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pH Stress Alters Cytoplasmic Membrane Fluidity and atpB Gene Expression in Streptococcus mutans

pH stress가 Streptococcus mutans의 형질막 유동성 및 atpB 유전자 발현에 미치는 영향

  • Cho, Chul Min (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Pusan National University) ;
  • Jung, Seung Il (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Pusan National University) ;
  • Kim, Myung Sup (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Pusan National University) ;
  • Lee, Sae A (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Pusan National University) ;
  • Kang, Jung Sook (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Pusan National University)
  • 조철민 (부산대학교 치의학전문대학원 구강생화학교실) ;
  • 정승일 (부산대학교 치의학전문대학원 구강생화학교실) ;
  • 김명섭 (부산대학교 치의학전문대학원 구강생화학교실) ;
  • 이새아 (부산대학교 치의학전문대학원 구강생화학교실) ;
  • 강정숙 (부산대학교 치의학전문대학원 구강생화학교실)
  • Received : 2016.10.25
  • Accepted : 2016.12.22
  • Published : 2017.01.30

Abstract

Streptococcus mutans (S. mutans), which plays a major role in the etiology of human dental caries, is able to tolerate exposure to acid shock in addition to its acidogenicity. We investigated the effects of pH stress on membrane fluidity, activities and expression levels of F-ATPase, and proton permeability in S. mutans. Using 1,6-diphenyl-1,3,5-hexatriene, we observed membrane ordering at pH 4.8 and pH 8.8. The ordering effects were larger at pH 4.8 in cytoplasmic membranes isolated from S. mutans (CMSM). Increasing pH resulted in a decrease in the activities and expression levels of F-ATPase. The proton permeability was decreased at both acidic and alkaline pHs, and the lowest permeability was observed at pH 4.8. The lower permeability at pH 8.8 than pH 6.8 is likely to be caused by the decreased proton influx due to the decreased CMSM fluidity. In addition, it seems to be evident that extremely low permeability at pH 4.8 was caused by the decreased proton influx due to the decreased CMSM fluidity as well as the increased proton efflux due to the increased activity and expression level of F-ATPase. It is likely that CMSM fluidity and F-ATPase activity are two major key factors that determine proton permeability in S. mutans. We suggest that CMSM fluidity plays an important role in the determination of proton permeability, which sheds light on the possibility of using nonspecific membrane fluidizers, e.g., ethanol, for anti-caries purposes.

치아우식의 주원인균인 Streptococcus mutans (S. mutans)는 산 생성 뿐 아니라 산에 대한 탁월한 저항성을 나타낸다. 본 연구에서는 S. mutans가 pH stress에 노출될 때 형질막 유동성, F-ATPase 활성과 발현 및 양성자 투과성 변화와 그 상관관계를 규명하였다. S. mutans로부터 형질막을 분리한 후 1,6-diphenyl-1,3,5-hexatriene을 사용하여 pH stress가 형질막 유동성 변화에 미치는 영향을 측정하였다. pH 4.8과 pH 8.8에서 배양한 S. mutans는 pH 6.8에서 배양한 S. mutans에 비하여 형질막 유동성이 감소되었다. F-ATPase 활성과 발현은 pH 4.8에서 가장 높았고, pH 8.8에서 가장 낮았다. 양성자 투과성은 pH 4.8과 pH 8.8에서 모두 감소되었으며, 특히 pH 4.8에서의 감소가 컸다. F-ATPase 활성만으로 양성자 투과성이 결정된다면 pH 8.8에서 가장 높아야 하나 pH 6.8보다 감소하는 것은 형질막 유동성 감소에 기인된 양성자 세포내 유입 감소와 관련된 것으로 추정한다. 또한 pH 4.8에서 양성자 투과성이 아주 낮은 것은 높은 F-ATPase 활성에 의한 양성자 세포외 유출 증가 뿐 아니라 형질막 유동성 감소에 의한 양성자 세포내 유입 감소에 기인된 것으로 추정한다. 따라서 pH stress에 의한 형질막 유동성 감소는 S. mutans가 세포내 pH 를 유지하는데 중요한 역할을 하는 것으로 생각되며 에탄올을 포함하여 비특이적으로 세포막 유동성을 증가시키는 약물들은 항우식제에 활용될 수 있을 것으로 추정한다.

