1887

Abstract

colonization of the respiratory tract is an essential precursor for pneumococcal disease. To colonize efficiently, bacteria must adhere to the epithelial-cell surface. possesses surface-associated exoglycosidases that are capable of sequentially deglycosylating human glycans. Two exoglycosidases, neuraminidase (NanA) and β-galactosidase (BgaA), have previously been shown to contribute to adherence to human epithelial cells, as deletion of either of these genes results in reduced adherence. It has been suggested that these enzymes may modulate adherence by cleaving sugars to reveal a receptor on host cells. Pretreatment of epithelial cells with exogenous neuraminidase restores the adherence of a mutant, whereas pretreatment with β-galactosidase does not restore the adherence of a mutant. These data suggest that BgaA may not function to reveal a receptor, and implicate an alternative role for BgaA in adherence. Here we demonstrate that β-galactosidase activity is not required for BgaA-mediated adherence. Addition of recombinant BgaA (rBgaA) to adherence assays and pretreatment of epithelial cells with rBgaA both significantly reduced the level of adherence of the parental strain, but not the BgaA mutant. One possible explanation of these data is that BgaA is acting as an adhesin and that rBgaA is binding to the receptor, preventing bacterial binding. A bead-binding assay demonstrated that BgaA can bind directly to human epithelial cells, supporting the hypothesis that BgaA is an adhesin. Preliminary characterization of the epithelial-cell receptor suggests that it is a glycan in the context of a glycosphingolipid. To further establish the relevance of this adherence mechanism, we demonstrated that BgaA-mediated adherence contributed to adherence of a recent clinical isolate to primary human epithelial cells. Together, these data suggest a novel role for BgaA as an adhesin and suggest that this mechanism could contribute to adherence of at least some pneumococcal strains .

Funding
This study was supported by the:
  • National Institutes of Health (Award R01AI076341-02)
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2011-08-01
2024-03-28
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References

  1. Bader D. E., Ring M., Huber R. E. ( 1988). Site-directed mutagenic replacement of glu-461 with gln in β-galactosidase (E. coli): evidence that glu-461 is important for activity. Biochem Biophys Res Commun 153:301–306 [View Article][PubMed]
    [Google Scholar]
  2. Bagnoli F., Moschioni M., Donati C., Dimitrovska V., Ferlenghi I., Facciotti C., Muzzi A., Giusti F., Emolo C. et al. ( 2008). A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells. J Bacteriol 190:5480–5492 [View Article][PubMed]
    [Google Scholar]
  3. Barthelson R., Mobasseri A., Zopf D., Simon P. ( 1998). Adherence of Streptococcus pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides. Infect Immun 66:1439–1444[PubMed]
    [Google Scholar]
  4. Bateman A., Holden M. T., Yeats C. ( 2005). The G5 domain: a potential N-acetylglucosamine recognition domain involved in biofilm formation. Bioinformatics 21:1301–1303 [View Article][PubMed]
    [Google Scholar]
  5. Berry A. M., Lock R. A., Thomas S. M., Rajan D. P., Hansman D., Paton J. C. ( 1994). Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli . Infect Immun 62:1101–1108[PubMed]
    [Google Scholar]
  6. Bongaerts R. J., Heinz H. P., Hadding U., Zysk G. ( 2000). Antigenicity, expression, and molecular characterization of surface-located pullulanase of Streptococcus pneumoniae . Infect Immun 68:7141–7143 [View Article][PubMed]
