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

ABSTRACT: 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 alpha-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)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU 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 [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-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.

SUBMITTER: Saksouk N

PROVIDER: S-EPMC1188263 | biostudies-other | 2005 Jul

REPOSITORIES: biostudies-other

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Streptococcus pneumoniae serotype 11D has a bispecific glycosyltransferase and expresses two different capsular polysaccharide repeating units.

Project description:Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain ?Glc as the fourth sugar. However, ?25% of 11D CPS RUs contain instead ?GlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much ?GlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.

| S-EPMC3724649 | biostudies-literature

Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking.

Project description:Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an alpha-helix configuration. In this paper we look at the immune response of CD8(+) T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8(+)CD28(-) T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8(+)CD28(-) T lymphocytes. The Sp1-induced CD8(+)CD28(-) T lymphocytes are CD122(low)CTLA-4(+)CD39(+). They synthesize IL-10 and TGF-beta. The Sp1-induced CD8(+)CD28(-) T cells exhibit immunosuppressive properties on CD4(+) T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8(+) T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8(+) T cell activation by enhancing crosslinking of TCR. The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8(+)CD28(-) T lymphocytes in vivo and in vitro. The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

| S-EPMC2742891 | biostudies-literature

Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype.

Project description:Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype.

| S-EPMC2446917 | biostudies-literature

The efficacy of pneumococcal capsular polysaccharide-specific antibodies to serotype 3 Streptococcus pneumoniae requires macrophages.

Project description:The efficacy of antibody immunity against Streptococcus pneumoniae stems from the ability of opsonic, serotype (ST)-specific antibodies to pneumococcal capsular polysaccharide (PPS) to facilitate killing of the homologous ST by host phagocytes. However, PPS-specific antibodies have been identified that are protective in mice, but do not promote opsonic killing in vitro, raising the question of how they mediate protection in vivo. To probe this question, we investigated the dependence of antibody efficacy against lethal systemic (intraperitoneal, i.p.) infection with Streptococcus pneumoniae serotype 3 (ST3) on macrophages and neutrophils for the following PPS3-specific monoclonal antibodies (MAbs) in survival experiments in mice using a non-opsonic human IgM (A7), a non-opsonic mouse IgG1 (1E2) and an opsonic mouse IgG1 (5F6). The survival of A7- and PPS3-specific and isotype control MAb-treated neutrophil-depleted and neutrophil-sufficient and macrophage-depleted and macrophage-sufficient mice were determined after i.p. challenge with ST3 strains 6303 and WU2. Neutrophils were dispensable for A7 and the mouse MAbs to mediate protection in this model, but macrophages were required for the efficacy of A7 and optimal mouse MAb-mediated protection. For A7-treated mice, macrophage-depleted mice had higher blood CFU, cytokines and peripheral neutrophil levels than macrophage-sufficient mice, and macrophage-sufficient mice had lower tissue bacterial burdens than control MAb-treated mice. These findings demonstrate that macrophages contribute to opsonic and non-opsonic PPS3-specific MAb-mediated protection against ST3 infection by enhancing bacterial clearance and suggest that neutrophils do not compensate for the absence of macrophages in the model used in this study.

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Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F.

Project description:The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

| S-EPMC8123889 | biostudies-literature

Transcriptional regulation of the capsular polysaccharide biosynthesis locus of streptococcus pneumoniae: a bioinformatic analysis.

Project description:The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor, which makes the bacterium resistant to phagocytosis. Expression of capsular polysaccharide must be adjusted at different stages of pneumococcal infection, thus, their transcriptional regulation appears to be crucial. To get insight into the existence of regulatory mechanisms common to most serotypes, a bioinformatic analysis of the DNA region located upstream of the capsular locus was performed. With the exception of serotype 37, the capsular locus is located between dexB and aliA on the pneumococcal chromosome. Up to 26 different sequence organizations were found among pneumococci synthesizing their capsule through a Wzy-polymerase-dependent mechanism, mostly varying according to the presence/absence of distinct insertion elements. As a consequence, only approximately 250 bp (including a 107 bp RUP_A element) was conserved in 86 sequences, although only a short (ca. 87 bp) region located immediately upstream of cpsA was strictly conserved in all the sequences analyzed. An exhaustive search for possible operator sequences was done. Interestingly, although the promoter region of serotype 3 isolates completely differs from that of other serotypes, most of the proteins proposed to regulate transcription in serotype 3 pneumococci were also predicted to function as possible regulators in non-serotype 3 S. pneumoniae isolates.

