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Vol. 42, No. 6, 2008
Issue release date: November 2008
Section title: Original Paper
Free Access
Caries Res 2008;42:435–443
(DOI:10.1159/000159607)

Effects of Sucrose on the Extracellular Matrix of Plaque-Like Biofilm Formed in vivo, Studied by Proteomic Analysis

Paes Leme A.F.a · Bellato C.M.b · Bedi G.c · Del Bel Cury A.A.a · Koo H.c · Cury J.A.a
aPiracicaba Dental School, UNICAMP, and bCENA/University of São Paulo, Piracicaba, Brazil; cUniversity of Rochester Medical Center, Rochester, N.Y., USA
email Corresponding Author

Abstract

Previous studies have shown that sucrose promotes changes in the composition of the extracellular matrix (ECM) of plaque-like biofilm (PLB), but its effect on protein expression has not been studied in vivo. Therefore, the protein compositions of ECM of PLB formed with and without sucrose exposure were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, a crossover study was conducted during two phases of 14 days each, during which a volunteer wore a palatal appliance containing eight enamel blocks for PLB accumulation. In each phase, a 20% sucrose solution or distilled and deionized water (control) were extraorally dripped onto the blocks 8×/day. On the 14th day, the PLB were collected, the ECM proteins were extracted, separated by two-dimensional gel electrophoresis, digested by in-gel trypsin and MALDI-TOF MS analyzed. In the ECM of PLB formed under sucrose exposure, the following changes compared with the control PLB were observed: (1) the presence of upregulated proteins that may be involved in bacterial response to environmental changes induced by sucrose and (2) the absence of calcium-binding proteins that may partly explain the low inorganic concentration found in ECM of PLB formed under sucrose exposure. The findings showing that sucrose affected the ECM protein composition of PLB in vivo provide further insight into the unique cariogenic properties of this dietary carbohydrate.

© 2008 S. Karger AG, Basel


  

Key Words

  • Dental plaque
  • Gel electrophoresis, two-dimensional
  • Mass spectrometry
  • Sucrose

References

  1. Abranches J, Candella MM, Wen ZT, Baker HV, Burne RA: Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. J Bacteriol 2006;188:3748–3756.
  2. Aires CP, Tabchoury CP, Del Bel Cury AA, Koo H, Cury JA: Effect of sucrose concentration on dental biofilm formed in situ and on enamel demineralization. Caries Res 2006;40:28–32.
  3. Bellato CM, Garcia AKM, Mestrinelli F, Tsai SM, Machado MA, Meinhardt LW: The induction of differentially expressed proteins of Xylella fastidiosa with citrus extract. Braz J Microbiol 2004;35:235–242.

