Predicted Functions and Linkage Specificities of the Products of the Streptococcus pneumoniae Capsular Biosynthetic Loci

ABSTRACT The sequences of the capsular biosynthetic (cps) loci of 90 serotypes of Streptococcus pneumoniae have recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlating cps gene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set of cps loci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similar cps gene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.

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Antibody Responses to Capsular Polysaccharide Backbone and O-Acetate Side Groups of Streptococcus pneumoniae Type 9V in Humans and Rhesus Macaques

Infection and Immunity ◽

10.1128/iai.66.8.3705-3710.1998 ◽

1998 ◽

Vol 66 (8) ◽

pp. 3705-3710 ◽

Cited By ~ 44

Author(s):

Tessie B. McNeely ◽

Joan M. Staub ◽

Cynthia M. Rusk ◽

Michael J. Blum ◽

John J. Donnelly

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Rhesus Macaques ◽

Acute Otitis Media ◽

Antibody Responses ◽

Side Chains ◽

Monkey Model ◽

Repeat Units ◽

Pneumococcal Bacteremia ◽

Infant Rhesus

ABSTRACT Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. α-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and β-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.

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The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

Biochemical Journal ◽

10.1042/bj20050217 ◽

2005 ◽

Vol 389 (1) ◽

pp. 63-72 ◽

Cited By ~ 10

Author(s):

Nehmé SAKSOUK ◽

Ludovic PELOSI ◽

Pierre COLIN-MOREL ◽

Manel BOUMEDIENNE ◽

Patricia L. ABDIAN ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Biochemical Characterization ◽

Repeat Unit ◽

Sugar Residue ◽

Functional Identification ◽

Polysaccharide Biosynthesis ◽

Sugar Addition ◽

Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

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Genetic, Biochemical, and Serological Characterization of a New Pneumococcal Serotype, 6H, and Generation of a Pneumococcal Strain Producing Three Different Capsular Repeat Units

Clinical and Vaccine Immunology ◽

10.1128/cvi.00647-14 ◽

2015 ◽

Vol 22 (3) ◽

pp. 313-318 ◽

Cited By ~ 40

Author(s):

In Ho Park ◽

K. Aaron Geno ◽

Jigui Yu ◽

Melissa B. Oliver ◽

Kyung-Hyo Kim ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Vaccine Coverage ◽

Repeat Unit ◽

Enzyme Specificity ◽

Content Type ◽

Repeat Units ◽

Novel Approach ◽

Serological Characterization ◽

Pneumococcal Strain ◽

Genes Encoding

ABSTRACTStreptococcus pneumoniaeclinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members.

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The glucosyl-1-phosphate transferase WchA (Cap8E) primes the capsular polysaccharide repeat unit biosynthesis of Streptococcus pneumoniae serotype 8

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.12.082 ◽

2005 ◽

Vol 327 (3) ◽

pp. 857-865 ◽

Cited By ~ 29

Author(s):

Ludovic Pelosi ◽

Manel Boumedienne ◽

Nehmé Saksouk ◽

Johannes Geiselmann ◽

Roberto A. Geremia

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Repeat Unit

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Structures of Capsular Polysaccharide Serotypes 35F and 35C of Streptococcus pneumoniae Determined by Nuclear Magnetic Resonance and Their Relation to Other Cross-Reactive Serotypes

Journal of Bacteriology ◽

10.1128/jb.00207-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2762-2769 ◽

Cited By ~ 5

Author(s):

C. Allen Bush ◽

John O. Cisar ◽

Jinghua Yang

Keyword(s):

Streptococcus Pneumoniae ◽

Magnetic Resonance ◽

Capsular Polysaccharide ◽

Genetic Relationships ◽

Repeat Unit ◽

Immune Selection ◽

Comprehensive Description ◽

Content Type ◽

Genetic Features

ABSTRACTThe structures ofStreptococcus pneumoniaecapsular polysaccharides (CPSs) are essential for defining the antigenic as well as genetic relationships between CPS serotypes. The four serotypes that comprise CPS serogroup 35 (i.e., types 35F, 35A, 35B, and 35C) are known to cross-react with genetically related type 20, 29, 34, 42, or 47F. While the structures of CPS serotype 35A (CPS35A) and CPS35B are known, those of CPS35F and CPS35C are not. In the present study, the serotypes of CPS35F and CPS35C were characterized by high-resolution heteronuclear magnetic resonance (NMR) spectroscopy and glycosyl composition analyses to reveal the following repeat unit structures:where OAc indicates O-acetylated. Importantly, CPS35F, the immunizing serotype for the production of group 35 serum, more closely resembles CPS34 and CPS47F than other members of serogroup 35. Moreover, CPS35C is distinct from either CPS35F or CPS35B but closely related to CPS35A and identical to de-O-acetylated CPS42. The findings provide a comprehensive view of the structural and genetic relations that exist between the members of CPS serogroup 35 and other cross-reactive serotypes.IMPORTANCECross-reactions of diagnostic rabbit antisera withStreptococcus pneumoniaecapsular polysaccharide serotypes are generally limited to members of the same serogroup. Exceptions do, however, occur, most notably among a group of nonvaccine serotypes that includes the members of serogroup 35 (i.e., types 35F, 35A, 35B, and 35C) and other genetically related types. The presently determined structures ofS. pneumoniaeserotypes 35F and 35C complete the structural characterization of serogroup 35 and thereby provide the first comprehensive description of how different members of this serogroup are related to each other and to types 29, 34, 42, and 47F. The structural and genetic features of these serotypes suggest the existence of three distinct capsular polysaccharide subgroups that presumably emerged by immune selection in the human host.

