Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A.

Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

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.

[Cloning and expression of the IGA-specific endopeptidase genes from Neisseria meningitidis].

IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.