Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide.

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gram-negative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identified as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1 → 5)-α-KDOp-(2 → partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

The ß-reducing end in α(2-8)-polysialic acid constitutes a unique structural motif.

Over the years, structural characterizations of α(2-8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is ß, which has the [I]CO2- group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2- group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2-8)-polySia near the reducing end, as the ß configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2-). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-ßOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2-8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2-8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.

Sialo-CEST: chemical exchange saturation transfer NMR of oligo- and poly-sialic acids and the assignment of their hydroxyl groups using selective- and HSQC-TOCSY.

Chemical exchange saturation transfer (CEST) is an NMR method that takes advantage of proton exchange between solute and solvent molecules in dynamic equilibrium, enabling the detection of the solute NMR signals with enhanced sensitivity. Herein, we report that the hydroxyl groups in a naturally occurring polysaccharide, α-2,8 polysialic acid in aqueous solution, yield very significant CEST effects even at 37°C where the resonances of the hydroxyl groups are not directly observed. We also report the assignments of the hydroxyl groups for the polymer and its oligomeric building blocks, from monomer to hexamer. We show that the same assignments can be made by either (1)H-(1)H TOCSY methods or (1)H-(13)C HSQC-TOCSY methods, to alleviate spectral overlap. Finally, we report the exchange rates of the OH groups with water and show how these rates can be used to select and fine-tune CEST effects.

Identification of an outer membrane lipoprotein involved in nasopharyngeal colonization by Moraxella catarrhalis in an animal model.

Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being upregulated and 291 being downregulated. Among the upregulated genes was one (ORF113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but it did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.