Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10.
ABSTRACT: Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.
Project description:In this report we describe studies on the structures of the O-linked carbohydrate units in cell-surface glycoproteins of epimastigote forms of the G-strain of Trypanosoma cruzi. Mild alkaline reductive degradation of the 38/43 kDa glycoproteins resulted in beta-elimination of glycosylated threonine and/or serine residues, and the liberation of N-acetylglucosaminitol, galactobiosyl-, galactotriosyl-, galactotetraosyl- and galactopentaosyl-N-acetylglucosaminitol. The structures of these oligosaccharide alditols were established by n.m.r. spectroscopy and methylation analysis as: Galf beta 1-4(Galp beta 1-6)GlcNAc-ol; Galp beta 1-3Galp beta 1-6(Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galp beta 1-2Galf beta 1-4)GlcNAc-ol.
Project description:UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-d-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.
Project description:Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfbeta1-6GalNAcbeta1-3Galalpha) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfbeta1-6GalNAcbeta1-3Galalpha rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a beta1-3 linkage between Galf and GalNAcbeta1-3Galalpha. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of beta1-6-linked Galf in recognition of adjacent GalNAcbeta1-3Galalpha in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.
Project description:A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [?3)-?-d-Galp-(1?3)-?-d-Galf-(1?]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [?3)-?-d-Galp-(1?3)-?-d-Galp-(1?] or O2c [?3)-?-d-GlcpNAc-(1?5)-?-d-Galf-(1?] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding ?-(1?3)-linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding ?-(1?3)-linked d-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.
Project description:Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: [structure: see text] Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the ?1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with ?1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with ?1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from ?-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity.
Project description:UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDP-Galp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a manner similar to that of UDP-Galp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the "open" mobile loop in UGM "closes" upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf and shed light on the mechanism of action of UGM.
Project description:Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumoniae The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-?-l-Rhap-[4-P-1-Gro]-(1-3)-?-d-Glcp-[(6-P-1)-Gro]-(1-3)-?-l-Rhap-[2-OAc]-(1-4)-?-d-Glcp-(1-; 16A, -3)-?-d-Galf-[2-OAc (70%)]-(1-3)-?-l-Rhap-(1-2)-?-l-Rhap-(1-3)-?-d-Galp-[(6-P-1)-Gro]-(1-3)-?-d-Galp-(1-4)-?-d-Glcp-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published cps gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of ?-l-Rhap-(1-3)-?-d-Glcp and ?-l-Rhap-(1-2)-?-l-Rhap, respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with factor antiserum 11c.IMPORTANCE The vaccine pressure against Streptococcus pneumoniae could result in a change of prevalence in carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve successful vaccination of the population, and similarly, it is important to increase the knowledge of even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen, and antigenic properties of CPS are based on the structure. Consequently, a better understanding of the structure, biosynthesis, and serology of the capsular polysaccharides can be of great importance toward developing future diagnostic tools and vaccines.
Project description:UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(?) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ? = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).
Project description:Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.
Project description:Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5'-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 A resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.