Molecular Cloning and Functional Characterization of Components of the Capsule Biosynthesis Complex of Neisseria meningitidis Serogroup A

Abstract

Background:The isolation of capsular polysaccharides from pathogenic bacteria for vaccine production is cost-intensive. Results: We describe the cloning, recombinant expression, and functional characterization of three enzymes from Neisseria meningitidis serogroup A that facilitate in vitro synthesis of the capsule polymer. Conclusion:The study presents a novel basis for efficient vaccine production. Significance: Economic vaccine production is prerequisite to combat meningococcal diseases.

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

“…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.…”

“…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.…”

“…Because the radioactive incorporation assay does not provide information on the composition and size of formed products, we used an HPLC-AEC assay that allows the recording of nucleotide sugar consumption and product formation (30). In a total volume of 75 l, 0.15 nmol of ⌬N37-CslB-His 6 were incubated with 4 mM UDP-GlcNAc.…”

“…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).…”

“…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).…”