Salmonella enteritidis (SE) has been recognized as an important zoonotic pathogen, and the prevention and control of salmonellosis has long been a conundrum. However, glycoconjugate vaccines seem to be a promising solution. Glycoproteins are conventionally synthesized by chemical cross-linking which features complex procedure and cost-intensiveness. Therefore, a stable biosynthesis method at lower cost is in urgent need. For the biosynthesis of SE O-antigen-based glycoproteins, we used CRISPR/Cas9 to develop the waaL-deleted SE strain ∆waaL. The synthesis of lipopolysaccharide (LPS) was detected based on silver staining. Circular polymerase extension cloning (CPEC) was employed to construct the plasmids expressing glycosyltransferase PglL, recombinant Pseudomonas aeruginosa exotoxin A (rEPA), and cholera toxin B subunit (CTB). Meanwhile, PilES45-K73 glycosylation motif was added to the N-terminal and C-terminal of rEPA and CTB, respectively. The recombinant plasmids were transformed into SE ∆waaL. After induction, the synthesis of glycoprotein was verified by Western blotting and the synthesized glycoprotein was purified by Ni-NTA column. The results showed that waaL deletion blocked the LPS synthesis of SE, and that rEPA and CTB proteins were expressed in SE ∆waaL. In addition, obvious glycosylation occurred to rEPA and CTB when PglL was expressed, and the glycosylated part was SE O antigen polysaccharide. In summary, after waaL deletion in SE, PglL can transfer its own O antigen polysaccharides (OPS) to the carrier proteins rEPA and CTB, resulting in OPS-rEPA and OPS-CTB glycoproteins. The result lays a basis for the biosynthesis of SE glycoprotein.
Keywords: CRISPR/Cas9; PglL; Salmonella enteritidis; biosynthesis; glycoprotein.