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

TY - JOUR

T1 - Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A

T2 - toward in vitro vaccine production

AU - Fiebig, Timm

AU - Freiberger, Friedrich

AU - Pinto, Vittoria

AU - Romano, Maria Rosaria

AU - Black, Alan

AU - Litschko, Christa

AU - Bethe, Andrea

AU - Yashunsky, Dmitry

AU - Adamo, Roberto

AU - Nikolaev, Andrei

AU - Berti, Francesco

AU - Gerardy-Schahn, Rita

PY - 2014/7/11

Y1 - 2014/7/11

N2 - 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)-a-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 1H NMR, 31P 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.

AB - 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)-a-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 1H NMR, 31P 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.

UR - https://www.scopus.com/pages/publications/84904208356

U2 - 10.1074/jbc.M114.575142

DO - 10.1074/jbc.M114.575142

M3 - Article

AN - SCOPUS:84904208356

SN - 0021-9258

VL - 289

SP - 19395

EP - 19407

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 28

ER -