Characterization of Pasteurella multocida type B capsule biosynthesis proteins

Pasteurella multocida causes several severe animal diseases, including hemorrhagic septicemia in ungulates. Strains of P. multocida are differentiated into five capsular and nine lipopolysaccharide genotypes. To date, only P. multocida strains that produce capsule types B and E have been recovered from cases of hemorrhagic septicemia. The structure of the type B capsule has recently been determined to be a repeating main chain of N-acetyl glucosamine (GlcNAc) and N-acetyl mannosaminuronic acid (ManNAcA), with a fructose and glycine side chain. In this study, we investigated the role of proteins involved in capsule biosynthesis in P. multocida strain M1404, a bison hemorrhagic septicemia isolate that produces a type B capsule. Using capsule absorbance assays, Alcian blue stains, and capsule Western blots to assess capsule production and export, as well as in-source collision-induced dissociation mass spectrometry to determine capsule structure, we show that BcbC is likely the capsule synthase; BcbE and BcbG are involved in fructose side chain addition, and BcbH is likely the glycine transferase. Furthermore, we show that inactivation of bcbE, bcbG, or bcbH significantly decreases overall capsule production in P. multocida strain M1404. Additionally, heterologous expression of bcbABCDI in a P. multocida strain VP161 (type A) capsule mutant allowed production of a repeating GlcNAc-ManNAcA monomer in this strain background that was indistinguishable from the type B main chain, indicating that these genes are necessary and sufficient for synthesis of the main repeating type B polysaccharide.