Journal of Microbiology and Biotechnology

  • 제17권2호
  • /
  • Pages.234-243
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  • 2007
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

IVET-based Identification of Virulence Factors in Vibrio vulnificus MO6-24/O

  • Lee, Ko-Eun (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Bang, Ji-Sun (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Baek, Chang-Ho (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Park, Dae-Kyun (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Hwang, Won (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University) ;
  • Choi, Sang-Ho (Department of Food Science and Technology, Seoul National University) ;
  • Kim, Kum-Soo (Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University)
  • 발행 : 2007.02.28
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초록

Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of ${\sim}18,000$ resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of $1.6{\times}10^6$. Two hours after infection, kanamycin was administered at $200{mu}g$ per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.

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참고문헌

  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database searching programs. Nucleic Acid Res. 25: 3389-3402 https://doi.org/10.1093/nar/25.17.3389
  2. Baek, C. H. and K. S. Kim. 2003. lacZ- and aph- Based reporter vectors for in vivo expression technology. J. Micribiol. Biotechnol. 13: 872-880
  3. Bina, J. E. and J. J. Mekalanos. 2001. Vibrio Cholerae tolC is required for bile resistance and colonization. Infect. Immun. 69: 4681-4685 https://doi.org/10.1128/IAI.69.7.4681-4685.2001
  4. Camilli, A., D. Beattie, and J. J. Mekalanos. 1995. Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 8: 143-157 https://doi.org/10.1111/j.1365-2958.1993.tb01211.x
  5. Cangelosi, G. A., E. A. Best, C. Martinetti, and E. W. Nester. 1991. Genetic analysis of Agrobacterium. Methods Enzymol. 204: 384-397 https://doi.org/10.1016/0076-6879(91)04020-O
  6. Chen, C. Y. et al. 2003. Comparative genome analysis of Vibrio vulnificus, a marine pathogen. Genome Res. 13: 2577-2587 https://doi.org/10.1101/gr.1295503
  7. Chiang, S. L., J. J. Mekalanos, and D. W. Holden. 1999. In vivo genetic analysis of bacterial virulence. Annu. Rev. Microbiol. 53: 129-154 https://doi.org/10.1146/annurev.micro.53.1.129
  8. De Lorenzo, V., M. Herrero, U. Jakubzik, and K. N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172: 6568- 6572 https://doi.org/10.1128/jb.172.11.6568-6572.1990
  9. Gray, L. D. and A. S. Kreger. 1985. Purification and characterization of extracellular cytolysin produced by Vibrio vulnificus. Infect. Immune. 48: 62-72
  10. Heidelberg, J. F. et al. 2002. DNA sequence of both chromosomes of the cholerae pathogen Vibrio cholerae. Nature 406: 477-483 https://doi.org/10.1038/35020000
  11. Hunt, M. L., D. J. Boucher, J. D. Boyce, and B. Adler. 2001. In vivo-expressed genes of Pasteurella multocida. Infect. Iimmun. 69: 3004-3012 https://doi.org/10.1128/IAI.69.5.3004-3012.2001
  12. Janakiraman, A. and J. M. Slauch. 2000. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium. Mol. Microbiol. 35: 1146-1155 https://doi.org/10.1046/j.1365-2958.2000.01783.x
  13. Jeong, H. S., J. E. Rhee, J. H. Lee, H. K. Choi, D. I. Kim, M. H. Lee, S. J. Park, and S. H. Choi. 2003 Identification of Vibrio vulnificus lrp and Its Influence on Survival under Various Stresses. J. Microbiol. Biotechnol 13: 159-163
  14. Kaniga, K., I. Dellor, and G. R. Cornelis. 1991. A wide-hostrange suicide vector for improving reverse genetics in Gramnegative bacteria: In activation of the blaA gene of Yersinia enterocolitica. Gene 20: 137-141
  15. Keen, N. T., S. Takami, and D. Kobayashi, and D. Trollinger. 1988. Improved broad-host range plasmids for DNA cloning in Gram-negative bacteria. Gene 70: 191-197 https://doi.org/10.1016/0378-1119(88)90117-5