    [Google Scholar]
  7. Burnaugh A. M., Frantz L. J., King S. J. ( 2008). Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases. J Bacteriol 190:221–230 [View Article][PubMed]
    [Google Scholar]
  8. Caines M. E., Zhu H., Vuckovic M., Willis L. M., Withers S. G., Wakarchuk W. W., Strynadka N. C. ( 2008). The structural basis for T-antigen hydrolysis by Streptococcus pneumoniae: a target for structure-based vaccine design. J Biol Chem 283:31279–31283 [View Article][PubMed]
    [Google Scholar]
  9. Cámara M., Boulnois G. J., Andrew P. W., Mitchell T. J. ( 1994). A neuraminidase from Streptococcus pneumoniae has the features of a surface protein. Infect Immun 62:3688–3695[PubMed]
    [Google Scholar]
  10. Cassidy J. T., Jourdian G. W., Roseman S. ( 1965). The sialic acids. VI. Purification and properties of sialidase from Clostridium perfringens . J Biol Chem 240:3501–3506[PubMed]
    [Google Scholar]
  11. Clarke V. A., Platt N., Butters T. D. ( 1995). Cloning and expression of the β-N-acetylglucosaminidase gene from Streptococcus pneumoniae. Generation of truncated enzymes with modified aglycon specificity. J Biol Chem 270:8805–8814[PubMed] [CrossRef]
    [Google Scholar]
  12. Conrady D. G., Brescia C. C., Horii K., Weiss A. A., Hassett D. J., Herr A. B. ( 2008). A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms. Proc Natl Acad Sci U S A 105:19456–19461 [View Article][PubMed]
    [Google Scholar]
  13. Cundell D., Masure H. R., Tuomanen E. I. ( 1995a). The molecular basis of pneumococcal infection: a hypothesis. Clin Infect Dis 21:(Suppl. 3)S204–S212 [View Article][PubMed]
    [Google Scholar]
  14. Cundell D. R., Gerard N. P., Gerard C., Idanpaan-Heikkila I., Tuomanen E. I. ( 1995b). Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377:435–438 [View Article][PubMed]
    [Google Scholar]
  15. Cundell D. R., Weiser J. N., Shen J., Young A., Tuomanen E. I. ( 1995c). Relationship between colonial morphology and adherence of Streptococcus pneumoniae . Infect Immun 63:757–761[PubMed]
    [Google Scholar]
  16. Cundell D. R., Gerard C., Idanpaan-Heikkila I., Tuomanen E. I., Gerard N. P. ( 1996). PAf receptor anchors Streptococcus pneumoniae to activated human endothelial cells. Adv Exp Med Biol 416:89–94[PubMed]
    [Google Scholar]
  17. Cupples C. G., Miller J. H. ( 1988). Effects of amino acid substitutions at the active site in Escherichia coli β-galactosidase. Genetics 120:637–644[PubMed]
    [Google Scholar]
  18. Cupples C. G., Miller J. H., Huber R. E. ( 1990). Determination of the roles of Glu-461 in β-galactosidase (Escherichia coli) using site-specific mutagenesis. J Biol Chem 265:5512–5518[PubMed]
    [Google Scholar]
  19. de Bentzmann S., Roger P., Dupuit F., Bajolet-Laudinat O., Fuchey C., Plotkowski M. C., Puchelle E. ( 1996). Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells. Infect Immun 64:1582–1588[PubMed]
    [Google Scholar]
  20. Denno D. M., Frimpong E., Gregory M., Steele R. W. ( 2002). Nasopharyngeal carriage and susceptibility patterns of Streptococcus pneumoniae in Kumasi, Ghana. West Afr J Med 21:233–236[PubMed]
    [Google Scholar]
  21. Edwards J. L., Brown E. J., Ault K. A., Apicella M. A. ( 2001). The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia. Cell Microbiol 3:611–622 [View Article][PubMed]
    [Google Scholar]
  22. Fine M. J., Smith M. A., Carson C. A., Mutha S. S., Sankey S. S., Weissfeld L. A., Kapoor W. N. ( 1996). Prognosis and outcomes of patients with community-acquired pneumonia. A meta-analysis. JAMA 275:134–141 [View Article][PubMed]
    [Google Scholar]
