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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-acetyineuraminidase 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 K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an [alpha]-2,8-1inked 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 ([DELTA]neuC), 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 [DELTA]neuC. 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.sup.-14]C]acetamidoglucal and [[N.sup.-14]C]acetylmannosamine (ManNAc) from UDP-[[.sup.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-GIcNAc 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.

Functional molecular mass of Escherichia coli K92 polysialyltransferase is determined by radiation inactivation and the membranes harboring NeuS in the presence and absence of other products of the K92 gene cluster are exposed to high-energy electrons. Results suggest that while the target size of the catalytic activity for K92 polysialyl transferase is larger than a monomer of NeuS, a large complex is not required for catalysis.

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. [ABSTRACT FROM AUTHOR]

Bacterial polysaccharides are components of glycoconjugate vaccines against encapsulated bacterial pathogens. Because these vaccines are prepared by random coupling methods the structure of the immunogens is difficult to characterize. As a model for the development of well-defined glycoconjugates we have devised a chemoenzymatic method for preparation of conjugatable oligosialic acids. In this scheme, chemically synthesized azide or alkyne derivatives of lactosides are sialylated by bacterial sialyltransferases. An HPLC method to follow the progress of the enzymatic reaction was developed based on fluorogenic coupling partners of the Huisgen 1,3-cycloaddition reaction, since the azide and alkyne aglycons are not convenient chromaphores. We have used this chemoenzymatic scheme to synthesize oligosialic acids of Neisseria meningitidis group C polysaccharide with a conjugatable aglycon. The chemoenzymatically synthesized oligosialic acids were coupled to bovine serum albumin with the 1,3-cycloaddition reaction. The resulting glycoconjugate was reactive with N. meningitidis group C-specific monoclonal antibody, thus confirming this as a possible pathway to glycoconjugate antigens. [ABSTRACT FROM AUTHOR]

We have chosen E. coli K92, whichproduces the alternating structure α(2-8)neuNAc α(2-9)neuNAc as a model system for studyingbacterial polysaccharide biosynthesis. We have shown that the polysialyltransferaseencoded by the K92 neuS gene can synthesize both α(2-8)and α(2-9) neuNAc linkages invivo by 13C-nuclear magnetic resonance analysisof polysaccharide isolated from a heterologous strain containingthe K92 neuS gene. The K92 polysialyltransferase is associated withthe membrane in lysates of cells harboring the neuS gene in expressionvectors. Although the enzyme can transfer sialic acid to the nonreducing endof oligosaccharides with either linkage, it is unable to initiate chainsynthesis without exogenously added polysialic acid. Thus, the polysialyltransferaseencoded by neuS is not sufficient for de novosynthesisof polysaccharide but requires another membrane component for initiation.The acceptor specificity of this polysialyltransferase was studiedusing sialic acid oligosaccharides of various structures as exogenousacceptors. The enzyme can transfer to the nonreducing end of allbacteria polysialic acids, but has a definite preference for α(2-8) acceptors. Gangliosides containingneuNAc α(2-8)neuNAc are elongated, whereas monsialylatedgangliosides are not. Disialylgangliosides are better acceptorsthan short oligosaccharides, suggesting a lipid-linked oligosaccharidemay be preferred in the elongation reaction. These studies showthat the K92 polysialyltransferase catalyzes an elongation reactionthat involves transfer of sialic acid from CMP–sialic acidto the nonreducing end of two different acceptor substrates. [ABSTRACT FROM PUBLISHER]

CMP‐β‐N‐acetylneuraminic acid (CMP‐neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP‐neuNAc synthetase catalyzes the formation of this substrate, CMP‐neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP‐neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS–PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I‐ASA‐CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP‐neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer. [ABSTRACT FROM PUBLISHER]