Lectins pp 295-311 | Cite as

Application of Lectin Microarray to Bacteria Including Lactobacillus casei/paracasei Strains

  • Emi YasudaEmail author
  • Tomoyuki Sako
  • Hiroaki Tateno
  • Jun Hirabayashi
Part of the Methods in Molecular Biology book series (MIMB, volume 1200)


Since 2005, lectin microarray technology has emerged as a simple and powerful technique for comprehensive glycan analysis. By using evanescent-field fluorescence detection technique, it has been applied for analysis of not only glycoproteins and glycolipids secreted by eukaryotic cells but also glycoconjugates on the cell surface of live eukaryotic cells. Bacterial cells are known to be decorated with polysaccharides, teichoic acids, and proteins in the peptide glycans of their cell wall and lipoteichoic acids in their phospholipid bilayer. Specific glycan structures are characteristic of many highly pathogenic bacteria, while polysaccharides moiety of lactic acid bacteria are known to play a role as probiotics to modulate the host immune response. However, the method of analysis and knowledge of glycosylation structure of bacteria are limited. Here, we describe the development of a simple and sensitive method based on lectin microarray technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. The entire procedure takes 3 h from putting labeled bacteria on the microarray to profiling its lectin binding affinity. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei/paracasei. The lectin binding profile of most strains was found to be unique. Our technique provides a novel strategy for rapid profiling of bacteria and enables us to differentiate numerous bacterial strains with relevance to the biological functions of surface glycosylation.

Key words

L. casei Polysaccharide Lectin Lactic acid bacteria Glycosylation CSA CSL SYTOX Orange 



We thank Yoshiko Kubo and Jinko Murakami of the Research Center for Medical Glycoscience at the National Institute of Advanced Industrial Science and Technology for help in preparation of the lectin microarray, Toshihiko Takada of the Yakult Central Institute for Microbiological Research for help with bacterial labeling methods and preparation of the electron microscopic images, and Dr. Koichi Watanabe for advice on choosing L. casei/paracasei strains. We deeply thank Mayumi Kiwaki and Tohru Iino of the Yakult Central Institute for Microbiological Research, Dr. Teruo Yokokura and the late Dr. Toshiaki Osawa, who always encouraged us and incited helpful discussions.