  23. Gebler J. C., Aebersold R., Withers S. G. ( 1992). Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coli . J Biol Chem 267:11126–11130[PubMed]
    [Google Scholar]
  24. Giefing C., Meinke A. L., Hanner M., Henics T., Bui M. D., Gelbmann D., Lundberg U., Senn B. M., Schunn M. et al. ( 2008). Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies. J Exp Med 205:117–131 [View Article][PubMed]
    [Google Scholar]
  25. Gould J. M., Weiser J. N. ( 2002). The inhibitory effect of C-reactive protein on bacterial phosphorylcholine platelet-activating factor receptor-mediated adherence is blocked by surfactant. J Infect Dis 186:361–371 [View Article][PubMed]
    [Google Scholar]
  26. Gruenert D. C., Finkbeiner W. E., Widdicombe J. H. ( 1995). Culture and transformation of human airway epithelial cells. Am J Physiol 268:L347–L360[PubMed]
    [Google Scholar]
  27. Hammerschmidt S. ( 2006). Adherence molecules of pathogenic pneumococci. Curr Opin Microbiol 9:12–20 [View Article][PubMed]
    [Google Scholar]
  28. Hammerschmidt S., Wolff S., Hocke A., Rosseau S., Müller E., Rohde M. ( 2005). Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect Immun 73:4653–4667 [View Article][PubMed]
    [Google Scholar]
  29. Henrichsen J. ( 1995). Six newly recognized types of Streptococcus pneumoniae . J Clin Microbiol 33:2759–2762[PubMed]
    [Google Scholar]
  30. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. ( 1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68 [View Article][PubMed]
    [Google Scholar]
  31. Hoskins J., Alborn W. E. Jr, Arnold J., Blaszczak L. C., Burgett S., DeHoff B. S., Estrem S. T., Fritz L., Fu D. J. et al. ( 2001). Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183:5709–5717 [View Article][PubMed]
    [Google Scholar]
  32. Iannelli F., Pearce B. J., Pozzi G. ( 1999). The type 2 capsule locus of Streptococcus pneumoniae . J Bacteriol 181:2652–2654[PubMed]
    [Google Scholar]
  33. Jeong J. K., Kwon O., Lee Y. M., Oh D. B., Lee J. M., Kim S., Kim E. H., Le T. N., Rhee D. K., Kang H. A. ( 2009). Characterization of the Streptococcus pneumoniae BgaC protein as a novel surface β-galactosidase with specific hydrolysis activity for the Galβ1–3GlcNAc moiety of oligosaccharides. J Bacteriol 191:3011–3023 [View Article][PubMed]
    [Google Scholar]
  34. Jin P., Kong F., Xiao M., Oftadeh S., Zhou F., Liu C., Russell F., Gilbert G. L. ( 2009). First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. J Infect Dis 200:1375–1380 [View Article][PubMed]
    [Google Scholar]
  35. Kaufman G. E., Yother J. ( 2007). CcpA-dependent and -independent control of β-galactosidase expression in Streptococcus pneumoniae occurs via regulation of an upstream phosphotransferase system-encoding operon. J Bacteriol 189:5183–5192 [View Article][PubMed]
    [Google Scholar]
  36. Kharat A. S., Tomasz A. ( 2003). Inactivation of the srtA gene affects localization of surface proteins and decreases adhesion of Streptococcus pneumoniae to human pharyngeal cells in vitro. Infect Immun 71:2758–2765 [View Article][PubMed]
    [Google Scholar]
  37. Kim J. O., Weiser J. N. ( 1998). Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae . J Infect Dis 177:368–377 [View Article][PubMed]
    [Google Scholar]
  38. Kim J. O., Romero-Steiner S., Sørensen U. B., Blom J., Carvalho M., Barnard S., Carlone G., Weiser J. N. ( 1999). Relationship between cell surface carbohydrates and intrastrain variation on opsonophagocytosis of Streptococcus pneumoniae . Infect Immun 67:2327–2333[PubMed]
    [Google Scholar]
  39. King S. J. ( 2010). Pneumococcal modification of host sugars: a major contributor to colonization of the human airway?. Mol Oral Microbiol 25:15–24 [View Article][PubMed]
