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Enzymatic Synthesis of Repeating Unit Oligosaccharides of Escherichia coli O104

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Bacterial Polysaccharides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1954))

Abstract

Escherichia coli serotype O104:H4 (ECO104) is a potent intestinal pathogen that causes severe bloody diarrhea and hemolytic-uremic syndrome. The O antigenic polysaccharides of ECO104 consist of repeating units with the structure [4Galα1-4Neu5,7,9Ac3α2-3Galβ1-3GalNAcβ1-]n. These repeating units are assembled sequentially by specific glycosyltransferases on a diphosphate-undecaprenol intermediate. Internal structures include mimics of the human T and sialyl-T antigen. This protocol describes the in vitro synthesis of the repeating unit by β1,3-Gal-transferase WbwC, α2,3-sialyltransferase WbwA, and α1,4-Gal-transferase WbwB. All of these enzymes require acceptor substrates based on GalNAc-diphosphate-lipid. These methods are applicable for the assembly of bacterial polysaccharides of gram-negative bacteria that require sugar-diphosphate intermediates and are a basis for vaccine synthesis.

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References

  1. Muniesa M, Hammeri J, Hertwig S et al (2012) Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology. Appl Environ Microbiol 78:4065–4073

    Article  CAS  Google Scholar 

  2. Raetz CRH, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700

    Article  CAS  Google Scholar 

  3. Gamian A, Romanowska E, Ulrich J et al (1992) The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide. Carbohydr Res 236:195–208

    Article  CAS  Google Scholar 

  4. Romanow A, Haselhorst T, Stummeyer K et al (2013) Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule. J Biol Chem 288:11718–11730

    Article  CAS  Google Scholar 

  5. Whitfield C, Trent MS (2014) Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem 83:99–128

    Article  CAS  Google Scholar 

  6. Wang L, Briggs CE, Rothemund D et al (2001) Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific get al enes. Gene 270:231–236

    Article  CAS  Google Scholar 

  7. Samuel G, Reeves P (2003) Biosynthesis of O-antigens: genes, and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly. Carbohydr Res 338:2503–2519

    Article  CAS  Google Scholar 

  8. Lehrer J, Vigeant KA, Tatar LD et al (2007) Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide. J Bacteriol 189:2618–2628

    Article  CAS  Google Scholar 

  9. Rush JS, Alaimo C, Robbiani R et al (2010) A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157. J Biol Chem 285:1671–1680

    Article  CAS  Google Scholar 

  10. Gao Y, Liu B, Strum S et al (2012) Biochemical characterization of WbdN, a beta1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157. Glycobiology 22:1092–1102

    Article  CAS  Google Scholar 

  11. Wang S, Hao Y, Lam JS et al (2015) Biosynthesis of the common polysaccharide antigen of Pseudomonas aeruginosa PAO1: characterization and role of GDP-D-rhamnose: GlcNAc/GalNAc-diphosphate-lipid alpha1,3-D-rhamnosyltransferase WbpZ. J Bacteriol 197:2012–2019

    Article  CAS  Google Scholar 

  12. Wang S, Czuchry D, Liu B et al (2014) Characterization of two UDP-gal: GalNAc-diphosphate-lipid β1,3-galactosyltransferases WbwC from Escherichia coli serotypes O104 and O5. J Bacteriol 196:3122–3133

    Article  Google Scholar 

  13. Riley JG, Menggad M, Montoya-Peleaz PJ et al (2005) The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-gal: GlcNAc{alpha}- pyrophosphate-R beta1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide. Glycobiology 15:605–613

    Article  CAS  Google Scholar 

  14. Xu C, Liu B, Hu B et al (2011) Biochemical characterization of UDP-gal:GlcNAc- pyrophosphate-lipid-1,4-Galactosyltransferase WfeD, a new enzyme from Shigella boydii type 14 that catalyzes the second step in O-antigen repeating-unit synthesis. J Bacteriol 193:449–459

    Article  CAS  Google Scholar 

  15. Brockhausen I, Hu B, Liu B et al (2008) Characterization of two beta-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152. J Bacteriol 190:4922–4932

    Article  CAS  Google Scholar 

  16. Chen C, Liu B, Xu Y et al (2016) Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3. Carbohydr Res 430:36–43

    Article  CAS  Google Scholar 

  17. Yi W, Shao J, Zhu L et al (2005) Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. J Am Chem Soc 127:2040–2041

    Article  CAS  Google Scholar 

  18. Hanisch FG, Baldus SE (1997) The Thomsen-Friedenreich (TF) antigen: a critical review on the structural, biosynthetic and histochemical aspects of a pancarcinoma-associated antigen. Histol Histopathol 12:263–281

    CAS  PubMed  Google Scholar 

  19. Czuchry D, Desormeaux P, Stuart M et al (2015) Identification and biochemical characterization of the novel alpha2,3-sialyltransferase WbwA from the pathogenic Escherichia coli serotype O104. J Bacteriol 197:3760–3768

    Article  CAS  Google Scholar 

  20. Czuchry D, Szarek WA, Brockhausen I (2017) Identification and biochemical characterization of WbwB, a novel UDP-gal: Neu5Ac-R alpha1,4-galactosyltransferase from the intestinal pathogen Escherichia coli serotype O104. Glycoconj J 35:65–76

    Article  Google Scholar 

  21. Si A, Misra AK (2016) Expedient synthesis of the pentasaccharide repeating unit of the polysaccharide O-antigen of Escherichia coli O11. Chemistry Open 5:47–50

    CAS  PubMed  Google Scholar 

  22. Bi N, Xiong C, Jin G et al (2017) Synthesis of a trisaccharide repeating unit of the O-antigen from Burkholderia cenocepacia and its dimer. Carbohydr Res 451:1–11

    Article  CAS  Google Scholar 

  23. Bhaumik I, Kar RK, Bhunia A et al (2016) Expedient synthesis of the pentasaccharide repeating unit of the O-antigen of Escherichia coli O86 and its conformational analysis. Glycoconj J 33:887–896

    Article  CAS  Google Scholar 

  24. Mitra A, Mukhopadhyay B (2016) Linear synthesis of the hexasaccharide related to the repeating unit of the O-antigen from Shigella flexneri serotype 1d (I: 7,8). Carbohydr Res 426:1–8

    Article  CAS  Google Scholar 

  25. Mandal PK (2014) Synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O117:K98:H4. Beilstein J Org Chem 10:2724–2728

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Inka Brockhausen & Diana Czuchry

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  1. Inka Brockhausen
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  2. Diana Czuchry
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Correspondence to Inka Brockhausen .

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Editors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Inka Brockhausen

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Brockhausen, I., Czuchry, D. (2019). Enzymatic Synthesis of Repeating Unit Oligosaccharides of Escherichia coli O104. In: Brockhausen, I. (eds) Bacterial Polysaccharides. Methods in Molecular Biology, vol 1954. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9154-9_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9154-9_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9153-2

  • Online ISBN: 978-1-4939-9154-9

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