  16. Lee, S. H., S. M. Butler, and A. Camilli. 2001. Selection for in vivo regulators of bacterial virulence. Proc. Nalt. Acad. Sci. USA 98: 6889-6894
  17. Lim, M. S., M. H. Lee, J. H. Lee, H. M. Ju, N. Y. Park, H. S. Jeong, J. E. Rhee, and S. H. Choi. 2005. Identification and Characterization of the Vibrio vulnificus malPQ Operon. J. Microbiol. Biotechnol. 15: 616-625
  18. Litwin, C. M., T. W. Rayback, and J. Skinner. 1996. Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect. Immun. 64: 2834-2838
  19. Lowe, A. M., D. T. Beattie, and R. L. Deresiewicz. 1998. Identification of novel staphylococcal virulence genes by in vivo expression technology. Mol. Microbiol. 27: 967-976 https://doi.org/10.1046/j.1365-2958.1998.00741.x
  20. Mahan, M. J., J. M. Slauch, and J. J. Mekalanos. 1993. Selection of bacterial virulence genes that are specifically induced in host tissue. Science 259: 686-688 https://doi.org/10.1126/science.8430319
  21. Makino, K. et al. 2003.Genome sequences of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of Vibrio cholerae. Lancet 361: 743-749 https://doi.org/10.1016/S0140-6736(03)12659-1
  22. McDougald, D., S. A. Rice, and S. Kjelleberg. 2000. The marine pathogen Vibrio vulnificus encodes a putative homologue of the Vibrio harveyi regulatory gene, luxR: A genetic and phylogenetic comparoson. Gene 248: 213-221 https://doi.org/10.1016/S0378-1119(00)00117-7
  23. Milton, D. L., R. O'Toole, P. Horstedt, and H. Wolf-Watz. 1996. Flagellin A in essential for the virulence of Vibrio angullarum. J. Bacteriol. 178: 1310-1319 https://doi.org/10.1128/jb.178.5.1310-1319.1996
  24. Park, K. J., S. H. H. Kim, M. G. Kim, D. H. Chung, S. D. Ha, K. S. Kim, D. J. Jahng, and K. H. Lee. 2004. Functional Complementation of Escherichia coli by the rpoS Gene of the Foodborne Pathogenic Vibrio vulnificus. J. Microbiol. Biotechnol. 14: 1063-1066
  25. Parsot, C., E. Taxman, and J. J. Mekalanos. 1991. ToxR regulates the production of lipoproteins and the expression of serum resistance in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 88: 1641-1645
  26. Reddy, G. P., U. Hayat, C. Abeygunawardana, C. Fox, A. C. Wright, D. R. Maneval, Jr., C. A. Bush, and J. G. Morris, Jr. 1992. Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus MO6-24. J. Bacterial. 174: 2620-2630
  27. Rhee, J. E., H. M. Ju, U. R. Park, B. C. Park, and S. H. Choi. 2004. Identification of the Vibrio vulnificus cadC and evaluation of its role in acid tolerance. J. Microbiol. Biotechnol. 14: 1093-1098
  28. Sambrook, J. and D. W. Russell. 2001. Molecular cloning: A laboratory manual, 3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y
  29. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/ Technology 1: 784-791 https://doi.org/10.1038/nbt1183-784
  30. Simpson, L. M., V. K. White, S. F. Zane, and J. D. Oliver. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect. Immun. 55: 269-272
  31. Sutcliff, I. C. and D. J. Harrington. 2002. Pattern searches for the identification of putative lipoprotein genes in Grampositive bacterial genomes. Microbiology 148: 2065-2077 https://doi.org/10.1099/00221287-148-7-2065
  32. Testa, J., L. W. Daniel, and A. S. Kreger. 1984. Extracellular phospholipase A2 and lysophospholipase produced by Vibrio vulnificus. Infect. Immun. 45: 777-793
  33. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Research 22: 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  34. Wang, J., A. Mushegian, S. Lory, and S. Jin. 1996. Largescale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection. Proc. Natl. Acad. Sci. USA 93: 10434-10438
  35. Young, G. M. and V. L. Miller. 1997. Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. Mol. Micriobiol. 25: 319-328 https://doi.org/10.1046/j.1365-2958.1997.4661829.x