Keywords

References

  1. Ajdic, D., McShan, W. M., McLaughlin, R. E., Savic, G., Chang, J., Carson, M. B., Primeaux, C., Tian, R., Kenton, S., Jia, H., Lin, S., Qian, Y., Li, S., Zhu, H., Najar, F., Lai, H., White, J., Roe, B. A. and Ferretti, J. J. 2002. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 99, 14434-14439. https://doi.org/10.1073/pnas.172501299
  2. Baker, J. L., Abranches, J., Faustoferri, R. C., Hubbard, C. J., Lemos, J. A., Courtney, M. A. and Quivey, R. G. 2015. Transcriptional profile of glucose-shocked and acid-adapted strains of Streptococcus mutans. Mol. Oral Microbiol. 30, 496-517. https://doi.org/10.1111/omi.12110
  3. Baker, J. L., Faustoferri, R. C. and Quivey, R. G. 2016. Acidadaptive mechanisms of Streptococcus mutans-the more we know, the more we don't. Mol. Oral Microbiol. doi: 10.1111/omi.12162.
  4. Ban, S. H., Kim, J. E., Pandit, S. and Jeon, J. G. 2012. Influences of Dryopteris crassirhizoma extract on the viability, growth and virulence properties of Streptococcus mutans. Molecules 17, 9231-9244. https://doi.org/10.3390/molecules17089231
  5. Belli, W. A. and Marquis, R. E. 1991. Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture. Appl. Environ. Microbiol. 57, 1134-1138.
  6. Bencini, D. A., Shanley, M. S., Wild, J. R. and O'Donovan, G. A. 1983. New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes. Anal. Biochem. 132, 259-264. https://doi.org/10.1016/0003-2697(83)90005-2
  7. Bender, G. R., Sutton, S. V. and Marquis, R. E. 1986. Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infect. Immun. 53, 331-338.
  8. Fozo, E. M. and Quivey, R. G. 2004. Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl. Environ. Microbiol. 70, 929-936. https://doi.org/10.1128/AEM.70.2.929-936.2004
  9. Gong, Y., Tian, X. L., Sutherland, T., Sisson, G., Mai, J., Ling, J. and Li, Y. H. 2009. Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple twocomponent systems involved in acid adaptation. Microbiology 155, 3322-3332. https://doi.org/10.1099/mic.0.031591-0
  10. Gutierrez, J. A., Crowley, P. J., Cvitkovitch, D. G., Brady, L. J., Hamilton, I. R., Hillman, J. D. and Bleiweis, A. S. 1999. Streptococcus mutans ffh, a gene encoding a homologue of the 54kDa subunit of the signal recognition particle, is involved in resistance to acid stress. Microbiology 145, 357-366. https://doi.org/10.1099/13500872-145-2-357
  11. Hamilton, I. R. and Buckley, N. D. 1991 Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol. Immun. 6, 65-71. https://doi.org/10.1111/j.1399-302X.1991.tb00453.x
  12. Hamilton, I. R. and St, Martin, E. J. 1982. Evidence for the involvement of proton motive force in the transport of glucose by a mutant of Streptococcus mutans strain DR0001 defective in glucose-phosphoenopyruvate phosphotransferase activity. Infect. Immun. 36, 567-575.
  13. Hwang, G., Liu, Y., Kim, D., Sun, V., Aviles-Reyes, A., Kajfasz, J. K., Lemos, J. A. and Koo, H. 2016. Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure. Scientific Reports. doi:10.1038/srep32841.
  14. Koo, H., Falsetta, M. L. and Klein, M. I. 2013. The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm. J. Dent. Res. 92, 1065-1073. https://doi.org/10.1177/0022034513504218
  15. Kuhnert, W. L., Zheng, G., Faustoferri, R. C. and Quivey, R. G. 2004. The F-ATPase operon promoter of Streptococcus mutans is transcriptionally regulated in response to external pH. J. Bacteriol. 186, 8524-8528. https://doi.org/10.1128/JB.186.24.8524-8528.2004
  16. Lemos, J. A., Abranches, J. and Burne, R. A. 2005. Responses of cariogenic streptococci to environmental stresses. Curr. Issues Mol. Biol. 7, 95-108.
  17. Loesche, W. J. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50, 353-380.
  18. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
  19. Mykytczuk, N. C., Trevors, J. T., Leduc, L. G. and Ferroni, G. D. 2007. Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog. Biophys. Mol. Biol. 95, 60-82. https://doi.org/10.1016/j.pbiomolbio.2007.05.001
  20. Petrackova, D., Becer, J., Svobodova, J. and Herman, P. 2010. Long-term adaptation of Bacillus subtilis 168 to extreme pH affects chemical and physical properties of the cellular membrane. J. Membr. Biol. 233, 73-83. https://doi.org/10.1007/s00232-010-9226-9
  21. Phan, T. N., Buckner, T., Sheng, J., Baldeck, J. D. and Marquis, R. E. 2004. Physiologic actions of zinc related to inhibition of acid and alkali production by oral streptococci in suspensions and biofilms. Oral Microbiol. Immunol. 19, 31-38. https://doi.org/10.1046/j.0902-0055.2003.00109.x
  22. Porter, E. V. and Chassy, B. M. 1982. Glucokinase from Streptococcus mutans. Methods Enzymol. 90, 25-30.
  23. Quivey, R. G., Faustoferri, R., Monahan, K. and Marquis, R. 2000. Shifts in membrane fatty acid profiles associated with acid adapation of Streptococcus mutans. FEMS Microbiol. Lett. 189, 89-92. https://doi.org/10.1111/j.1574-6968.2000.tb09211.x
  24. Quivey, R. G., Grayhack, E. J., Faustoferri, R. C., Hubbard, C. J., Baldeck, J. D., Wolf, A. S., MacGilvray, M. E., Rosalen, P. L., Scott-Anne, K., Santiago, B., Gopal, S., Payne, J. and Marquis, R. E. 2015. Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants. Mol. Oral Microbiol. 30, 474-495. https://doi.org/10.1111/omi.12107
  25. Quivey, R. G., Kuhnert, W. L. and Hahn, K. 2000. Adaptation of oral streptococci to low pH. Adv. Microb. Physiol. 42, 239-274.
  26. Zhang, G. J., Liu, H. W., Yang, L., Zhong, Y. G. and Zheng, Y. Z. 2000. Influence of membrane physical state on the lysosomal proton permeability. J. Membr. Biol. 175, 53-62. https://doi.org/10.1007/s002320001054

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