3 (soluble); lane 3: ApxIAD1; lane 4: ApxIAD2; lane 5: MBP-ApxIAD1 fusion; lane 6: MBP_Nlinker-ApxIAD1 fusion; lane 7: ApxIAD3 Ni-NTA affinity purified; lane 8 ApxIAD3 (whole cell lysate); lane 9: ApxIAD1; lane 10: ApxIAD2; lane 11: MBP-ApxIAD1 fusion; lane 12: MBP_Nlinker-ApxIAD1 fusion

Fig. 3

Glycosylation of ApxIAD3 (Ca²⁺-binding domain). a- Schematic representation of ApxIAD3. The peptide sequence was engineered to include N- and C-terminal NAT sequons as well as three separate internal modifications. The three ApxIAD3 variants were expressed in E. coli with and without chromosomally encoded ngt and agt. The fragments were purified by Ni-NTA affinity chromatography, resolved by SDS-PAGE and detected by either coomassie stain (b) or the presence of glycoprotein detected by PAS staining (c)

Fig. 4

Glycosylation of ApxIVAC1, ApxIVAC2 and ApxIIAD3 (Ca²⁺-binding domain). a- Schematic representation of ApxIVAC1, ApxIVAC2 and ApxIAD3. Each peptide was engineered to include N- and C-terminal NAT sequons. ApxIAD3 was further modified to include an internal NDT motif. b- The toxin fragments were expressed in E. coli with and without chromosomally encoded ngt and agt. The fragments were purified by Ni-NTA affinity chromatography, resolved by SDS-PAGE and detected by coomassie staining. c- The following Ni-NTA purified fragments were resolved by SDS-PAGE and PAS stained: Lane 1: ApxIA (no NX(S/T) sequons); Lane 2: ApxIA (G71 T), no ngt and agt Lane 3: ApxIA (G71 T) plus chromosomally encoded ngt and agt; Lane 4: ApxIIA, no ngt and agt; Lane 5: ApxIIA plus chromosomally encoded ngt and agt; Lane 6: AtaC, plasmid-encoded ngt and agt

Fig. 5

Apx toxin fragments expressed in E. coli with and without chromosomally encoded ngt and agt, were resolved by SDS-PAGE and detected by immunoblot analysis probed with anti-6xHis (green) and anti-dextran (red) antibody. Lanes 2/3: ApxIAD3 (G71 T); lanes 4/5: ApxIAD3 (V83 T); lanes 6/7: ApxIAD3 (G114 T); lanes 8/9: ApxIIAD3 (G64 T); lanes 10/11: ApxIAD1 (whole cell lysate); lane 12: E. coli expressing AtaC_1866–2428 and plasmid encoded ngt and agt

Fig. 6

In-tact MALDI-MS analysis of purified ApxIA (a and c) and ApxIIA (b and d) expressed from wild type E. coli (upper panels) and with ngt and agt encoded on the chromosome (lower panels). a and b are peaks corresponding to singly charged ions, c and d are peaks corresponding to doubly charged ions. The mass shift in the peaks observed in the lower panel in each figure correspond to the addition of hexose units