DOI QR코드

DOI QR Code

정지기 Salmonella typhimurium 세포에서 특이적으로 발현되는 세포질 단백질의 동정 및 발현조절에 대한 연구

Identification and Chararterization of Stationary-phase Specific Cytosolic Protein in Salmonella typhimurium

  • 유아영 (부산대학교 생명과학부 미생물학과) ;
  • 김영희 (부산대학교 생명과학부 미생물학과) ;
  • 유종언 (부산대학교 생명과학부 미생물학과) ;
  • 김삼웅 (부산대학교 생명과학부 미생물학과) ;
  • 백형석 (부산대학교 생명과학부 미생물학과) ;
  • 강호영 (부산대학교 생명과학부 미생물학과)
  • Yoo, Ah-Young (Division of Biological Sciences, Pusan National University) ;
  • Kim, Young-Hee (Division of Biological Sciences, Pusan National University) ;
  • Yu, Jong-Earn (Division of Biological Sciences, Pusan National University) ;
  • Kim, Sam-Woong (Division of Biological Sciences, Pusan National University) ;
  • Baik, Hyung-Suk (Division of Biological Sciences, Pusan National University) ;
  • Kang, Ho-Young (Division of Biological Sciences, Pusan National University)
  • 발행 : 2007.02.28

초록

Salmoenella는 대표적인 intracellular pathogen으로 숙주의 면역 세포인 macrophage 내에서 살아남아 이들을 매개로 숙주의 몸 전체를 이동해 가면서 전신성 감염을 일으킨다. 살모넬라는 숙주 세포 내부의 이러한 극한 환경을 극복하기 위해서 다양한 방어 기작을 가진다. 본 연구에서는 복합적인 스트레스가 작용하는 정지기 Salmonella에서 특이적으로 발현되는 단백질에 주목하였다. 정지기 상태의 Salmonella에서 약 20 kDa의 단백질이 특이적으로 많이 발현되었으며, 세포질 분획을 통해 이 단백질이 세포질 부분에 존재함을 알 수 있었다. MALDI-TOF 분석을 통해 이 단백질이 $\b{D}NA$ binding $\b{p}rotein$ in $\b{s}tationary$ phase (Dps) 단백질임을 확인하였다. Dps 단백질은 스트레스가 주어진 상황에서 DNA에 비특이적으로 결합하여 DNA가 안정한 형태를 유지하도록 하여 스트레스로부터 염색체를 보호하는 역할을 하는 것으로 알려져 있다. 이후의 연구를 위하여 과발현하여 정제한 Dps 단백질을 토끼에 주사하여 Dps 특이적인 항체를 제조하였다. dps 발현에 영향을 미치는 조절자 단백질을 알기 위하여 다양한 S. typhimrium 돌연변이주들 내에서의 Dps 단백질양을 조사하였다.

Salmonella is facultative intracellular pathogen that can survive and replicate in macrophages even though these cells are equipped with a plethora of anti-microbial mechanisms. To survive in this hostile intracellular environment, Salmonella has evolved numerous defense mechartisms. An approximately 20 kDa protein was detected as a stationary-phase specific protein band in cytosolic fraction. It was identified as a DNA binding protein in stationary phase (Dps) by analysis of MALDI-TOF assay. It has been known that Dps, the protein produced in the stationary phase of bacteria, allows DNA to form chromatin by binding to DNA nonspecifically and protects DNA from reactive oxidative species (ROS). For further study, Dps specific polyclonal antibodies were generated by injection of purified Dps protein into rabbit. To examine the Finfluence of several regulatory proteins in the expression dps gene, Dps protein level in various S. typhimurium mutants defecting regulatory proteins were investigated by Westernblot using Dps specific polyclonal antibodies.

