Effect of Acceptor Chain Length and Hydrophobicity on Polymerization Kinetics of the Neisseria meningitidis Group C Polysialyltransferase.

Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in virulence make the polysialyltransferases (PST) targets for therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here, we have generated recombinant bovine nucleotide monophosphate kinase to facilitate steady state kinetic assays of the PST. We have characterized the N. meningitidis group C (NmC) PST kinetically, using substrate analogues to describe the polymerization reaction. We observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased, indicating a tighter binding of longer oligomers. In addition, we observed a biphasic relationship between kcat and chain length, which can be attributed to a switch in the mechanism of transfer of sialic acid from distributive to processive as the chain length increased above six sialic acid units. Substitution of donor substrate with the analogue CMP-9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose that this decrease in kcat is caused by a destabilization of the transition state and/or an increase affinity of the product due to presence of the fluoro substituent. The acceptor's hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned over at a faster rate than the α-2,9 polysialic acid substrates lacking the hydrophobic end. This finding suggests the presence of a secondary ligand binding site that tethers the acceptor substrate to the enzyme active site.

Characterization of the meningococcal serogroup X capsule N-acetylglucosamine-1-phosphotransferase.

Neisseria meningitidis serogroups A, B, C, Y, W135 and X are responsible for most cases of meningococcal meningitis. Neisseria meningitidis serogroup X has recently emerged as a contributor to outbreaks of disease in Africa, but there is currently no vaccine against serogroup X. Understanding of the biosynthesis of the serogroup X capsular polysaccharide would provide useful tools for vaccine production. The serogroup X polysaccharide is a homopolymer of (α1→4)-linked N-acetylglucosamine (GlcNAc)-1-phosphate. It has been shown that the gene cluster xcbABC encodes synthesis of this polysaccharide. The xcbA gene product has significant homology with sacB, which is responsible for synthesis of the Neisseria serogroup A capsular polysaccharide, an (α1→6)-N-acetylmannosamine-1-phosphate homopolymer. The xcbA protein also shares homology with the catalytic domain of human N-acetylglucosamine-1-phosphoryltransferase, a key enzyme in the mannose-6-phosphate receptor pathway. In this study, we show that xcbA in the appropriate background is sufficient for the synthesis of N. meningitidis serogroup X polysaccharide. By ELISA we detected polysaccharide in fractions of Escherichia coli expressing the xcbA gene. We isolated polysaccharide from an E. coli strain expressing XcbA and demonstrated that this polysaccharide has a (13)C-NMR spectrum identical to that of polysaccharide isolated from N. meningitidis Group X. We also demonstrate that the purified XcbA protein is an N-acetylglucosamine-1-phosphotransferase that transfers N-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 4-hydroxyl of an N-acetylglucosamine-1-phosphate oligosaccharide. Oligosaccharides fluorescently labeled at the aglycon are extended by XcbA only after the 4-phosphate occupying the non-reducing GlcNAc has been removed. The minimum size of fluorescent acceptors is a trisaccharide.

Characterization and acceptor preference of a soluble meningococcal group C polysialyltransferase.

Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.

In vitro generation of polysialylated cervical mucins by bacterial polysialyltransferases to counteract cytotoxicity of extracellular histones.

Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone-mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell-based experiments demonstrated the activity of elongated mucins against histone-mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.

Successive glycosyltransfer of sialic acid by Escherichia coli K92 polysialyltransferase in elongation of oligosialic acceptors.

