Willie F. Vann

Predominance of two newly described capsular polysaccharide types among clinical isolates of Staphylococcus aureus

A capsular polysaccharide typing schema for Staphylococcus aureus, based upon the preparation of rabbit typing sera with eight prototype strains, has been reported ( Karakawa and Vann , 1982). These antisera were used to classify the capsular polysaccharides of 246 S. aureus isolates from patients in a survey of hospitals in several countries and 49 consecutive blood isolates obtained over a 17-month period in a clinical study at the Boston Veterans Administration Medical Center. Two capsular types, 5 and 8, accounted for about 70% of these isolates; most of the remaining strains could not be typed with the available antisera. The clinical study of bacteremia identified capsular types 5 and 8 among both community-acquired and nosocomial isolates and showed that strains bearing these two types caused the patterns of disease reported for staphylococcal bacteremia. There was an association between the phage type and the capsular type of these bacteremic strains. The capsular types of the classic encapsulated strains of S. aureus, M (type 1) and Smith (type 2), were not observed among blood isolates in this study. The observation that most clinical isolates of S. aureus belong to two recently defined capsular types provides a new focus for investigations into the virulence of this common nosocomial pathogen and suggests the potential for protective acquired immunity against staphylococcal bacteremia.

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.

Cloning and Expression of the HumanN-Acetylneuraminic Acid Phosphate Synthase Gene with 2-Keto-3-deoxy-d-glycero- d-galacto-nononic Acid Biosynthetic Ability

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia colisialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results inN-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme usesN-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.

Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in α‐2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane‐associated polysialyltransferase (NmB‐polyST). Here we present a comprehensive characterization of NmB‐polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB‐polyST was expressed, purified and shown to synthesize long polySia chains in a non‐processive manner in vitro. Subsequent structure–function analyses of NmB‐polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E‐D/E‐G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP‐Neu5Ac binding was demonstrated by mutational analysis of NmB‐polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure–function relations and for phylogenetic sorting of these important enzymes.

An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides

A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.

Cross-reactive antigens and immunity to diseases caused by encapsulated bacteria

Antigenic structures may be shared among naturally occurring polymers, including proteins and polysaccharides. Proteins are polymers of amino acids. Cross-reactions between proteins are due to similarities in their overall shape rather than their individual amino acid components. Cross-reactions have been demonstrated among proteins with similar evolutionary development and structure, such as serum albumins or immunoglobulins. Polysaccharides are polymers of monosaccharides. In contrast to proteins, antigenic specificities may be conferred by mono-, di-, and trisaccharides. Since there are about 150 known naturally occurring monosaccharides, it is not unexpected that cross-reactions are demonstrable between polysaccharides from widely divergent sources.

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.

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

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.

Gene Products Required for De Novo Synthesis of Polysialic Acid in Escherichia coli K1

Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the alpha(2-8)-polysialic acid NeuNAc(alpha2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.

The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase

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-linked 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 (DeltaneuC), 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 DeltaneuC. 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-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(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-GlcNAc 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.