  1. 1.
    Akira S, Takeda K (2004) Toll-like receptor signaling. Nat Rev Immunol 4:499–511PubMedCrossRefGoogle Scholar
  2. 2.
    Inohara N, Nuñes G (2003) NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol 3:371–382PubMedCrossRefGoogle Scholar
  3. 3.
    Lebeer S, Vanderleyden SJ, De Keersmaecker SCJ (2008) Genes and molecules of L. supporting probiotic action. Microbiol Mol Biol Rev 72:728–764PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Baik YS, Cheong WJ (2007) Development of SPE for recovery of polysaccharides and its application to the determination of monosaccharide composition of the polysaccharide sample of a lactobacillus KLB 58. J Sep Sci 30:1509–1515PubMedCrossRefGoogle Scholar
  5. 5.
    Kullberg MC (2008) Soothing intestinal sugars. Nature 453:602–604PubMedCrossRefGoogle Scholar
  6. 6.
    Liu CH, Lee SM, VanLare JM et al (2008) Regulation of surface architecture by symbiotic bacteria mediates host colonization. Proc Natl Acad Sci U S A 105:3951–3956PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Yasuda E, Serata M, Sako T (2008) Suppressive effect on activation of macrophages by Lactobacillus casei Strain Shirota genes determining the synthesis of cell wall-associated polysaccharides. Appl Environ Microbiol 74:4746–4755PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Coyne MJ, Tzianabos AO, Mallory BC et al (2001) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis. Infect Immun 69(7):4342–4350PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Cobb BA, Wang Q, Tzianabos AO et al (2004) Polysaccharide processing and presentation by the MHCII pathway. Cell 117:677–687PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Mazmanian SK, Liu CH, Tzianabos AO et al (2005) An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122:107–118PubMedCrossRefGoogle Scholar
  11. 11.
    Vaningelgem F, Zamfir M, Mozzi F et al (2004) Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics. Appl Environ Microbiol 70:900–912PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Cieslewicz MJ, Chaffin D, Glusman G et al (2005) Structural and genetic diversity of group B Streptococcus capsular polysaccharides. Infect Immun 73:3096–3103PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Bentley SD, Aanensen DM, Mavroidi A et al (2006) Genetic analysis of the capsular biosynthetic locus from all 90 Pneumococcal serotypes. PLoS Genet 2:e31PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Feng Y, Xiao-Min W (2007) Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen indentified by cDNA microarray. World J Gastroenterol 13:3369–3373Google Scholar
  15. 15.
    Pretzer G, Snel J, Molenaar D et al (2005) Biodiversity-based identification and functional characterization of the mannose-specific adhesion of Lactobacillus plantarum. J Bacteriol 187:6128–6136PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Fang Y, Cao H, Cover TL et al (2007) Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Gastroenterology 132:562–575CrossRefGoogle Scholar
  17. 17.
    Kankainen M, Paulin L, Tynkkynwn S et al (2009) Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein. Proc Natl Acad Sci U S A 106:17193–17198PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Angeloni S, Ridet JL, Kusy N et al (2005) Glycoprofiling with micro-arrays of glycoconjugates and lectins. Glycobiology 15:31–41PubMedCrossRefGoogle Scholar
  19. 19.
    Hsu K-L, Mahal LK (2006) A lectin microarray approach for the rapid analysis of bacterial glycans. Nat Protoc 1:543–549PubMedCrossRefGoogle Scholar
  20. 20.
    Hsu K-L, Pilobello KT, Mahal LK (2006) Analyzing the dynamic bacterial glycome with a lectin microarray approach. Nat Chem Biol 2:153–157PubMedCrossRefGoogle Scholar
  21. 21.
    Uchiyama N, Kuno A, Koseki‐Kuno S et al (2006) Development of a lectin microarray based on an Evanescent-field fluorescence principle: a new strategy for glycan profiling. Method Enzymol 415:341–351CrossRefGoogle Scholar
  22. 22.
    Tateno H, Uchiyama N, Kuno A et al (2007) A novel strategy for mammalian cell surface glycome profiling using lectin microarray. Glycobiology 17:1138–1146PubMedCrossRefGoogle Scholar
  23. 23.
    Hsu K-L, Gildersleeve JC, Mahal LK (2008) A simple strategy for the creation of a recombinant lectin microarray. Mol BioSyst 4:654–662PubMedCrossRefGoogle Scholar
  24. 24.
    Hirabayashi J, Yamada M, Kuno A et al (2013) Lectin microarrays: concept, principle and applications. Chem Soc Rev 42:4443–4458PubMedCrossRefGoogle Scholar
  25. 25.
    Yasuda E, Tateno H, Hirabayashi J et al (2011) Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides. Appl Environ Microbiol 77:4539–4546PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Dicks LM, Plessis EM, Dellaglio F et al (1996) Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosus ATCC 15820 as Lactobacillus zeae nom. rev., designation of ATCC 334 as the neotype of L. casei subsp. casei, and rejection of the name Lactobacilllus paracasei. Int J Syst Bacteriol 46:337–340PubMedCrossRefGoogle Scholar
  27. 27.
    Yan X, Habbersett RC, Cordek JM et al (2000) Development of a mechanism-based, DNA staining protocol using SYTOX Orange nucleic acid stain and DNA fragment sizing flow cytometry. Anal Biochem 286:138–148PubMedCrossRefGoogle Scholar
  28. 28.
    Yan X, Habbersett RC, Yoshida TM et al (2005) Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis. Anal Chem 77:3554–3562PubMedCrossRefGoogle Scholar
  29. 29.
    Shida K, Kiyoshima-Shibata J, Nagaoka M et al (2006) Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion. J Dairy Sci 89:3306–3317PubMedCrossRefGoogle Scholar
  30. 30.
    Tateno H (2010) SUEL-related lectins, a lectin family widely distributed throughout organisms. Biosci Biotechnol Biochem 74(6):1141–1144PubMedCrossRefGoogle Scholar
  31. 31.
    Nagaoka M, Muto M, Nomoto K et al (1990) Structure of polysaccharide-peptidoglycan complex from the cell wall of Lactobacillus casei YIT 9018. J Biochem 108:568–571PubMedGoogle Scholar
  32. 32.
    Šimelyte E, Rimpiläinen M, Lehtonen L et al (2000) Bacterial cell wall-induced arthritis: chemical composition and tissue distribution of four Lactobacillus strains. Infect Immun 68:3535–3540PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Yokokura T, Kodaira S, Ishiwa H et al (1974) Lysogeny in Lactobacilli. J Gen Mirobiol 84:277–284CrossRefGoogle Scholar
  34. 34.
    Shirai T, Watanabe Y, Lee M et al (2009) Structure of Rha-binding lectin CSL3: unique pseudo-tetrameric architecture of a pattern recognition protein. J Mol Biol 391:390–403PubMedCrossRefGoogle Scholar
  35. 35.
    Watanabe Y, Abolhassani M, Tojo Y et al (2009) The function of Rha-binding lectin in innate immunity by restricted binding Gb3. Dev Comp Immunol 33:187–197PubMedCrossRefGoogle Scholar
  36. 36.
    Schweizer D (1981) Counterstain-enhanced chromosome banding. Hum Genet 57:1–14PubMedGoogle Scholar
  37. 37.
    Takada T, Matsumoto K, Nomoto K (2004) Development of multi-color FISH method for analysis of seven Bifidobacterium species in human feces. J Microbiol Methods 58:413–421PubMedCrossRefGoogle Scholar
  38. 38.
    Yasuno S, Kokubo K, Kamei M (1999) New method for determining the sugar composition of glycoproteins, glycolipids, and oligosaccharides by high-performance liquid chromatography. Biosci Biotechnol Biochem 63:1353–1359PubMedCrossRefGoogle Scholar
  39. 39.
    Shimizu-Kadota M, Kiwaki M, Sawaki S et al (2000) Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei Shirota (S-1): characterization of attachment sites and integrase gene. Gene 249:127–134PubMedCrossRefGoogle Scholar
  40. 40.
    Yuki N, Watanabe K, Mike A et al (1999) Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: Selective isolation from feces and identification using monoclonal antibodies. Int J Food Microbiol 48:51–57PubMedCrossRefGoogle Scholar
  41. 41.
    Annuk H, Hynes SO, Hirmo S et al (2001) Characterisation and differentiation of lactobacilli by lectin typing. J Med Microbiol 50:1069–1074PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Emi Yasuda
    • 1
    Email author
  • Tomoyuki Sako
    • 2
  • Hiroaki Tateno
    • 3
  • Jun Hirabayashi
    • 3
  1. 1.Yakult Central Institute for Microbiological ResearchTokyoJapan
  2. 2.Yakult Europe B.V.AlmereThe Netherlands
  3. 3.Research Center for Stem Cell EngineeringNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan

Personalised recommendations