The Group B Streptococcal Sialic Acid O-Acetyltransferase Is Encoded by neuD, a Conserved Component of Bacterial Sialic Acid Biosynthetic Gene Clusters

Abstract

Nearly two dozen microbial pathogens have surface polysaccharides or lipo-oligosaccharides that contain sialic acid (Sia), and several Sia-dependent virulence mechanisms are known to enhance bacterial survival or result in host tissue injury. Some pathogens are also known to O-acetylate their Sias, although the role of this modification in pathogenesis remains unclear. We report that neuD, a gene located within the Group B Streptococcus (GBS) Sia biosynthetic gene cluster, encodes a Sia O-acetyltransferase that is itself required for capsular polysaccharide (CPS) sialylation. Homology modeling and site-directed mutagenesis identified Lys-123 as a critical residue for Sia O-acetyltransferase activity. Moreover, a single nucleotide polymorphism in neuD can determine whether GBS displays a "high" or "low" Sia O-acetylation phenotype. Complementation analysis revealed that Escherichia coli K1 NeuD also functions as a Sia O-acetyltransferase in GBS. In fact, NeuD homologs are commonly found within Sia biosynthetic gene clusters. A bioinformatic approach identified 18 bacterial species with a Sia biosynthetic gene cluster that included neuD. Included in this list are the sialylated human pathogens Legionella pneumophila, Vibrio parahemeolyticus, Pseudomonas aeruginosa, and Campylobacter jejuni, as well as an additional 12 bacterial species never before analyzed for Sia expression. Phylogenetic analysis shows that NeuD homologs of sialylated pathogens share a common evolutionary lineage distinct from the poly-Sia O-acetyltransferase of E. coli K1. These studies define a molecular genetic approach for the selective elimination of GBS Sia O-acetylation without concurrent loss of sialylation, a key to further studies addressing the role(s) of this modification in bacterial virulence.Sialic acids (Sias) 3 are a family of related nine-carbon acidic sugars (nonulosonic acids) that are found at the outer ends of glycan chains on all vertebrate cells. Over 20 microbial pathogens are known to use surface Sia decoration as a form of molecular mimicry that is often crucial to virulence (1). Several convergent strategies for microbial Sia decoration have been identified, including endogenous biosynthesis, scavenging, or direct transfer of host Sias to their own surface glycans (1). Heavily sialylated bacteria often cause sepsis and meningitis by exhibiting Sia-dependent serum resistance due to deactivation of the alternative complement pathway (2-4). In addition, bacterial Sias can enhance intracellular survival (5), participate in biofilm formation (6), or mask underlying antibody epitopes (7). Antibodies elicited against sialylated bacterial glycolipids can also cross-react with host peripheral nerve axons, leading to the debilitating autoimmune disorder called GuillainBarré syndrome (8, 9). In short, there are multiple Sia-dependent virulence mechanisms that enhance bacterial survival and/or result in host tissue injury.Several pathogenic bacteria are known to modify Sia residues by O-acetylation (10 -18). In other contexts, ...

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).…”

“…A polymorphism within the GBS O-acetyltransferase encoded by neuD is at least partially responsible for controlling the degree of O-acetylation in GBS, where 88C corresponds to the "low-O-Ac" phenotype (Ͻ5% O-Ac) and 88F corresponds to the "high-O-Ac" phenotype (Ͼ30% Sia-O-Ac) (18). Here we show that in high O-Ac GBS strains, NeuA prevents even greater levels O-acetylation (Ͼ70%) as observed in the NeuA esterase-inactive mutant (Fig.…”

“…Recently, we described the biochemical and genetic basis of capsular Sia O-acetylation in GBS (17,18), a modification unrecognized during earlier GBS capsule studies. The O-acetyl modification of Sias and related molecules is not unique to GBS; rather, it is characteristic of many bacterial pathogens (17, 19 -26) as well as many vertebrate cell types (27)(28)(29)(30)(31)(32).…”

“…That Maintain the NeuA CMP-Sia Synthetase Activity-The GBS O-acetyltransferase, NeuD, acts at the level of free intracellular Sia (17,18). We hypothesized that the NeuA C-terminal esterase domain could act on free intracellular O-acetylated Sias to regulate capsular O-acetylation on the bacterial surface.…”

Sialic acid utilization by bacterial pathogens

“…The only Grampositive bacterium reported to produce a sialic acidcontaining capsule is S. agalactiae, which possesses a sialyltransferase (CpsK) that adds a terminal a-2,3-linked Neu5Ac to galactose within the capsule's oligosaccharide repeat (Chaffin et al, 2005). Again, recent work has shown that this Neu5Ac residue can be modified by O-acetylation (Lewis et al, 2006).…”