Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A: toward in vitro vaccine production.

The Journal of biological chemistry 2014 Jul 11

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The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [ → 6)-α-D-ManNAc-(1 → OPO3 (-)→]n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-D-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-D-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by (1)H NMR, (31)P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Citation

Timm Fiebig, Friedrich Freiberger, Vittoria Pinto, Maria Rosaria Romano, Alan Black, Christa Litschko, Andrea Bethe, Dmitry Yashunsky, Roberto Adamo, Andrei Nikolaev, Francesco Berti, Rita Gerardy-Schahn. Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A: toward in vitro vaccine production. The Journal of biological chemistry. 2014 Jul 11;289(28):19395-407