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Comparative Genetics of Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Types Belonging to Serogroup 19

Journal of Bacteriology ◽

10.1128/jb.181.17.5355-5364.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5355-5364 ◽

Cited By ~ 42

Author(s):

Judy K. Morona ◽

Renato Morona ◽

James C. Paton

Keyword(s):

Streptococcus Pneumoniae ◽

Genetic Basis ◽

Capsular Polysaccharide ◽

Single Gene ◽

Repeat Unit ◽

Structural Difference ◽

Structural Diversity ◽

Polysaccharide Biosynthesis ◽

Flanking Sequences ◽

The Individual

ABSTRACT The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aIand cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19Kto -O) common to all four serogroup 19 members. Thesecps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.

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Membrane linkage of a Streptococcus pneumoniae Wzy capsular polysaccharide occurs through an acylglycerol

10.1101/2020.09.16.299636 ◽

2020 ◽

Author(s):

Thomas R. Larson ◽

Janet Yother

Keyword(s):

Streptococcus Pneumoniae ◽

Pathogenic Bacteria ◽

Capsular Polysaccharide ◽

Repeat Unit ◽

Capsular Polysaccharides ◽

Outer Face ◽

Gram Positive ◽

Bacterial Surface ◽

Gram Positive Bacteria ◽

Undecaprenyl Phosphate

ABSTRACTCapsular polysaccharides (capsules) protect bacteria from environmental insults and can contribute to virulence in pathogenic bacteria. Their appropriate display on the bacterial surface is critical to their functions. In Gram-positive bacteria, most capsules are synthesized by the Wzy polymerase-dependent pathway, which is also utilized in the synthesis of many capsules and O-antigens of Gram-negative bacteria. Synthesis of capsule repeat units initiates on undecaprenyl-phosphate on the inner face of the cytoplasmic membrane, with polymerization occurring on the outer face of the membrane. In Gram-positive bacteria, the capsule can be transferred to peptidoglycan, as in Streptococcus pneumoniae where a direct glycosidic bond to the peptidoglycan N-acetylglucosamine occurs. In S. pneumoniae, capsule can also be detected on the membrane, and this has generally been assumed to reflect polysaccharide that is linked to undecaprenyl-phosphate and in the process of synthesis. We provide evidence here, however, that final membrane linkage occurs through an acylglycerol, and essentially all of the polysaccharide is transferred from the initial undecaprenyl-phosphate acceptor to an alternate acceptor. This step allows for recycling of undecaprenyl-phosphate and represents an additional terminal step in capsule synthesis. In this regard, capsule synthesis resembles that of the wall- and lipoteichoic acids of S. pneumoniae, wherein a common repeat unit and polymer structure are synthesized by the Wzy pathway with divergence at the terminal step that results in linkages to peptidoglycan and a membrane acylglycerol anchor.IMPORTANCELinkage of capsular polysaccharides to the bacterial cell surface is a critical step in assuring the ability of these polymers to fulfill their functions, such as the resistance to complement-mediated phagocytosis that can be essential for pathogenic organisms to survive in host environments. Knowledge of the mechanisms by which these linkages occur is incomplete. In this study, we provide evidence for linkage of an S. pneumoniae Wzy capsule to an acylglycerol, the most abundant class of lipids in the membrane. This linkage provides a terminal acceptor for capsule that occurs in addition to that of peptidoglycan. Transfer to these terminal receptors is an essential step in CPS synthesis, as failure to do so can be lethal for the cell.

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Adsorption Isotherm Predictions for Multiple Molecules in MOFs Using the Same Deep Learning Model

10.26434/chemrxiv.9894224.v1 ◽

2019 ◽

Author(s):

Ryther Anderson ◽

Achay Biong ◽

Diego Gómez-Gualdrón

Keyword(s):

Neural Network ◽

Machine Learning ◽

Molecular Simulation ◽

Large Scale ◽

Learning Model ◽

Operating Conditions ◽

Small Subset ◽

Screening Methods ◽

Large Set ◽

Metal Organic

<div>Tailoring the structure and chemistry of metal-organic frameworks (MOFs) enables the manipulation of their adsorption properties to suit specific energy and environmental applications. As there are millions of possible MOFs (with tens of thousands already synthesized), molecular simulation, such as grand canonical Monte Carlo (GCMC), has frequently been used to rapidly evaluate the adsorption performance of a large set of MOFs. This allows subsequent experiments to focus only on a small subset of the most promising MOFs. In many instances, however, even molecular simulation becomes prohibitively time consuming, underscoring the need for alternative screening methods, such as machine learning, to precede molecular simulation efforts. In this study, as a proof of concept, we trained a neural network as the first example of a machine learning model capable of predicting full adsorption isotherms of different molecules not included in the training of the model. To achieve this, we trained our neural network only on alchemical species, represented only by their geometry and force field parameters, and used this neural network to predict the loadings of real adsorbates. We focused on predicting room temperature adsorption of small (one- and two-atom) molecules relevant to chemical separations. Namely, argon, krypton, xenon, methane, ethane, and nitrogen. However, we also observed surprisingly promising predictions for more complex molecules, whose properties are outside the range spanned by the alchemical adsorbates. Prediction accuracies suitable for large-scale screening were achieved using simple MOF (e.g. geometric properties and chemical moieties), and adsorbate (e.g. forcefield parameters and geometry) descriptors. Our results illustrate a new philosophy of training that opens the path towards development of machine learning models that can predict the adsorption loading of any new adsorbate at any new operating conditions in any new MOF.</div>

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