Separate Pathways for O Acetylation of Polymeric and Monomeric Sialic Acids and Identification of Sialyl O -Acetyl Esterase in Escherichia coli K1

Abstract

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.Escherichia coli K1 is a versatile human and animal facultative pathogen that causes a variety of extraintestinal diseases including sepsis, meningitis, cystitis, pyelonephritis, cellulitis, pneumonia, and postoperative infections. The ability of E. coli K1 to invade and traverse the mammalian epithelial cell barrier also may contribute to inflammatory bowel syndromes such as Crohn's disease. A primary virulence determinant in these diseases is the polysialic acid capsule or K1 antigen, a homopolymer of 2-keto-3-deoxy-5-acetamido-7,8,9-D-glycero-D-galacto-nonulosonic, or N-acetylneuraminic acid (Neu5Ac, the most common sialic acid), residues connected by ␣2,8-glycoketosidic linkages (36). The kps and neu genes needed for polysialic acid synthesis and export map to a 17-kb accretion domain inserted near pheV (7). Mutation of neu (biosynthetic) genes generally results in no capsular polysaccharide produced while kps mutations usually result in intracellular accumulation of unexported polysaccharides (46, 47). Polysialic acid is also found on the mammalian neural cell adhesion molecule and comprises the group B meningococcal, Pasteurella haemolytica A2, and Moraxella nonliquefaciens capsular polysaccharides (41). Mammalian polysialic acid regulates cell migration, axon pathfinding and targeting, and plasticity in the embryonic and adult nervous system (6). Molecular mimicry of this antigen by the bacterial capsules is thought to account for the relatively low immunogenicity of microbial polysialic acid, which has limited the attempts to produce safe and effective capsulebased vaccines ...

NeuA Sialic Acid O-Acetylesterase Activity Modulates O-Acetylation of Capsular Polysaccharide in Group B Streptococcus

“…This behavior has also been noted for Sias released from the GBS surface by mild acid hydrolysis (17,18). of O-acetyltransferase-and O-acetylesterase-encoding genes (18,38), which may themselves produce enzymes with varied mechanisms and/or activity levels (Fig. 4) (51).…”

“…Initial experiments on the bifunctional enzymes employed 4-nitrophenyl-acetate and (acetylated) platelet-activating factor as substrates of the esterase reaction (36,37). More recently, work by Vimr and coworkers (38) suggest that in E. coli, the native substrate of the NeuA esterase domain is O-acetylated Sia. Based on these studies and our earlier observations, we hypothesized that GBS NeuA acts natively as a Sia-O-acetylesterase, an intracellular process that could mitigate overall O-acetylation of the sialylated capsule in vivo.…”

Sialic acid utilization by bacterial pathogens

“…In E. coli the Oacetyltransferases NeuO and NeuD modify PSA and monomeric Neu5Ac, respectively, the latter of which can be deacetylated by NeuA acting as a bifunctional enzyme (Fig. 1) (Steenbergen et al, 2006;Deszo et al, 2005;Bergfeld et al, 2007). These new discoveries highlight the huge potential diversity of capsule structures that may be presented on the cell surface by varying the patterns of acetylated sialic acid (King et al, 2007).…”

“…An initial step in PSA synthesis is thought to include the addition of Neu5Ac by the NeuE protein to some asyet-unidentified initiator molecule, presumed to be a lipid (Vimr et al, 2004). Once synthesized, sialic acid residues in the PSA capsule of both N. meningitidis and E. coli can be modified by O-acetylation (Claus et al, 2004;Steenbergen et al, 2006;Deszo et al, 2005), an emerging and exciting new feature of sialic acid metabolism also seen in unencapsulated organisms (see below). In E. coli the Oacetyltransferases NeuO and NeuD modify PSA and monomeric Neu5Ac, respectively, the latter of which can be deacetylated by NeuA acting as a bifunctional enzyme (Fig.…”

“…The site of interaction of factor H (fH) on the gonococcal cell surface is deliberately indicated in an ambiguous manner (black arrow) as it is still unclear whether the sialylated LPS is an actual binding site for fH. The monomeric O-acetylated Neu5Ac produced by E. coli NeuD is believed to enter the normal PSA biosynthetic pathway via NeuA/NeuS (Steenbergen et al, 2006); hence the omission of the activated intermediate from the figure. The light-green discontinuous arrow represents the pathways (mostly not defined) by which the LPS is exposed on the outer membrane.…”