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.

The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

“…Activity Testing of CslA and CslB by Use of a Radioactive Assay System-Enzymatic activity was analyzed using an adapted radioactive incorporation assay previously described for the CPs of N. meningitidis serogroups X and A (30,31 14 C]GlcNAc (American Radiolabeled Chemicals). The assay was performed in the presence and absence of 2 l of heat-inactivated N. meningitidis serogroup L lysate.…”

“…4B, 37°C, overnight incubation) and 10 mM MgCl 2 (Fig. 4, C-E, 37°C, 45-min incubation) because Mg 2ϩ is known to promote Stealth activity (30,31,47) and was found to stabilize UDP-Glc and UDP-Gal. In general, all enzymes as well as all other reactants were used in the same concentrations as in the radioactive incorporation assay described above.…”

“…All of the 1 H and 31 P NMR experiments were recorded as described previously (31). To detect the 13 C chemical shifts (Table 3), a 1 H, 13 C heteronuclear multiple quantum coherence experiment was recorded on a BrukerAvance III 400-MHz spectrometer equipped with a 5-mm broadband probe (Bruker).…”

“…In this context, we focused our work on capsule polymerases (CPs) of both pathogenic and rarely occurring meningococcal serogroups and thereby follow two research lines: (i) exploitation of recombinant CPs for in vitro production of glycoconjugate vaccines (30,31) and (ii) delineation of structure-function relationships as a basis for drug target evaluation (32)(33)(34)(35)(36)(37)(38)(39). Recently, we cloned and functionally expressed the CPs from serogroups A (CsaB) and X (CsxA) (30,31).…”

“…Recently, we cloned and functionally expressed the CPs from serogroups A (CsaB) and X (CsxA) (30,31). Both enzymes belong to the Stealth protein family that exclusively consists of D-hexose-1-phosphate transferases (40).…”