    [Google Scholar]
  40. King S. J., Hippe K. R., Gould J. M., Bae D., Peterson S., Cline R. T., Fasching C., Janoff E. N., Weiser J. N. ( 2004). Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway. Mol Microbiol 54:159–171 [View Article][PubMed]
    [Google Scholar]
  41. King S. J., Hippe K. R., Weiser J. N. ( 2006). Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae . Mol Microbiol 59:961–974 [View Article][PubMed]
    [Google Scholar]
  42. Krivan H. C., Roberts D. D., Ginsburg V. ( 1988). Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc beta 1-4Gal found in some glycolipids. Proc Natl Acad Sci U S A 85:6157–6161 [View Article][PubMed]
    [Google Scholar]
  43. Lanie J. A., Ng W. L., Kazmierczak K. M., Andrzejewski T. M., Davidsen T. M., Wayne K. J., Tettelin H., Glass J. I., Winkler M. E. ( 2007). Genome sequence of Avery’s virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J Bacteriol 189:38–51 [View Article][PubMed]
    [Google Scholar]
  44. Leiberman A., Leibovitz E., Piglansky L., Raiz S., Press J., Yagupsky P., Dagan R. ( 2001). Bacteriologic and clinical efficacy of trimethoprim–sulfamethoxazole for treatment of acute otitis media. Pediatr Infect Dis J 20:260–264 [View Article][PubMed]
    [Google Scholar]
  45. Marchler-Bauer A., Anderson J. B., Derbyshire M. K., DeWeese-Scott C., Gonzales N. R., Gwadz M., Hao L., He S., Hurwitz D. I. et al. ( 2007). CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res 35:Database issueD237–D240 [View Article][PubMed]
    [Google Scholar]
  46. McCool T. L., Weiser J. N. ( 2004). Limited role of antibody in clearance of Streptococcus pneumoniae in a murine model of colonization. Infect Immun 72:5807–5813 [View Article][PubMed]
    [Google Scholar]
  47. Moschioni M., Donati C., Muzzi A., Masignani V., Censini S., Hanage W. P., Bishop C. J., Reis J. N., Normark S. et al. ( 2008). Streptococcus pneumoniae contains 3 rlrA pilus variants that are clonally related. J Infect Dis 197:888–896 [View Article][PubMed]
    [Google Scholar]
  48. Muramatsu H., Tachikui H., Ushida H., Song X., Qiu Y., Yamamoto S., Muramatsu T. ( 2001). Molecular cloning and expression of endo-β-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides. J Biochem 129:923–928[PubMed] [CrossRef]
    [Google Scholar]
  49. Paterson G. K., Mitchell T. J. ( 2006). The role of Streptococcus pneumoniae sortase A in colonisation and pathogenesis. Microbes Infect 8:145–153 [View Article][PubMed]
    [Google Scholar]
  50. Richard J. P., Huber R. E., Lin S., Heo C., Amyes T. L. ( 1996). Structure–reactivity relationships for β-galactosidase (Escherichia coli, lac Z). 3. Evidence that Glu-461 participates in Brønsted acid–base catalysis of β-d-galactopyranosyl group transfer. Biochemistry 35:12377–12386 [View Article][PubMed]
    [Google Scholar]
  51. Robertson G. T., Ng W. L., Foley J., Gilmour R., Winkler M. E. ( 2002). Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence. J Bacteriol 184:3508–3520 [View Article][PubMed]
    [Google Scholar]
  52. Romero-Steiner S., Caba J., Rajam G., Langley T., Floyd A., Johnson S. E., Sampson J. S., Carlone G. M., Ades E. ( 2006). Adherence of recombinant pneumococcal surface adhesin A (rPsaA)-coated particles to human nasopharyngeal epithelial cells for the evaluation of anti-PsaA functional antibodies. Vaccine 24:3224–3231 [View Article][PubMed]
    [Google Scholar]
  53. Song X. M., Connor W., Hokamp K., Babiuk L. A., Potter A. A. ( 2008). Streptococcus pneumoniae early response genes to human lung epithelial cells. BMC Res Notes 1:64 [View Article][PubMed]
    [Google Scholar]
