Leann L. MacLean

Structural and Genetic Analyses of O Polysaccharide from Actinobacillus actinomycetemcomitans Serotype f

ABSTRACT The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed ofl-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five knownA. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitansserotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

A plasmid‐encoded rfbO:54 gene cluster is required for biosynthesis of the O:54 antigen in Salmonella enterica serovar Borreze

Previous studies demonstrated that the presence of a 7–8 kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in several Salmonella enterica serovars. In this study, a 6.7 kb plasmid from a field isolate of S. enterica serovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O‐polysaccharide‐substituted LPS molecules and O:54 serotype. SDS‐PAGE analysis of other 0:54 isolates indicated that the O:54 O‐polysaccharide can be co‐expressed with an additional O‐polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N‐acetylmannosamine (D‐ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure:

Structural variation in the O‐specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes

The O‐polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose‐containing homopolymers. Structural analysis, using chemical and high‐field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units

The structure of the antigenic lipopolysaccharide O-chains produced by Salmonella urbana and Salmonella godesberg

The lipopolysaccharides of Salmonella urbana and Salmonella godesberg, which belong in group N (O:30) of the Kauffmann-White system, were shown by SDS-PAGE electrophoresis, glycose analysis, periodate oxidation, methylation, and 1H- and 13C-n.m.r. analyses to have identical O-chains composed of repeating, branched pentasaccharide units having the structure: [----4)-beta-D-Glcp-(1----3)-alpha-D-GalNAcp-(1----2)-alpha-D-P erNAcp-(1----3)-alpha-L-Fucp-(1----]n 4 increases 1 beta-D-Glcp. The serological cross-reactivity of S. urbana and S. godesberg with Brucella abortus and Yersinia enterocolitica (O:9) can now be related to the presence of a 1,2-glycosylated N-acyl derivative of 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective lipopolysaccharide O-chains.

The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270

Strains of the Gram-negative bacterium Cronobacter (formerly known as Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and they have been particularly associated with meningitis in neonates where infant milk formulae have been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D 1H and 13C NMR, and MS methods revealed that the O-polysaccharide produced by Cronobacter muytjensii strain 3270, isolated from powdered infant formula from Denmark, was a linear unbranched polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-galactose (d-GalNAc), 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 3-acetamido-3-deoxy-d-quinovose (d-Qui3NAc), l-rhamnose (l-Rha), and d-glucuronic acid (d-GlcA) in equimolar ratio, and has the structure -->3)-alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->6)-alpha-D-GlcpNAc-(1-->4)-beta-D-GlcpA-(1--> The specific structural characteristics of the O-polysaccharides of C. muytjensii may be of value in the identification and tracking of the bacterial pathogen.

Application of 1D and 2D NMR techniques to the structure elucidation of the O-polysaccharide from Proteus mirabilis O: 57

SummaryThe LPS O-polysaccharide (O-PS) produced by Proteus mirabilis serotype O: 57 (ATCC 49995) was shown by NMR spectroscopy and chemical analysis to be a high-molecular-weight acidic branched polymer of pentasaccharide repeating units, composed of d-glucose, d-galactose, 2-acetamido-2-deoxy-d-galactose and glycerophosphate residues (1:2:2:2:1). Application of one-and two-dimensional NMR methods allowed the complete assignment of notoriously crowded 1H and 13C spectra of the O-PS, leading to the determination of its structure. Several of the NMR techniques used were applied for the first time to the structure elucidation of polysaccharides. The resonances of galactose H5, H6 and H6′ were identified by a 1D analog of 3D NOESY-TOCSY and 2D {1H, 1H} triple-quantum experiments. The position and the nature of the phosphate group were determined from 2D 31P (ω1)-half-filtered COSY and 2D 31P-relayed COSY spectra. 2D HMQC-TOCSY and 2D single-quantum proton-carbon long-range correlation techniques were used to overcome the difficulties of severe overlap and higher order effects in the 1H NMR spectrum of the O-PS. The latter technique, together with 2D NOESY, enabled us to identify the substitution positions, the anomeric configurations and the sequence of the component glycose residues in the O-PS.

Structure of the antigenic repeating pentasaccharide unit of the LPS O-polysaccharide of Cronobacter sakazakii implicated in the Tennessee outbreak

Strains of the Gram-negative bacterium Cronobacter (Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and have been particularly associated with meningitis in neonates where infant-milk formulae has been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D 1H and 13C NMR, and MS methods revealed that the O-polysaccharide produced by C. sakazakii (3290), a clinical strain from the Tennessee outbreak, was a branched polymer of repeating pentasaccharide units composed of 2-acetamido-2-deoxy-D-galactose, 3-(N-acetyl-L-alanylamido)-3-deoxy-D-quinovose, D-glucuronic acid, and D-glucose present in the molar ratio 1:1:1:2 and had the structure:The O-PS structure provides a unique specific structurally defined marker for the clinical tracking of this pathogen.

The structure of the polysaccharide produced by Proteus vulgaris (ATCC 49990)

An exocellular polysaccharide produced by a clinical isolate of Proteus vulgaris (ATCC 49990) was shown by composition, methylation, periodate oxidation, and nuclear magnetic resonance analyses to be composed of repeating trisaccharide units containing D-galactose, 2-acetamido-2-deoxy-D-glucose and pyruvic acid (2:1:1), and having the structure: [formula: see text] The homologus smooth lipopolysaccharide of the P. vulgaris strain was determined to have an O-polysaccharide component having the same structure as the above extracellular polysaccharide.

Characterization of the O-antigen in the lipopolysaccharide of Cronobacter (Enterobacter) malonaticus 3267

Cronobacter malonaticus, a Gram-negative bacterium previously known as Enterobacter sakazakii, is an opportunistic pathogen known to cause serious infection in infants and neonates. To provide aid for the serological and chemical identification of clinical, environmental, or food isolates of this emerging pathogen, the characterization of the lipopolysaccharide (LPS) O-polysaccharide (O-PS) antigens of Cronobacter spp. is being undertaken. The structural analysis of the O-PS, obtained by hydrazinolysis of the lipopolysaccharide produced by Cronobacter malonaticus HPB 3267, was investigated by composition, methylation, and two-dimensional high-resolution nuclear magnetic resonance methods, and was found to be a polymer of a branched pentasaccharide unit. This unit is composed of D-glucose (D-Glc), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues (1: 1: 1: 1: 1), forms the repeating oligosaccharide in the O-PS antigen, and has the structure: [structure: see text].

The structure of the O-antigen of Cronobactersakazakii HPB 2855 isolate involved in a neonatal infection

Strains of Cronobactersakazakii (previously known as Enterobactersakazakii) are medically recognized important Gram-negative bacterial pathogens that cause enterocolitis, septicemia, and meningitis, with a high mortality rate in neonates. The structure of their O-antigens, that form part of their somatic lipopolysaccharide (LPS) components, is of interest for their chemical and serological identification and their relationship to virulence. The O-polysaccharide (O-PS) of C.sakazakii HPB 2855 (SK 81), a strain isolated from an infant at the Hospital for Sick Children in Toronto in 1981, was shown to be a polymer of a partially O-acetylated-repeating hexasaccharide unit composed of d-glucose, d-galacturonic acid, 2-acetamido-2-deoxy-d-galactose, and l-rhamnose (1:1:1:3). From composition and methylation analysis, and the application of 1D and 2D (1)H and (13)C NMR spectroscopy, the O-PS was determined to be a polymer of a repeating oligosaccharide unit having the structure: