Conformational differences between linear alpha (2----8)-linked homosialooligosaccharides and the epitope of the group B meningococcal polysaccharide

The alpha-(2----8)-linked sialic acid oligosaccharides (NeuAc)n exhibit an unusual degree of heterogeneity in the conformation of their linkages. This was diagnosed by observation in their 13C NMR spectra of an equivalent and unique heterogeneity in the chemical shifts of their anomeric carbons and subsequently confirmed by more comprehensive 1H and 13C NMR studies. In these studies both one-dimensional and two-dimensional experiments were carried out on the trisaccharide (NeuAc)3 and colominic acid. In addition to the unambiguous assignment of the signals in the spectra, these experiments demonstrated that both linkages of (NeuAc)3 differed in conformation from each other and from the inner linkages of colominic acid. The NMR data indicate that these conformational differences extend to both terminal disaccharides of oligosaccharides larger than (NeuAc)5, a result that has considerable physical and biological significance. In the context of the group B meningococcal polysaccharide, it provides an explanation for the conformational epitope of the group B meningococcal polysaccharide, which was proposed on the evidence that (NeuAc)10, larger than the optimum size of an antibody site, was the smallest oligosaccharide able to bind to group B polysaccharide specific antibodies. Because the two terminal disaccharides of (NeuAc)10 differ in conformation to its inner residues, the immunologically functional part of (NeuAc)10 resides in its inner six residues. This number of residues is now consistent with the maximum size of an antibody site.     

Determinant specificities of the groups B and C polysaccharides of Neisseria meningitidis

A meningococcal group B-specific horse antiserum contains at least two distinct populations of antibodies with specificities for determinants on the group B capsular polysaccharide antigen. These two populations were differentiated on the basis of the ability of only one of them to be absorbed from the antiserum by the structurally related colominic acid. The nature of the colominic acid-specific determinant was elucidated by a radioimmunoassay inhibition technique with the use of a series of linear alpha-(2----8)-linked oligomers of sialic acid as inhibitors. Colominic acid was labeled by prior removal of its N-acetyl groups, followed by their replacement with the use of [3H]acetic anhydride. The conformational nature of the determinant was proposed because of the unusually large size (10 sialic acid residues) of the oligomer required to function as an efficient inhibitor. The structure of the determinant responsible for the second population of group B-specific antibodies has not been determined, but it is obviously based on an as yet undefined conformational or structural feature peculiar to the group B meningococcal polysaccharide. In contrast to the colominic acid-specific group B determinant, the determinant responsible for the group C polysaccharide-specific rabbit antibodies proved to be more conventional. Inhibitory properties of the alpha-(2----9)-linked oligomers maximized with those containing four or five sialic acid residues, which is consistent with the approximate estimated maximal size of an antibody site.     

Structural studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12.

A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.     

Control of meningococcal meningitis with meningococcal vaccines.

The development of effective meinigococcal vaccines was based upon the finding that immunity to the meningococcus was directly correlated with serum bactericidal antibodies. Purified high molecular weight capsular polysaccharides of serogroups A and C meningococci stimulated the production of humoral antibodies which had group specific bactericidal activity. In controlled field trials in Army recruits, group C polysaccharide vaccines were highly effective in preventing group C disease. Following its use as a routine immunization in recruits in October 1971 group C meningococcal disease has been almost completely eliminated from Army training centers. Group A vaccine has been field tested in Egyptian school children with great success. Group B polysaccharide has failed to induce bactericidal antibodies in humans and, therefore, new research is underway to attempt to develop a cell wall protein antigen as a vaccine against group B disease.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.     

Kinetic parameters of a beta-D-galactoside alpha 2 leads to 6 sialyltransferase from embryonic chicken liver

A beta-D-galactoside alpha 2 leads to 6 sialyltransferase was purified 500-fold in 14% yield from 14-day embryonic chicken liver. Characterization of the product of the sialyltransferase catalysis was accomplished by separation and permethylation of double-labelled ([14C]NeuAc, [3H]Gal) oligosaccharides following their release from the glycoprotein fetuin by hydrazinolysis. The enzyme transfers NeuAc to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to)R-terminated oligosaccharides; no activity was found towards Gal(beta 1 leads to 3)GalNAc(alpha 1 leads to)R structures. The trisaccharide. NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)Glc, was shown to be a good inhibitor of the sialyltransferase. Kinetic investigations of the enzyme indicate it to have a sequential, random bi-bi mechanism.     

一种新的CTAB加入方法对A群脑膜炎球菌荚膜多糖分子大小的影响

目的探讨CTAB不同的加入方法对A群脑膜炎球菌荚膜多糖分子大小的影响。方法采用分次加入手动搅拌和持续加入机械快速搅拌两种CTAB加入方法,纯化获得荚膜多糖粗糖,分别编为B组和C组。将两组荚膜多糖粗糖分别纯化获得精糖,分别编为D组和E组。以Sepharose CL-4B凝胶层析纯化获得荚膜多糖并检测其KD值。结果 B组荚膜多糖粗糖的KD值介于0.34~0.35之间,C组荚膜多糖粗糖的KD值介于0.03~0.05,进一步用苯酚纯化获得精糖后KD值D组介于0.34~0.36之间,E组介于0.22~0.28之间。两组相比KD值显著降低。结论CTAB的加入过程对A群脑膜炎球菌荚膜多糖的分子大小有明显的影响,CTAB沉淀时进行快速而充分的搅拌,纯化获得的荚膜多糖相对分子质量更大。     

Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis

The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.     

Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.

High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.     

CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.     

Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.     

Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10^?3. Measurement of enzymatic activities involved in the biosynthesis of the α-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)₇ residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out-break of meningococcal disease.     

Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination

Capsule-deficient mutants of Neisseria meningitidis serogroup B strain B1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule-deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C, W135, Y and Z.     

Substrate specificity of two bacteriophage-associated endo-N-acetylneuraminidases.

For Escherichia coli Bos12 (O16:K92:H-), a bacteriophage (phi 92) has been isolated which carries a depolymerase active on the K92 capsular polysaccharide. As seen under the electron microscope, phi 92 belongs to Bradley's morphology group A and is different from the phage phi 1.2 previously described (Kwiatkowski et al., J. Virol. 43:697-704, 1982), which grows on E. coli K235 (O1:K1:H-), depolymerizes colominic acid, and belongs to morphology group C. The specificity of the phi 1.2- and phi 92-associated endo-N-acetylneuraminidases has been studied with respect to the following substrates (all alkali treated, and where NeuNAc represents N-acetylneuraminic acid): (i) [-alpha-NeuNAc-(2 leads to 8)-]n (colominic acid), (ii) [-alpha-NeuNAc-(2 leads to 8)-alpha-NeuNAc-(2 leads to 9)-]n (E. coli K92 polysaccharide), and (iii) [-alpha-NeuNAc-(2 leads to 9)-]n (Neisseria meningitidis type C capsular polysaccharide). The increase in periodate consumption of these glycans upon incubation with purified phi 1.2 or phi 92 particles was measured, and the split products obtained from all substrates after exhaustive degradation were analyzed by gel chromatography. It was found that the Neisseria polysaccharide is not appreciably affected by either virus enzyme and that phi 1.2 only depolymerizes a small fraction of the K92 glycan. Colominic acid, however, is completely degraded by both agents, phi 92 yielding smaller fragments (one to six NeuNAc residues) than phi 1.2 (two to seven). Phage phi 92 additionally depolymerizes the K92 glycan, essentially to oligosaccharides of two, four, and six residues. The size distribution of these K92 oligosaccharides indicates that the phi 92 enzyme predominantly cleaves the alpha(2 leads to 8) linkages in this polymer.     

The metabolism of sialic acids in isolated rat colonic mucosal cells.

The activities of ten enzymes involved in sialic acid metabolism were measured in colonic mucosal cells from rats and compared with those in liver. A methodology was devised that enabled all ten enzyme activities to be evaluated in a single rat colon preparation. Enzyme assays with radioactively labelled substrates were developed for maximum sensitivity, and the identification of substrates and products was carefully checked to assess the contribution of contaminants to enzyme reactions with low activity. The activities of most enzymes involved in the biosynthesis of N-acetyl-D-neuraminic acid (NeuAc) from UDP-N-acetyl-D-glucosamine were found to be more than 20-fold lower than those in liver. The activities of CMP-NeuAc synthase, N-acetyl-D-glucosamine 2-epimerase, N-acetyl-D-glucosamine kinase, sialyltransferase and sialidase were similar to or 2-4-fold lower than in liver. The biosynthesis of NeuAc via its 9-phosphate was demonstrated in the 100 000 g supernatant of colonic-cell homogenates by enzymic assay and precursor experiments with N-acetyl[14C]-mannosamine. No alternative route for NeuAc formation could be detected. The 100 000g supernatant fractions of liver, kidney and colonic mucosal cells utilized N-acetyl[14C]mannosamine with differing efficiencies. Radioactive products identified as sialic acid biosynthetic intermediates amounted to 49%, 0.04% and 5.6% of added precursor in liver, kidney and colon respectively. Catabolism of labelled precursor to non-hexosamine products was high in kidney and colonic mucosal-cell fractions.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl alpha-D-mannopyranoside and appeared to be alpha-mannan, while that which passed through the column was virtually all beta-glucan. Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the alpha-mannan contained predominantly alpha-(1 leads to 2)-linked side chains branching from an alpha-(1 leads to 6)-linked backbone at the (1 leads to 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The beta-glucan seemed to contain mainly (1 leads to 6)-linked side chains branching from a (1 leads to 3)-linked backbone at the (1 leads to 3,6)-linked glucopyranosyl residues.     

Localization of acetyl groups in macromolecules of glucomannan from Eremurus zangezuricus roots

Water-soluble glucomannan from roots of Eremurus zangezuricus Mikheev was studied. The polysaccharide contains D-glucose, D-mannose, and acetyl groups in the molecular ratio of 1:2.8:0.38. 13C NMR studies showed that the polysaccharide under study is a linear, partly acetylated 1,4-beta-D-glucomannan. The acetyl groups are attached to the C2 and C3 of mannopyranose units. The polymer contains, on average, one acetyl group per seven mannose units. The glucomannan of E. zangezuricus has the following parameters: [alpha]D = -37.5 degrees, [eta] = 3.73 dl/g, and Mw = 151 kDa.     

High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature

The capsular polysaccharide of Escherichia coli K92 consists of a linear polymer of Neu5Ac with alternating alpha(2-8) and alpha(2-9) linkages. It accumulates when the bacterium is grown at 37 degrees C in a defined medium containing D-xylose and L-asparagine as carbon and nitrogen sources. Release of the capsular polymer into the medium was maximal (450 micrograms x ml-1) in the stationary phase of growth (76 h). This medium could be useful for obtaining sufficient polymer to develop effective vaccines. The enzyme, CMP-Neu5Ac synthetase, was not detected in cells grown at 20 degrees C. The lack of this enzyme explains the absence of polymer biosynthesis when the bacterium was grown at 20 degrees C.     

Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.