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Time-course Analysis of Biofilm Formation in Quorum Sensing-deficient Bacteria

Quorum sensing 결핍 세균에서 생물막 형성의 시간적 추이 분석

  • Kim, Soo-Kyoung (Department of Pharmacy, College of Pharmacy, Pusan National University) ;
  • Lee, Mi-Nan (Department of Pharmacy, College of Pharmacy, Pusan National University) ;
  • Lee, Joon-Hee (Department of Pharmacy, College of Pharmacy, Pusan National University)
  • 김수경 (부산대학교 약학대학 약학과 미생물학 연구실) ;
  • 이미난 (부산대학교 약학대학 약학과 미생물학 연구실) ;
  • 이준희 (부산대학교 약학대학 약학과 미생물학 연구실)
  • Received : 2014.05.07
  • Accepted : 2014.06.05
  • Published : 2014.06.30

Abstract

Pseudomonas aeruginosa and Vibrio vulnificus are Gram-negative human pathogens, which exert their virulence through quorum sensing (QS) regulation. The infection of these pathogens have been known to be mediated by biofilm formation in many cases and this study carried out the time-course analysis of biofilm formation depending on the QS regulation in P. aeruginosa and V. vulnificus. In P. aeruginosa, our results demonstrated that QS-deficient mutant better attached to surface at initial stage of biofilm formation, but poorly proceeded to the maturation of the biofilm structure, while wild type less attached at initial stage but developed highly structured biofilm at late stage. Because of this, the quantitative comparison of biofilm formation between wild type and the QS mutant showed the reversion; the QS mutant formed more biofilm until 10 h after inoculation than wild type, but wild type formed much more biofilm after 10 h than QS mutant. V. vulnificus has been reported to form more biofilm with the mutation on QS system. When we performed the same time-course analysis of the V. vulnificus biofilm formation, the reversion was not detected even with prolonged culture for 108 h and the QS mutant always forms more biofilm than wild type. These results indicate that the QS regulation negatively affects the attachment at early stage but positively facilitates the biofilm maturation at late stage in P. aeruginosa, while the QS regulation has a negative effect on the biofilm formation throughout the biofilm development in V. vulnificus. Based on our results, we suggest that the developmental stage of biofilm and bacterial species should be considered when the QS system is targeted for biofilm control.

녹농균(Pseudomonas aeruginosa)과 비브리오 불니피쿠스균(Vibrio vulnificus)은 그람 음성의 병원균들로써, quorum sensing(QS) 기전을 통해 병원성을 발현하는 세균들이다. 이들 병원균의 감염은 많은 경우 생물막 형성에 의해 매개된다고 알려져 있는데, 이에 본 연구에서는 P. aeruginosa와 V. vulnificus를 대상으로 QS 기전의 유무에 따른 생물막 형성의 시간적 추이를 분석해 보았다. 그 결과 P. aeruginosa의 경우 QS 기전이 결핍된 균주가 야생형에 비해 초기 부착은 더 잘 하였으나, 이후 생물막 구조의 성숙 능력은 야생형에 비해 현저히 떨어짐을 알 수 있었다. 이러한 특성 때문에 야생형과 QS 결핍 균주의 생물막 형성을 시간의 추이에 따라 정량적으로 비교해 보면 초기 10시간 정도 까지는 QS 결핍 균주가 더 많은 생물막을 형성하다가, 이후 야생형이 더 많이 생물막을 형성하는 역전 현상이 관찰되었다. V. vulnificus는 P. aeruginosa와는 달리 QS 결핍 균주가 야생형보다 더 많은 생물막을 형성한다고 보고된 균주이다. 이 균주에서 같은 방식으로 생물막 형성을 조사해 본 결과, 108시간의 장시간 동안에도 항상 QS 결핍 균주가 야생형 보다 더 많은 생물막을 형성하여, 역전 현상은 관찰되지 않았다. 이 결과는 P. aeruginosa의 경우에는 QS 기전이 초기 부착은 저해하는 방향으로, 성숙과정은 촉진시키는 방향으로 작용하며, V. vulnificus에서는 일관되게 생물막 형성을 저해하는 방향으로 작용함을 보여주는 것이다. 따라서 생물막 제어를 위한 타겟으로 QS기전을 이용할 때에는 제어하고자 하는 생물막 형성 단계와 세균 종을 함께 고려하여야 한다고 제안한다.

