Glycoengineering the <i>Pseudomonas</i> exotoxin A for multi-sequon integration and enhanced bioconjugation efficiency by PglS

Glycoconjugate vaccines, also known as polysaccharide protein conjugate vaccines, consist of bacterial polysaccharides covalently linked to immunogenic carrier proteins. Bioconjugate vaccines are a type of glycoconjugate produced by oligosaccharyltransferases that catalyze the en bloc transfer of polysaccharides to specific amino acid motifs, called sequons, engineered into carrier proteins. Designing carrier proteins that are highly glycosylated by a specific oligosaccharyltransferase is critical for scalable bioconjugation platforms. Here, we describe the development of improved Pseudomonas aeruginosa exotoxin A (EPA) carrier proteins for glycosylation by the Acinetobacter baylyi ADP1 O-linking oligosaccharyltransferase PglS. Using a structure-guided approach, we integrated sequons at the termini or on surface-exposed loops of EPA and quantified the glycosylation of each site. Most sequons were 50% glycosylated on average, but glycosylation ranged from 20-75% suggesting a preference by PglS for certain sites. We then combined the best-glycosylated sites to design 3- and 6-sequon-containing EPA carriers and used capillary immunoassay electrophoresis to quantify EPA glycoforms. Using E. coli and Streptococcus glycans, we show that EPA carriers containing six sequons (EPA6) exhibit 1.5- to 5-fold higher glycosylation than carriers with fewer sequons. Furthermore, EPA6 could be comparably glycosylated with Klebsiella O2β O-antigen when secreted to the periplasm in an unfolded state via either the Sec or SRP pathways. However, no conjugates were produced when EPA6 was routed through the Tat pathway that secretes folded protein. Our results lay the groundwork for a general glycoengineering strategy for developing future bioconjugate vaccine carrier proteins as well as methods to evaluate such proteins.