  54. Stoner G. D., Kikkawa Y., Kniazeff A. J., Miyai K., Wagner R. M. ( 1975). Clonal isolation of epithelial cells from mouse lung adenoma. Cancer Res 35:2177–2185[PubMed]
    [Google Scholar]
  55. Sung C. K., Li H., Claverys J. P., Morrison D. A. ( 2001). An rpsL cassette, Janus, for gene replacement through negative selection in Streptococcus pneumoniae . Appl Environ Microbiol 67:5190–5196 [View Article][PubMed]
    [Google Scholar]
  56. Tettelin H., Nelson K. E., Paulsen I. T., Eisen J. A., Read T. D., Peterson S., Heidelberg J., DeBoy R. T., Haft D. H. et al. ( 2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae . Science 293:498–506 [View Article][PubMed]
    [Google Scholar]
  57. Tong H. H., McIver M. A., Fisher L. M., DeMaria T. F. ( 1999). Effect of lacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adherence of otitis media-associated serotypes of Streptococcus pneumoniae to chinchilla tracheal epithelium. Microb Pathog 26:111–119 [View Article][PubMed]
    [Google Scholar]
  58. Uchiyama S., Carlin A. F., Khosravi A., Weiman S., Banerjee A., Quach D., Hightower G., Mitchell T. J., Doran K. S., Nizet V. ( 2009). The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion. J Exp Med 206:1845–1852 [View Article][PubMed]
    [Google Scholar]
  59. Umemoto J., Bhavanandan V. P., Davidson E. A. ( 1977). Purification and properties of an endo-α-N-acetyl-d-galactosaminidase from Diplococcus pneumoniae . J Biol Chem 252:8609–8614[PubMed]
    [Google Scholar]
  60. Vickerman M. M., Iobst S., Jesionowski A. M., Gill S. R. ( 2007). Genome-wide transcriptional changes in Streptococcus gordonii in response to competence signaling peptide. J Bacteriol 189:7799–7807 [View Article][PubMed]
    [Google Scholar]
  61. Watson D. A., Musher D. M. ( 1990). Interruption of capsule production in Streptococcus pneumoniae serotype 3 by insertion of transposon Tn916. Infect Immun 58:3135–3138[PubMed]
    [Google Scholar]
  62. Watt J. P., O’Brien K. L., Katz S., Bronsdon M. A., Elliott J., Dallas J., Perilla M. J., Reid R., Murrow L. et al. ( 2004). Nasopharyngeal versus oropharyngeal sampling for detection of pneumococcal carriage in adults. J Clin Microbiol 42:4974–4976 [View Article][PubMed]
    [Google Scholar]
  63. Whatmore A. M., Barcus V. A., Dowson C. G. ( 1999). Genetic diversity of the streptococcal competence (com) gene locus. J Bacteriol 181:3144–3154[PubMed]
    [Google Scholar]
  64. Zähner D., Hakenbeck R. ( 2000). The Streptococcus pneumoniae β-galactosidase is a surface protein. J Bacteriol 182:5919–5921 [View Article][PubMed]
    [Google Scholar]
  65. Zartler E. R., Porambo R. J., Anderson C. L., Chen L. H., Yu J., Nahm M. H. ( 2009). Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes. J Biol Chem 284:7318–7329 [View Article][PubMed]
    [Google Scholar]
  66. Zeleny R., Altmann F., Praznik W. ( 1997). A capillary electrophoretic study on the specificity of β-galactosidases from Aspergillus oryzae, Escherichia coli, Streptococcus pneumoniae, and Canavalia ensiformis (jack bean). Anal Biochem 246:96–101 [View Article][PubMed]
    [Google Scholar]
  67. Zhang J. R., Mostov K. E., Lamm M. E., Nanno M., Shimida S., Ohwaki M., Tuomanen E. ( 2000). The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. Cell 102:827–837 [View Article][PubMed]
    [Google Scholar]
  68. Zysk G., Bongaerts R. J., ten Thoren E., Bethe G., Hakenbeck R., Heinz H. P. ( 2000). Detection of 23 immunogenic pneumococcal proteins using convalescent-phase serum. Infect Immun 68:3740–3743 [View Article][PubMed]
    [Google Scholar]
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