Keywords

References

  1. Cos, P., Tote, K., Horemans, T., and Maes, L. 2010. Biofilms: an extra hurdle for effective antimicrobial therapy. Curr. Pharm. Des. 16, 2279-2295. https://doi.org/10.2174/138161210791792868
  2. Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., and Greenberg, E.P. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298. https://doi.org/10.1126/science.280.5361.295
  3. Fazli, M., Almblad, H., Rybtke, M.L., Givskov, M., Eberl, L., and Tolker-Nielsen, T. 2014. Regulation of biofilm formation in Pseudomonas and Burkholderia species. Environ. Microbiol. DOI 10.1111/1462-2920.12448.
  4. Henke, J.M. and Bassler, B.L. 2004. Bacterial social engagements. Trends Cell. Biol. 14, 648-656. https://doi.org/10.1016/j.tcb.2004.09.012
  5. Heydorn, A., Ersboll, B., Kato, J., Hentzer, M., Parsek, M.R., Tolker-Nielsen, T., Givskov, M., and Molin, S. 2002. Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression. Appl. Environ. Microbiol. 68, 2008-2017. https://doi.org/10.1128/AEM.68.4.2008-2017.2002
  6. Huq, A., Whitehouse, C.A., Grim, C.J., Alam, M., and Colwell, R.R. 2008. Biofilms in water, its role and impact in human disease transmission. Curr. Opin. Biotechnol. 19, 244-247. https://doi.org/10.1016/j.copbio.2008.04.005
  7. Hurley, M.N., Camara, M., and Smyth, A.R. 2012. Novel approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Eur. Respir. J. 40, 1014-1023. https://doi.org/10.1183/09031936.00042012
  8. Jeong, H.S., Lee, M.H., Lee, K.H., Park, S.J., and Choi, S.H. 2003. SmcR and cyclic AMP receptor protein coactivate Vibrio vulnificus vvpE encoding elastase through the RpoS-dependent promoter in a synergistic manner. J. Biol. Chem. 278, 45072-45081. https://doi.org/10.1074/jbc.M308184200
  9. Jones, M.K. and Oliver, J.D. 2009. Vibrio vulnificus: disease and pathogenesis. Infect. Immun. 77, 1723-1733. https://doi.org/10.1128/IAI.01046-08
  10. Kim, S.M., Park, J.H., Lee, H.S., Kim, W.B., Ryu, J.M., Han, H.J., and Choi, S.H. 2013. LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells. Infect. Immun. 81, 3721-3730. https://doi.org/10.1128/IAI.00561-13
  11. Lee, J.H., Lequette, Y., and Greenberg, E.P. 2006. Activity of purified QscR, a Pseudomonas aeruginosa orphan quorum-sensing transcription factor. Mol. Microbiol. 59, 602-609. https://doi.org/10.1111/j.1365-2958.2005.04960.x
  12. Lequette, Y., Lee, J.H., Ledgham, F., Lazdunski, A., and Greenberg, E.P. 2006. A distinct QscR regulon in the Pseudomonas aeruginosa quorum-sensing circuit. J. Bacteriol. 188, 3365-3370. https://doi.org/10.1128/JB.188.9.3365-3370.2006
  13. Matsumoto-Mashimo, C., Guerout, A.M., and Mazel, D. 2004. A new family of conditional replicating plasmids and their cognate Escherichia coli host strains. Res. Microbiol. 155, 455-461. https://doi.org/10.1016/j.resmic.2004.03.001
  14. Page, M.G. and Heim, J. 2009. Prospects for the next anti-Pseudomonas drug. Curr. Opin. Pharmacol. 9, 558-565. https://doi.org/10.1016/j.coph.2009.08.006
  15. Parsek, M.R., and Greenberg, E.P. 2005. Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol. 13, 27-33. https://doi.org/10.1016/j.tim.2004.11.007
  16. Pearson, J.P., Pesci, E.C., and Iglewski, B.H. 1997. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacteriol. 179, 5756-5767. https://doi.org/10.1128/jb.179.18.5756-5767.1997
  17. Purevdorj, B., Costerton, J.W., and Stoodley, P. 2002. Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 68, 4457-4464. https://doi.org/10.1128/AEM.68.9.4457-4464.2002
  18. Reading, N.C. and Sperandio, V. 2006. Quorum sensing: the many languages of bacteria. FEMS Microbiol. Lett. 254, 1-11. https://doi.org/10.1111/j.1574-6968.2005.00001.x
  19. Roh, J.B., Lee, M.A., Lee, H.J., Kim, S.M., Cho, Y., Kim, Y.J., Seok, Y.J., Park, S.J., and Lee, K.H. 2006. Transcriptional regulatory cascade for elastase production in Vibrio vulnificus: LuxO activates luxT expression and LuxT represses smcR expression. J. Biol. Chem. 281, 34775-34784. https://doi.org/10.1074/jbc.M607844200
  20. Schuster, M. and Greenberg, E.P. 2006. A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int. J. Med. Microbiol. 296, 73-81. https://doi.org/10.1016/j.ijmm.2006.01.036
  21. Schuster, M., Lostroh, C.P., Ogi, T., and Greenberg, E.P. 2003. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185, 2066-2079. https://doi.org/10.1128/JB.185.7.2066-2079.2003
  22. Walters, M.C., Roe, F., Bugnicourt, A., Franklin, M.J., and Stewart, P.S. 2003. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob. Agents Chemother. 47, 317-323. https://doi.org/10.1128/AAC.47.1.317-323.2003
  23. Welch, M., Mikkelsen, H., Swatton, J.E., Smith, D., Thomas, G.L., Glansdorp, F.G., and Spring, D.R. 2005. Cell-cell communication in Gram-negative bacteria. Mol. Biosyst. 1, 196-202. https://doi.org/10.1039/b505796p
  24. Whiteley, M., Lee, K.M., and Greenberg, E.P. 1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96, 13904-13909. https://doi.org/10.1073/pnas.96.24.13904
  25. Williams, P. 2007. Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology 153, 3923-3938. https://doi.org/10.1099/mic.0.2007/012856-0