Hijacking bacterial glycosylation for the production of glycoconjugates, from vaccines to humanised glycoproteins

Abstract

Objectives: Glycosylation or the modification of a cellular component with a carbohydrate moiety has been demonstrated in all three domains of life as a basic post-translational process important in a range of biological processes. This review will focus on the latest studies attempting to exploit bacterial N-linked protein glycosylation for glycobiotechnological applications including glycoconjugate vaccine and humanised glycoprotein production. The challenges that remain for these approaches to reach full biotechnological maturity will be discussed.

Key findings: Oligosaccharyltransferase-dependent N-linked glycosylation can be exploited to make glycoconjugate vaccines against bacterial pathogens. Few technical limitations remain, but it is likely that the technologies developed will soon be considered a cost-effective and flexible alternative to current chemical-based methods of vaccine production. Some highlights from current glycoconjugate vaccines developed using this in-vivo production system include a vaccine against Shigella dysenteriae O1 that has passed phase 1 clinical trials, a vaccine against the tier 1 pathogen Francisella tularensis that has shown efficacy in mice and a vaccine against Staphylococcus aureus serotypes 5 and 8. Generation of humanised glycoproteins within bacteria was considered impossible due to the distinct nature of glycan modification in eukaryotes and prokaryotes. We describe the method used to overcome this conundrum to allow engineering of a eukaryotic pentasaccharide core sugar modification within Escherichia coli. This core was assembled by combining the function of the initiating transferase WecA, several Alg genes from Saccharomyces cerevisiae and the oligosaccharyltransferase function of the Campylobacter jejuni PglB. Further exploitation of a cytoplasmic N-linked glycosylation system found in Actinobacillus pleuropneumoniae where the central enzyme is known as N-linking glycosyltransferase has overcome some of the limitations demonstrated by the oligosaccharyltransferase-dependent system.

Summary: Characterisation of the first bacterial N-linked glycosylation system in the human enteropathogen Campylobacter jejuni has led to substantial biotechnological applications. Alternative methods for glycoconjugate vaccine production have been developed using this N-linked system. Vaccines against both Gram-negative and Gram-positive organisms have been developed, and efficacy testing has thus far demonstrated that the vaccines are safe and that robust immune responses are being detected. These are likely to complement and reduce the cost of current technologies thus opening new avenues for glycoconjugate vaccines. These new markets could potentially include glycoconjugate vaccines tailored specifically for animal vaccination, which has until today thought to be non-viable due to the cost of current in-vitro chemical conjugation methods. Utilisation of N-linked glycosylation to generate humanised glycoproteins is also close to becoming reality. This 'bottom up' assembly mechanism removes the heterogeneity seen in current humanised products. The majority of developments reported in this review exploit a single N-linked glycosylation system from Campylobacter jejuni; however, alternative N-linked glycosylation systems have been discovered which should help to overcome current technical limitations and perhaps more systems remain to be discovered. The likelihood is that further glycosylation systems exist and are waiting to be exploited.

Keywords: PglB; glycoconjugates; humanised glycoproteins; vaccines.

Figure 1

Example of chemical conjugation procedure used to generate glycoconjugate vaccines. In this example, production requires purification of lipopolisaccharide (LPS) from Francisella tularensis subs. tularensis, endotoxin cleavage, further purification, chemical activation and linkage to carrier protein CRM₁₉₇.

Figure 2

Simplified working model of T-cell activation by glycoconjugate vaccines. The glycan portion of the glycoconjugate binds to the B cell receptor (BCR), the glycoprotein is internalised into an endosome where the action of reactive oxygen species (ROS) and proteases process the glycoprotein into smaller glycopeptides, with shorter polysaccharide repeat units. The peptide portion of the newly formed glycopeptide binds to major histocompatibility complex (MHC) class II and enables presentation of the hydrophilic carbohydrate to the αβ T-cell receptor (αβTCR). Downstream processes enable maturation of a B cell to become a memory B cell and produce carbohydrate specific IgG antibodies (adapted from Avci et al.^[15]). P, peptide; carbohydrate component shown as black circles.

Figure 3

Utilisation of PglB to make glycoconjugate vaccines. (a) Typical O-antigen assembly model; glycosyltransferases (GTs) sequentially build polysaccharide on undecaprenyl pyrophosphate (black circles), this is flipped into the periplasmic compartment of the cell where a ligase (WaaL) transfers the polysaccharide onto a lipid A core, before this is transported and presented on the surface of the bacterium. (b) Model of N-linked glycoprotein assembly in Campylobacter jejuni; GTs sequentially build a heptasaccharide on undecaprenyl pyrophosphate; this is flipped into the periplasmic compartment of the cell before PglB using the UndPP linked sugar as a substrate, transfers it onto an acceptor protein (Peb3) within the acceptor sequon D/E-X₁-N-X₂-S/T. (c) Protein glycan coupling technology; plasmids coding for a carrier protein (CP), the OTase (PglB) and a polysaccharide of choice (PS) are transformed into a laboratory strain of E. coli, expressing the genetic contents of the three plasmids within an E. coli strain lacking WaaL allows for glycoconjugate vaccine production (GP). Cy, cytoplasm; IM, inner membrane; P, periplasm; OM, outer membrane; (n) indicates polymerisation status of polysaccharide.

Figure 4

Major classes of N-linked glycans. N indicates the asparagine residues onto which the reducing end sugar is attached. GlcNAc is N-acetyl glucosamine; NeuAc is N-acetyl neuraminic acid. Boxed area indicates core pentasaccharide structure present in all eukaryotic glycans.

Figure 5

Schematic demonstrating prokaryotic assembly of the eukaryotic glycan pentasaccharide core and transfer to an acceptor protein. WecA attaches GlcNAc to UndP, Alg13/14 add a second GlcNAc residue, before Alg1 and Alg2 attach three mannose residues to generate GlcNAc₂Man₃ core. The Escherichia coli flippase, Wzx, then moves the UndPP pentasaccharide to the periplasmic compartment of the cell where upon recognition by PglB, the glycan moiety is transferred to GP within the acceptor sequon D-X-N-X-S/T. GP, glycoprotein.