    External Resources

  4. Black C, Allan I, Ford SK, Wilson M, McNab R: Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis. Arch Oral Biol 2004;49:295–304.
  5. Blum H, Beier H, Gross HJ: Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987;8:93–99.
  6. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
  7. Caldas TD, Yaagoubi AEI, Richarme G: Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 1998;273:11478–11482.
  8. Ccahuana-Vásquez RA, Tabchoury CP, Tenuta LM, Del Bel Cury AA, Vale GC, Cury JA: Effect of frequency of sucrose exposure on dental biofilm composition and enamel demineralization in the presence of fluoride. Caries Res 2007;41:9–15.
  9. Cury JA, Rebello MAB, Del Bel Cury AA: In situ relationship between sucrose exposure and the composition of dental plaque. Caries Res 1997;31:356–360.
  10. Cury JA, Rebelo MAB, Del Bel Cury AA, Derbyshire MTVC, Tabchoury CPM: Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res 2000;34:491–497.
  11. Fox DJ, Dawes C: The extraction of protein matrix from human dental plaque. Arch Oral Biol 1970;15:1069–1077.
  12. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM: Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601–605.
  13. Hara AT, Queiroz CS, Paes Leme AF, Serra MC, Cury JA: Caries progression and inhibition in human and bovine root dentine in situ. Caries Res 2003;37:339–344.
  14. Huang CM: Comparative proteomic analysis of human whole saliva. Arch Oral Biol 2004;49:951–962.
  15. Iacono VJ, Mackay BJ, Pollock JJ, Bolot PR, Laqqenhein S, Grossbard BL, Rochon ML: Roles of lysozyme in the host response to periodontopathic organisms; in Genco RJ, Mergenhagen SE (eds): Host-Bacterial Interactions in Periodontal Diseases. Washington, American Society for Microbiology, 1982, pp 318–342.
  16. Jayaraman GC, Penders JE, Burne RA: Transcriptional analysis of the Streptococcus mutanshrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification. Mol Microbiol 1997;25:329–341.
  17. Joe A, Murray CS, McBride BC: Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant. Infect Immun1994;62:1358–1368.
  18. Len ACL, Cordwell SJ, Harty DWS, Jacques NA: Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat. Proteomics 2003;3:627–646.
  19. Len ACL, Harty DWS, Jacques NA: Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance. Microbiology 2004a;150:1339–1351.
  20. Len ACL, Harty DWS, Jacques NA: Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance. Microbiology 2004b;150:1353–1366.
  21. Mukhopadyay SV, Kapatral V, Xu W, Chakrabarty AM: Characterization of a Hank’s type serine/threonine kinases and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. J Bacteriol 1999;181:6615–6622.
  22. Nandakumar R, Nandakumar MP, Marten MR, Ross JM: Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus. J Proteom Res 2005;4:250–257.
  23. Paddick JS, Brailsford SR, Rao S, Soares RF, Kidd EA, Beighton D, Homer KA: Effect of biofilm growth on expression of surface proteins of Actinomyces naeslundii genospecies 2. Appl Environ Microbiol 2006;72:3774–3779.
  24. Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA: The role of sucrose in cariogenic dental biofilm formation – new insight. J Dent Res 2006;85:878–887.
  25. Pancholi V, Fischetti VA: α-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 1998;273:14503–14515.
  26. Pappin DJC, Hojrup P, Bleasby AJ: Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 1993;3:327–332.
  27. Pecharki GD, Cury JA, Paes Leme AF, Tabchoury CPM, Del Bel Cury AA, Rosalen PL, Bowen WH: Effect of sucrose containing iron (II) on dental biofilm and enamel demineralization in situ. Caries Res 2005;39:123–129.
  28. Quivey RG Jr, Kuhnert WL, Hahn K: Adaptation of oral streptococci to low pH. Adv Microb Physiol 2000;42:239–274.
  29. Ribeiro CCC, Tabchoury CPM, Del Bel Cury AA, Tenuta LMA, Rosalen PL, Cury JA: Effect of starch on the cariogenic potential of sucrose. Br J Nutr 2005;94:44–50.
  30. Rose RK, Dibdin GH, Shellis RP: A quantitative study of calcium binding and aggregation in selected oral bacteria. J Dent Res 1993;72:78–84.
  31. Sutherland IW: The biofilm matrix – an immobilized but dynamic microbial environment. Trends Microbiol 2001;9:222–227.
  32. Svensäter G, Sjögreen B, Hamilton IR: Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins. Microbiology 2000;146:107–117.
  33. Tenuta LM, Ricomini Filho AP, Del Bel Cury AA, Cury JA: Effect of sucrose on the selection of mutans streptococci and lactobacilli in dental biofilm formed in situ. Caries Res 2006;40:546–549.
  34. Vale GC, Tabchoury CP, Arthur RA, Del Bel Cury AA, Paes Leme AF, Cury JA: Temporal relationship between sucrose-associated changes in dental biofilm composition and enamel demineralization. Caries Res 2007;41:406–412.
  35. Vijay K, Brody MS, Freudlund E, Price CW: A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis. Mol Microbiol 2000;35:180–188.
  36. Welin J, Wilkins JC, Beighton D, Wrzesinski K, Fey SJ, Mose-Larsen P, Hamilton IR, Svensäter G: Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans. FEMS Microbiol Lett 2003;227:287–293.
  37. Wilkins JC, Beighton D, Homer KA: Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis. Appl Environ Microbiol 2003;69:5290–5296.
  38. Wilkins JC, Homer KA, Beighton D: Altered protein expression of Streptococcusoralis cultured at low pH revealed by two-dimensional gel electrophoresis. Appl Environ Microbiol 2001;67:3396–3405.
  39. Wilkins JC, Homer KA, Beighton D: Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions. Appl Environ Microbiol 2002;68:2382–2390.