Escherichia coli K92 produces a capsular polysialic acid with alternating alpha2,8 alpha2,9 NeuNAc linkages. This polysaccharide is cross-reactive with the neuroinvasive pathogen Neisseria meningitidis Group C. The K92 polysialyltransferase (PST) catalyzes the synthesis of the polysialic acid with alternating linkages by the transfer of NeuNAc from CMP-NeuNAc to the nonreducing end of the growing polymer. We used a fluorescent-based high-performance liquid chromatography assay to characterize the process of chain extension. The PST elongates the acceptor GT3-FCHASE in a biphasic fashion. The initial phase polymers are characterized by accumulation of product containing 1-8 additional sialic acid residues. This phase is followed by a very rapid formation of high-molecular weight (MW) polymer as the accumulated oligosaccharides containing 8-10 sialic acids are consumed. The high-MW polymer contains 90-100 sialic acids and is sensitive to degradation by periodate and K1-5 endoneuraminidase, suggesting that the polymer contains the alternating structure. The polymerization reaction does not appear to be strictly processive, since oligosaccharides of each intermediate size were detected before accumulation of high-molecular weight polymer. Synthesis can be blocked by CMP-9-azido-NeuNAc. These results suggest that the K92 PST forms both alpha2,8 and alpha2,9 linkages in a successive and nonprocessive fashion.

Extracellular structure of polysialic acid explored by on cell solution NMR.

The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, alpha(2 --> 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13C,15N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.

Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases.

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

Functional molecular mass of Escherichia coli K92 polysialyltransferase as determined by radiation target analysis.

The polysialyltransferase of Escherichia coli K92 catalyzes the transfer of sialic acid from CMP-sialic acid to a growing chain of polysialic acid at the nonreducing end. The enzyme encoded by the neuS gene is membrane-associated and has been suggested to be organized within a complex of several proteins encoded by the K92 gene cluster. Attempts to prepare a soluble active NeuS enzyme have been unsuccessful. Recent results suggest that de novo synthesis of polysialic acid requires coexpression of four genes from the cluster: neuS, neuE, kpsC, and kpsS. However, elongation of preexisting polysialic acid chains only requires expression of neuS. The molecular organization of the catalytic unit of bacterial polysialyltransferases has not been described. We used radiation inactivation to measure the size of the minimum functional unit catalyzing the polysialyltransferase chain extension and de novo reactions. Membranes harboring NeuS in the presence and absence of other products of the K92 gene cluster were exposed to high-energy electrons. The rate of loss of polysialyltransferase activity reveals the mass of the molecules essential for catalytic activity. We observed that the transfer of neuNAc from CMP-neuNAc to a polysialic acid acceptor is catalyzed by a complex with a target size larger than that of monomeric NeuS. The target size of the unit catalyzing the extension of existing polysialic acid chains does not differ significantly from the size of the unit catalyzing transfer of sialic acid to the endogenous acceptor. Parallel samples of membranes containing NeuS and a green fluorescent protein (GFP) chimera were compared by target analysis. The target size of this structural unit was estimated by analysis of the rate of decay of the GFP-NeuS chimera band migrating in the immunoblots. The target size of the structural unit is larger than expected for a monomer. The results of these experiments show that while the target size of the catalytic activity for K92 polysialyltransferase is larger than a monomer of NeuS, a large complex is not required for catalysis.

Separate pathways for O acetylation of polymeric and monomeric sialic acids and identification of sialyl O-acetyl esterase in Escherichia coli K1.

O acetylation at carbon positions 7 or 9 of the sialic acid residues in the polysialic acid capsule of Escherichia coli K1 is catalyzed by a phase-variable contingency locus, neuO, carried by the K1-specific prophage, CUS-3. Here we describe a novel method for analyzing polymeric sialic acid O acetylation that involves the release of surface sialic acids by endo-N-acetylneuraminidase digestion, followed by fluorescent labeling and detection of quinoxalinone derivatives by chromatography. The results indicated that NeuO is responsible for the majority of capsule modification that takes place in vivo. However, a minor neuO-independent O acetylation pathway was detected that is dependent on the bifunctional polypeptide encoded by neuD. This pathway involves O acetylation of monomeric sialic acid and is regulated by another bifunctional enzyme, NeuA, which includes N-terminal synthetase and C-terminal sialyl O-esterase domains. A homologue of the NeuA C-terminal domain (Pm1710) in Pasteurella multocida was also shown to be an esterase, suggesting that it functions in the catabolism of acetylated environmental sialic acids. Our combined results indicate a previously unexpected complexity in the synthesis and catabolism of microbial sialic and polysialic acids. These findings are key to understanding the biological functions of modified sialic acids in E. coli K1 and other species and may provide new targets for drug or vaccine development.

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase.

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.