Multi-Enzymatic Cascade Synthesis of Oligosaccharide Haptens from Streptococcus suis Serotype 2 and 1/2 Capsular Polysaccharides and Antigenicity Evaluation

Streptococcus suis is a globally distributed zoonotic pathogen that poses a serious threat to both livestock husbandry and human public health, with serotypes 2 and 1/2 being the primary pathogenic variants. Capsular polysaccharide (CPS)-based vaccines represent a promising preventive and control strategy against S. suis infections; however, progress in this area remains relatively limited. In this study, six key glycosyltransferasesCps2G, Cps2H, Cps2J, Cps2K, Cps1/2K, and Cps2Ninvolved in the biosynthesis of the CPS heptasaccharide repeating units from S. suis serotypes 2 and 1/2 were successfully heterologously expressed and purified under optimized in vitro conditions. Their catalytic functions, biochemical properties, and substrate specificities were systematically characterized. Utilizing these recombinant enzymes, we implemented a one-pot multienzyme (OPME) synthesis approach to efficiently produce 13 oligosaccharide derivatives related to the heptasaccharide repeating units, including both α-galactosylated and nongalactosylated forms, in high yields. These oligosaccharide haptens were subsequently conjugated to bovine serum albumin via chemical cross-linking to generate coating antigens for antigenicity evaluation. The results revealed that pentasaccharide 5c, which lacks both the terminal sialic acid residue and α-galactose branch but retains the majority of the native CPS backbone structure, displayed the strongest binding affinity toward S. suis-positive mouse and rabbit antisera. This study establishes an efficient in vitro enzymatic platform as a viable alternative to traditional chemical synthesis and extraction for accessing CPS-derived oligosaccharide haptens, enabling the identification of promising immunoreactive epitopes for anti-S. suis vaccine design.