title: Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development.
abstract: Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany.
authors.forename: Timm
authors.initials: T
authors.lastname: Fiebig
authors.forename: Francesco
authors.initials: F
authors.lastname: Berti
authors.forename: Friedrich
authors.initials: F
authors.lastname: Freiberger
authors.forename: Vittoria
authors.initials: V
authors.lastname: Pinto
authors.forename: Heike
authors.initials: H
authors.lastname: Claus
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Romano
authors.forename: Daniela
authors.initials: D
authors.lastname: Proietti
authors.forename: Barbara
authors.initials: B
authors.lastname: Brogioni
authors.forename: Katharina
authors.initials: K
authors.lastname: Stummeyer
authors.forename: Monika
authors.initials: M
authors.lastname: Berger
authors.forename: Ulrich
authors.initials: U
authors.lastname: Vogel
authors.forename: Paolo
authors.initials: P
authors.lastname: Costantino
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D016667:Bacterial Capsules / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D055808:Drug Discovery; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008585:Meningitis, Meningococcal / Q000276:immunology / Q000517:prevention & control; D022401:Meningococcal Vaccines / Q000096:biosynthesis*; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D022401:Meningococcal Vaccines; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase
keywords: NMR; Neisseria meningitidis serogroup X; capsule polymerases; recombinant protein production; vaccine development