Characterization of cpsF and its product CMP‐ N ‐acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

1996 ◽  
Vol 19 (3) ◽  
pp. 555-563 ◽  
Author(s):  
Rachel F. Haft ◽  
Michael R. Wessels ◽  
Mary Fisk Mebane ◽  
Neil Conaty ◽  
Craig E. Rubens
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Polysaccharide Biosynthesis ◽  
Acetylneuraminic Acid ◽  
Group B

scholarly journals L-Ficolin/Mannose-Binding Lectin-Associated Serine Protease Complexes Bind to Group B Streptococci Primarily through N-Acetylneuraminic Acid of Capsular Polysaccharide and Activate the Complement Pathway

2007 ◽  
Vol 76 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Youko Aoyagi ◽  
Elisabeth E. Adderson ◽  
Craig E. Rubens ◽  
John F. Bohnsack ◽  
Jin G. Min ◽  
...  
Keyword(s):  
Serine Protease ◽  
Specific Antibody ◽  
Capsular Polysaccharide ◽  
Mannose Binding Lectin ◽  
Lectin Pathway ◽  
Group B Streptococci ◽  
Acetylneuraminic Acid ◽  
Mannose Binding ◽  
Group B ◽  
Binding Lectin

ABSTRACT Group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis. Most infants who are colonized with GBS at birth do not develop invasive disease, although many of these uninfected infants lack protective levels of capsular polysaccharide (CPS)-specific antibody. The lectin pathway of complement is a potential mechanism for initiating opsonization of GBS with CPS-specific antibody-deficient serum. In this study, we determined whether mannose-binding lectin (MBL)/MBL-associated serine protease (MASP) complexes and L-ficolin/MASP complexes bind to different strains of GBS to activate the lectin pathway, and we identified the molecules recognized by lectins on the GBS surface. We found that MBL did not bind to any GBS examined, whereas L-ficolin bound to GBS cells of many serotypes. L-ficolin binding to GBS cells correlated with the CPS content in serotypes Ib, III (restriction digestion pattern types III-2 and III-3), and V but not with the group B-specific polysaccharide (GBPS) content or with the lipoteichoic acid (LTA) content. L-ficolin bound to purified CPS and GBPS in a concentration-dependent manner but not to purified LTA. All strains to which L-ficolin/MASP complexes bound consumed C4. When N-acetylneuraminic acid (NeuNAc) was selectively removed from GBS cells by treatment with neuraminidase, the reduction in L-ficolin binding was correlated with the amount of NeuNAc removed. Additionally, L-ficolin was able to bind to wild-type strains but was able to bind only weakly to unencapsulated mutants and a mutant strain in which the CPS lacks NeuNAc. We concluded that L-ficolin/MASP complexes bind to GBS primarily through an interaction with NeuNAc of CPS.

scholarly journals Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide

2003 ◽  
Vol 374 (3) ◽  
pp. 767-772 ◽  
Author(s):  
Elisabet ROMAN ◽  
Ian ROBERTS ◽  
Kerstin LIDHOLT ◽  
Marion KUSCHE-GULLBERG
Keyword(s):  
Escherichia Coli ◽  
Capsular Polysaccharide ◽  
Glucose Dehydrogenase ◽  
Fold Increase ◽  
Extra Copy ◽  
Polysaccharide Biosynthesis ◽  
Polysaccharide Production ◽  
Significant Difference ◽  
K5 Polysaccharide

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAβ1,4-GlcNAcα1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-d-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

scholarly journals NeuA Sialic Acid O-Acetylesterase Activity Modulates O-Acetylation of Capsular Polysaccharide in Group B Streptococcus

2007 ◽  
Vol 282 (38) ◽  
pp. 27562-27571 ◽  
Author(s):  
Amanda L. Lewis ◽  
Hongzhi Cao ◽  
Silpa K. Patel ◽  
Sandra Diaz ◽  
Wesley Ryan ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Capsular Polysaccharide ◽  
Group B Streptococcus ◽  
Site Directed Mutagenesis ◽  
Synthetase Activity ◽  
Bacterial Surface ◽  
Acetylneuraminic Acid ◽  
Single Polypeptide ◽  
Group B

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) was enhanced by CTP and Mg2+, the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac2 followed by CMP activation of Neu5Ac or activation of Neu5,9Ac2 followed by de-O-acetylation of CMP-Neu5,9Ac2. Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

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

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
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.

scholarly journals Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7

2011 ◽  
Vol 20 (7) ◽  
pp. 1208-1219 ◽  
Author(s):  
Erumbi S. Rangarajan ◽  
Karen M. Ruane ◽  
Ariane Proteau ◽  
Joseph D. Schrag ◽  
Ricardo Valladares ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Enzymatic Characterization ◽  
Acetylneuraminic Acid

scholarly journals Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides

2009 ◽  
Vol 191 (6) ◽  
pp. 1891-1900 ◽  
Author(s):  
Jinghua Yang ◽  
Mary Ritchey ◽  
Yasuo Yoshida ◽  
C. Allen Bush ◽  
John O. Cisar
Keyword(s):  
Dental Plaque ◽  
Molecular Basis ◽  
Capsular Polysaccharide ◽  
Polysaccharide Biosynthesis ◽  
Streptococcus Oralis ◽  
High Resolution Nmr ◽  
Close Relationship ◽  
Plaque Biofilm ◽  
Biofilm Community

ABSTRACT The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2ribitol-5-PO4 −-6Galfβ1-3Galβ1]n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

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