  

Author Contacts

Prof. Jaime A. Cury
Piracicaba Dental School, PO Box 52
13414-903 Piracicaba, SP (Brazil)
Tel./Fax +55 19 2106 5302
E-Mail jcury@fop.unicamp.br

  

Article Information

Received: February 6, 2008
Accepted after revision: August 6, 2008
Published online: October 3, 2008
Number of Print Pages : 9
Number of Figures : 2, Number of Tables : 2, Number of References : 39

  

Publication Details

Caries Research

Vol. 42, No. 6, Year 2008 (Cover Date: November 2008)

Journal Editor: Shellis R.P. (Bristol)
ISSN: 0008–6568 (Print), eISSN: 1421–976X (Online)

For additional information: http://www.karger.com/CRE


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Previous studies have shown that sucrose promotes changes in the composition of the extracellular matrix (ECM) of plaque-like biofilm (PLB), but its effect on protein expression has not been studied in vivo. Therefore, the protein compositions of ECM of PLB formed with and without sucrose exposure were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, a crossover study was conducted during two phases of 14 days each, during which a volunteer wore a palatal appliance containing eight enamel blocks for PLB accumulation. In each phase, a 20% sucrose solution or distilled and deionized water (control) were extraorally dripped onto the blocks 8×/day. On the 14th day, the PLB were collected, the ECM proteins were extracted, separated by two-dimensional gel electrophoresis, digested by in-gel trypsin and MALDI-TOF MS analyzed. In the ECM of PLB formed under sucrose exposure, the following changes compared with the control PLB were observed: (1) the presence of upregulated proteins that may be involved in bacterial response to environmental changes induced by sucrose and (2) the absence of calcium-binding proteins that may partly explain the low inorganic concentration found in ECM of PLB formed under sucrose exposure. The findings showing that sucrose affected the ECM protein composition of PLB in vivo provide further insight into the unique cariogenic properties of this dietary carbohydrate.

© 2008 S. Karger AG, Basel


  

Author Contacts

Prof. Jaime A. Cury
Piracicaba Dental School, PO Box 52
13414-903 Piracicaba, SP (Brazil)
Tel./Fax +55 19 2106 5302
E-Mail jcury@fop.unicamp.br

  

Article Information

Received: February 6, 2008
Accepted after revision: August 6, 2008
Published online: October 3, 2008
Number of Print Pages : 9
Number of Figures : 2, Number of Tables : 2, Number of References : 39

  

Publication Details

Caries Research

Vol. 42, No. 6, Year 2008 (Cover Date: November 2008)

Journal Editor: Shellis R.P. (Bristol)
ISSN: 0008–6568 (Print), eISSN: 1421–976X (Online)

For additional information: http://www.karger.com/CRE


Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: 2/6/2008
Accepted: 8/6/2008
Published online: 10/3/2008
Issue release date: November 2008

Number of Print Pages: 9
Number of Figures: 2
Number of Tables: 2

ISSN: 0008-6568 (Print)
eISSN: 1421-976X (Online)

For additional information: http://www.karger.com/CRE


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Abranches J, Candella MM, Wen ZT, Baker HV, Burne RA: Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. J Bacteriol 2006;188:3748–3756.
  2. Aires CP, Tabchoury CP, Del Bel Cury AA, Koo H, Cury JA: Effect of sucrose concentration on dental biofilm formed in situ and on enamel demineralization. Caries Res 2006;40:28–32.
  3. Bellato CM, Garcia AKM, Mestrinelli F, Tsai SM, Machado MA, Meinhardt LW: The induction of differentially expressed proteins of Xylella fastidiosa with citrus extract. Braz J Microbiol 2004;35:235–242.

