Biochemical Characterization of Thepolysialic Acid-specific O-Acetyltransferase NeuO of Escherichia coli K1

Abstract

Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of ␣2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5 -coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (k cat /K m donor ) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel ␤-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed ␤-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.Escherichia coli K1 is one of the main organisms causing bacterial sepsis and meningitis during the neonatal period (1-3). The pathogenesis of E. coli meningitis is a complex process involving colonization of the gastrointestinal tract, intestinal translocation, bacteremia, passage of the blood-brain barrier, and invasion of the arachnoidal space (4). Cell culture experiments indicated that transfer across the blood-brain barrier is mediated by transcytosis through brain endothelial cells (5, 6) and that, in contrast to other bacterial agents causing meningitis, E. coli K1 invades meningioma cells directly, leading to rapid cell death before an inflammatory response can be induced (7). Despite advances in diagnostics and therapeutics of neonatal infections, E. coli neonatal meningitis is still characterized by high rates of mortality and a high incidence of permanent neurological sequelae in those that survive (8, 9).E. coli K1 is protected by a thick layer of capsular polysaccharide, which is the major virulence factor important for serum resistance and vital passage of the blood-brain barrier (5, 10, 11). The K1 capsule consists of polysialic acid (polySia) 2 formed by the most prevalent sialic acid of humans, 5-N-acetyl neuraminic acid (Neu5Ac). Up to 200 residues are joined in ␣2,8-glycosidic linkages, resulting in a linear homopolymer that is structurally iden...

The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases

“…Similar observations have been made for NeuO, a prophage-encoded sialate O-acetyltransferase found in E. coli K1 strains that harbor the CUS-3 phage (25). NeuO catalyzes the O-acetylation of K1-CPS, and the enzyme acts exclusively on ␣2,8-linked polySia with at least 14 sialic acid residues (26,60).…”

“…Silver staining, Coomassie staining, and Western blot analysis with anti-penta-His antibody (Qiagen) were performed as described previously (26).…”

“…Since 2004, several genes encoding sialate O-acetyltransferases have been identified in bacteria that contain sialic acid as a component of their CPS or lipo-oligosaccharide, such as Escherichia coli K1, Campylobacter jejuni, and type III group B streptococci (25)(26)(27)(28). Notably, all these enzymes belong to the hexapeptide repeat family, suggesting that the specificity for sialic acid evolved exclusively within this protein family.…”

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).…”

“…Some pathogens, such as Neisseria spp., H. influenzae and C. jejuni, regulate expression of cellsurface features by stochastic genetic mechanisms relying on rapid and reversible ON to OFF switching of the expression of key biosynthetic genes, the so-called 'phase variation ' (van der Woude & Baumler, 2004). Phase variation also modulates O-acetylation of the PSA capsule of E. coli K1 (Deszo et al, 2005;Bergfeld et al, 2007). Such variable expression makes it likely that any population of bacteria will be expressing alternative glycoforms and that any one expression pattern might confer a fitness advantage to the relevant bacteria under any given conditions.…”