키워드

참고문헌

  1. Abigail A. S. and D. D. Whitt. 2002. Sallmonella Species, pp. 381-397. In Abigail A. Salyers and Dixie D. Whitt (ed.), Bacterial pathogenesis: A molecular approach. American Society for Microbiology, Washington, D.C
  2. Almiron, M., A. J. Link, D. Furlong and R. Kolter. 1992. A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev. 6, 2646-2654 https://doi.org/10.1101/gad.6.12b.2646
  3. Altuvia, S., M. Almiron, G. Huisman, R. Kolter and G. Storz. 1994. The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase. Mol. Microbiol. 13, 265-272 https://doi.org/10.1111/j.1365-2958.1994.tb00421.x
  4. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293-300
  5. Buchmeier, N. A., C. J. Lipps, M. Y. So and F. Heffron. 1993. Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol. Micobiol. 7, 933-936 https://doi.org/10.1111/j.1365-2958.1993.tb01184.x
  6. Farr, S. B. and T. Kogoma. 1991. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 55, 561-585
  7. Fields, P. I., V. Swansom, C. G. Haidaris and F. Heffron. 1986. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl. Acad. Sci. USA. 83, 5189-5193 https://doi.org/10.1073/pnas.83.14.5189
  8. Grant, R. A., D. J. Filman, S. E. Finkel, R. Kolter and J. M. Hogle. 1998. The crustal structure of Dps, a ferritin homolog that binds and protects DNA. Nat. Struct. Biol. 5, 294-303 https://doi.org/10.1038/nsb0498-294
  9. Groisman E. A. and H. Ochman. 1997 How Salmonella became a pathogen. Trends Microbiol. 5, 343-349 https://doi.org/10.1016/S0966-842X(97)01099-8
  10. Groote, M. A., U. A. Ochsner, M. U. Shiloh, C. Hathan, J. M. McCord, M. C. Dinauer, S. J. Libby, A. Vazqurez-Torres, Y. Xu and F. C. Fang. 1997. Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase. Proc. Natl. Acad. Sci. USA. 94, 13997-14001 https://doi.org/10.1073/pnas.94.25.13997
  11. Grove, A. and S. P. Wilinson. 2005. Differential DNA binding and protection by dimeric and dodecameric forms of the ferritin homolog Dps from Deinococcus radiodurans. J. Mol. Biol. 347, 495-508 https://doi.org/10.1016/j.jmb.2005.01.055
  12. Gulig, P. A. and R. Curtiss III. 1987. Plasmid-associated virulence of Salmonella typhimurium. Infect. Immun. 55, 2891-2901
  13. Jeong, K. C., D. J. Baumler and C. W. Kaspar. 2006. dps expression in Escherichia coli O157:H7 requires an extended-10 region and is affected by the cAMP receptor protein. Biochim. Biophys. Acta. 1759, 51-59 https://doi.org/10.1016/j.bbaexp.2006.02.001
  14. Lai, E. M., U. Nair, N. D. Phadke and J. R. Maddock. 2004. Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli. Mol. Microbiol. 52, 1029-1044 https://doi.org/10.1111/j.1365-2958.2004.04040.x
  15. Libby, S. J., W. Goebel, A. Ludwig, N. Buchmeier, F. C. Fang, D. G. Guiney, J. G. Songer and F. Heffron. 1994. A cytolysin encoded by Salmonella is required for survival within macrophages. Proc. Natl. Acad. Sci. USA. 91, 489-493 https://doi.org/10.1073/pnas.91.2.489
  16. Loewen P. C., B. Hu, J. Strutinsky and R. Sparling. 1998. Regulation in the rpoS regulon of Escherichia coli. Can. J. Microbiol. 44, 707-17 https://doi.org/10.1139/cjm-44-8-707
  17. Mastroeni, P., A. Vazquez-Torres, F. C. Fang, Y. Xu, S. Khan, C. E. Hormaeche and G. Gougan. 2000. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J. Exp. Med. 192, 237-248 https://doi.org/10.1084/jem.192.2.237
  18. Miller, R. A. and B. E. Britigan. 1997, Role of oxidants in microbial pathophysiology. Clinic. Microbiol. Rev. 10, 1-18
  19. Nair, S. and S. E. Finkel. 2004. Dps protects cells against multiple stresses during stationary phase. J. Bacteriol. 186, 4192-4198 https://doi.org/10.1128/JB.186.13.4192-4198.2004
  20. Parry C. M., T. T. Hien, G. Dougan, N. J. White and J. J. Farrar. 2002. Typhoid fever. N. Engl. J Med. 347, 1770-1782 https://doi.org/10.1056/NEJMra020201
  21. Pomposiello, P. J., M. H. Bennik and B. Demple. 2001. Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J. Bacteriol. 183, 3890-3902 https://doi.org/10.1128/JB.183.13.3890-3902.2001
  22. Reindel S., C. L. Schmidt, S. Anemuller and B. F. Matzanke. 2005. Expression and regulation pattern of ferritin-like DpsA in the Archaeon Halobacterium salinarum. BioMetals. 18, 387-397 https://doi.org/10.1007/s10534-005-3713-y
  23. Roschenthaler, R., P. Kindler, P. Herrlich and J. Igbokwe. 1970. The action of nitrofurantoin: inhibition of growth of Escherichia coli K 12 and of IPTG-induced beta-galaotosidase synthesis. Zentralbl Bakteriol. 215, 203-11
  24. Sambrook, J. and David W. Russel. 2001. Molecular Cloning a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  25. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  26. Spector, M. P. The starvation-stress response (SSR) of Salmonella. Adv. Microb. Physiol. 40, 233-279
  27. Towbin, H., T. Staehelin and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354 https://doi.org/10.1073/pnas.76.9.4350
  28. Tsolis, R. M., A. J. Baumler and F. Heffron. 1995. Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages. Infect. Immun. 63, 1739-1744
  29. Valdivia, R. H. and S. Falkow. 1996. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol. Microbiol. 22, 367-378 https://doi.org/10.1046/j.1365-2958.1996.00120.x
  30. Vazquez-Torres, A., J. Jones-Carson, P. Mastroeni, H. Ischiropoulos and F. C. Fang. 2000. Antimicrobial actions of the NAPDH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J. Exp. Med. 192, 227-236 https://doi.org/10.1084/jem.192.2.227
  31. Wolf, S. G., D. Frenkiel, T. Arad, S. E. Finkel, R. Kolter and A. Minsky. 1999. DNA protection by stress- induced biocrystallization. Nature. 400, 83-85 https://doi.org/10.1038/21918
  32. Zeth, K., S. Offermann, L. O. Essen and D. Oesterhelt. 2004. Iron-oxo clusters biomineralizing on protein surfaces: Structural analysis of Halobacterium salinarum DpsA in its low- and high-iron states. Proc. Natl. Acad. Sci. 101, 13780-13785 https://doi.org/10.1073/pnas.0401821101
  33. Zhao, G., P. Ceci, A. Hari, L. Giangiacomo, T. M. Laue, E. Chiancone and N. D. Chasteen. 2002. Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. J. Biol. Chem. 277, 27689-27696 https://doi.org/10.1074/jbc.M202094200
  34. Zheng M., X. Wang, L. J. Templeton, D. R. Smulski, R. A. LaRossa and G. Storz. 2001. DNA microarray-mediated transcriptional profiling of the Escherichia coli responses to hydrogen peroxide. J. Bacteriol. 183, 4562-4570 https://doi.org/10.1128/JB.183.15.4562-4570.2001