    External Resources

  4. Black C, Allan I, Ford SK, Wilson M, McNab R: Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis. Arch Oral Biol 2004;49:295–304.
  5. Blum H, Beier H, Gross HJ: Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987;8:93–99.
  6. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
  7. Caldas TD, Yaagoubi AEI, Richarme G: Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 1998;273:11478–11482.
  8. Ccahuana-Vásquez RA, Tabchoury CP, Tenuta LM, Del Bel Cury AA, Vale GC, Cury JA: Effect of frequency of sucrose exposure on dental biofilm composition and enamel demineralization in the presence of fluoride. Caries Res 2007;41:9–15.
  9. Cury JA, Rebello MAB, Del Bel Cury AA: In situ relationship between sucrose exposure and the composition of dental plaque. Caries Res 1997;31:356–360.
  10. Cury JA, Rebelo MAB, Del Bel Cury AA, Derbyshire MTVC, Tabchoury CPM: Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res 2000;34:491–497.
  11. Fox DJ, Dawes C: The extraction of protein matrix from human dental plaque. Arch Oral Biol 1970;15:1069–1077.
  12. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM: Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601–605.
  13. Hara AT, Queiroz CS, Paes Leme AF, Serra MC, Cury JA: Caries progression and inhibition in human and bovine root dentine in situ. Caries Res 2003;37:339–344.
  14. Huang CM: Comparative proteomic analysis of human whole saliva. Arch Oral Biol 2004;49:951–962.
  15. Iacono VJ, Mackay BJ, Pollock JJ, Bolot PR, Laqqenhein S, Grossbard BL, Rochon ML: Roles of lysozyme in the host response to periodontopathic organisms; in Genco RJ, Mergenhagen SE (eds): Host-Bacterial Interactions in Periodontal Diseases. Washington, American Society for Microbiology, 1982, pp 318–342.
  16. Jayaraman GC, Penders JE, Burne RA: Transcriptional analysis of the Streptococcus mutanshrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification. Mol Microbiol 1997;25:329–341.
  17. Joe A, Murray CS, McBride BC: Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant. Infect Immun1994;62:1358–1368.
  18. Len ACL, Cordwell SJ, Harty DWS, Jacques NA: Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat. Proteomics 2003;3:627–646.
  19. Len ACL, Harty DWS, Jacques NA: Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance. Microbiology 2004a;150:1339–1351.
  20. Len ACL, Harty DWS, Jacques NA: Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance. Microbiology 2004b;150:1353–1366.
  21. Mukhopadyay SV, Kapatral V, Xu W, Chakrabarty AM: Characterization of a Hank’s type serine/threonine kinases and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. J Bacteriol 1999;181:6615–6622.
  22. Nandakumar R, Nandakumar MP, Marten MR, Ross JM: Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus. J Proteom Res 2005;4:250–257.
  23. Paddick JS, Brailsford SR, Rao S, Soares RF, Kidd EA, Beighton D, Homer KA: Effect of biofilm growth on expression of surface proteins of Actinomyces naeslundii genospecies 2. Appl Environ Microbiol 2006;72:3774–3779.
  24. Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA: The role of sucrose in cariogenic dental biofilm formation – new insight. J Dent Res 2006;85:878–887.
  25. Pancholi V, Fischetti VA: α-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 1998;273:14503–14515.
  26. Pappin DJC, Hojrup P, Bleasby AJ: Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 1993;3:327–332.
  27. Pecharki GD, Cury JA, Paes Leme AF, Tabchoury CPM, Del Bel Cury AA, Rosalen PL, Bowen WH: Effect of sucrose containing iron (II) on dental biofilm and enamel demineralization in situ. Caries Res 2005;39:123–129.
  28. Quivey RG Jr, Kuhnert WL, Hahn K: Adaptation of oral streptococci to low pH. Adv Microb Physiol 2000;42:239–274.
  29. Ribeiro CCC, Tabchoury CPM, Del Bel Cury AA, Tenuta LMA, Rosalen PL, Cury JA: Effect of starch on the cariogenic potential of sucrose. Br J Nutr 2005;94:44–50.
  30. Rose RK, Dibdin GH, Shellis RP: A quantitative study of calcium binding and aggregation in selected oral bacteria. J Dent Res 1993;72:78–84.
  31. Sutherland IW: The biofilm matrix – an immobilized but dynamic microbial environment. Trends Microbiol 2001;9:222–227.
  32. Svensäter G, Sjögreen B, Hamilton IR: Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins. Microbiology 2000;146:107–117.
  33. Tenuta LM, Ricomini Filho AP, Del Bel Cury AA, Cury JA: Effect of sucrose on the selection of mutans streptococci and lactobacilli in dental biofilm formed in situ. Caries Res 2006;40:546–549.
  34. Vale GC, Tabchoury CP, Arthur RA, Del Bel Cury AA, Paes Leme AF, Cury JA: Temporal relationship between sucrose-associated changes in dental biofilm composition and enamel demineralization. Caries Res 2007;41:406–412.
  35. Vijay K, Brody MS, Freudlund E, Price CW: A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis. Mol Microbiol 2000;35:180–188.
  36. Welin J, Wilkins JC, Beighton D, Wrzesinski K, Fey SJ, Mose-Larsen P, Hamilton IR, Svensäter G: Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans. FEMS Microbiol Lett 2003;227:287–293.
  37. Wilkins JC, Beighton D, Homer KA: Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis. Appl Environ Microbiol 2003;69:5290–5296.
  38. Wilkins JC, Homer KA, Beighton D: Altered protein expression of Streptococcusoralis cultured at low pH revealed by two-dimensional gel electrophoresis. Appl Environ Microbiol 2001;67:3396–3405.
  39. Wilkins JC, Homer KA, Beighton D: Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions. Appl Environ Microbiol 2002;68:2382–2390.