Molecular cloning and functional characterization of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A: toward in vitro vaccine production.
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.
From the Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Timm
T
Fiebig
From the Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Friedrich
F
Freiberger
Novartis Vaccines, Research, Via Fiorentina 1, 53100 Siena, Italy.
Vittoria
V
Pinto
Novartis Vaccines, Research, Via Fiorentina 1, 53100 Siena, Italy.
Maria Rosaria
MR
Romano
College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom, and.
Alan
A
Black
From the Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Christa
C
Litschko
From the Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Andrea
A
Bethe
the Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya Street 10, Moscow 119832, Russian Federation.
Dmitry
D
Yashunsky
Novartis Vaccines, Research, Via Fiorentina 1, 53100 Siena, Italy.
Roberto
R
Adamo
College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, United Kingdom, and.
Andrei
A
Nikolaev
Novartis Vaccines, Research, Via Fiorentina 1, 53100 Siena, Italy, francesco.berti@novartis.com.
Francesco
F
Berti
From the Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany, gerardy-schahn.rita@mh-hannover.de.
Rita
R
Gerardy-Schahn
D016667:Bacterial Capsules / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D022401:Meningococcal Vaccines; D038381:Neisseria meningitidis, Serogroup A / Q000201:enzymology* / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D022401:Meningococcal Vaccines; D011135:Polysaccharides, Bacterial
Acetyltransferase; Capsule Polymerase; Enzyme Catalysis; Epimerase; Gram-negative Bacteria; Neisseria meningitidis Serogroup A; Nuclear Magnetic Resonance (NMR); Polysaccharide; Recombinant Protein Expression; Vaccine Development