| S-EPMC2695774 | biostudies-literature

Comparative genetics of capsular polysaccharide biosynthesis in Streptococcus pneumoniae types belonging to serogroup 19.

Project description: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 19 cps 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 (cps19aI and 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 cps19K to -O) common to all four serogroup 19 members. These cps 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 19 cps 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.

| S-EPMC94042 | biostudies-literature

An antibody to Streptococcus pneumoniae capsular polysaccharide enhances quorum sensing

Project description:The primary mechanism by which pneumococcal capsular polysaccharide-based vaccines are believed to mediate protection is by induction of serotype-specific opsonic antibodies that facilitate bacterial killing by phagocytes (opsonophagocytosis). However, antibodies that are protective against experimental pneumococcal pneumonia in mice but do not promote opsonophagocytic killing in vitro have also been identified 1-3. Such non-opsonic antibodies are associated with bacterial clearance in vivo, but the mechanism by which this occurs is unknown. In this letter, we demonstrate that a protective, non-opsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibody (MAb) enhances quorum sensing, which results in competence induction and fratricide of serotype 3 pneumococcus. Gene expression profile analysis revealed that the MAb together with the pneumococcal autoinducer, competence stimulating peptide 2 (CSP2), augments differential expression of competence (com) related bacteriocin-like peptide (blp) genes that are known to be involved in pneumococcal fratricide. Taken together, these findings reveal a previously unsuspected mechanism of antibody action, namely, enhancement of quorum sensing and bacterial fratricide. Given that this activity does not require phagocytes, antibodies that function accordingly may hold promise as adjuncts to current vaccines or as desired products of next generation pneumococcal vaccines.

2011-03-01 | GSE27258 | GEO

Biochemical activities of Streptococcus pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor mutations affecting the initiating glycosyltransferase Cps2E.

Project description:The capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Its synthesis requires multiple enzymes, and defects that block completion of the pathway can be lethal in the absence of secondary suppressor mutations. In this study, we examined the functions of three capsular glycosyltransferases (Cps2F, Cps2G, and Cps2I) involved in serotype 2 CPS synthesis, whose deletions select for secondary mutations. We demonstrate that Cps2F is a rhamnosyltransferase that catalyzes addition of the third and fourth sugars in the capsule repeat unit, while Cps2G adds the fifth sugar (glucose). Addition of the terminal residue (glucuronic acid) could not be detected; however, activities of the other glycosyltransferases together with bioinformatic analyses suggest that this step is mediated by Cps2I. Most of the secondary suppressor mutations resulting from loss of these enzymes occur in cps2E, the gene encoding the initiating glycosyltransferase. Examination of the 69 S. pneumoniae serotypes containing Cps2E homologues yielded a consensus amino acid sequence for this protein and demonstrated that there is a highly significant association between the residues that are 100% conserved and those altered by suppressor mutations. Cps2E contains an extracytoplasmic loop whose function is unknown. Among our collection of mutants, six contained missense mutations affecting amino acids in the extracytoplasmic loop. These residues are highly conserved among S. pneumoniae Cps2E homologues, and mutations therein severely reduced CPS synthesis and Cps2E activity. The critical functions of these amino acids suggest a role for the Cps2E extracytoplasmic loop in initiation, and possibly regulation, of capsule synthesis.

| S-EPMC3889599 | biostudies-literature

Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene.

Project description:As part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated '33F-1'). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results. Whole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence. All ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence. We have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.

| S-EPMC6218050 | biostudies-literature

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