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. 1988 Jan;56(1):225–229. doi: 10.1128/iai.56.1.225-229.1988

Identification of regions on a 230-kilobase plasmid from enteroinvasive Escherichia coli that are required for entry into HEp-2 cells.

P L Small 1, S Falkow 1
PMCID: PMC259260  PMID: 2826335

Abstract

Certain strains of Escherichia coli can cause an invasive diarrheal disease in humans which clinically resembles shigellosis. These strains share with Shigella species the ability to enter and replicate within colonic epithelial cells and the ability to bind Congo red dye in vitro when grown at 37 degrees C. Like shigellae, they contain a large plasmid essential for virulence. A 230-kilobase (kb) plasmid from enteroinvasive E. coli was genetically marked with a transposon and mobilized into an E. coli K-12 background. This plasmid conferred upon E. coli K-12 the ability to enter and multiply within cultured epithelial cells, as well as the ability to bind Congo red. Expression of these phenotypes required growth at 37 degrees C. Transposon mutagenesis was used to identify regions on the 230-kb plasmid required for virulence. All transposon insertions which resulted in loss of the ability to enter epithelial cells, as well as the ability to bind Congo red dye, were mapped to a single 25-kb BamHI fragment. Subclones from this 25-kb region were tested for the ability to complement invasion in noninvasive derivatives. A subclone containing about 8 kb of the left end of the 25-kb BamHI fragment was capable of complementing noninvasive mutants with Tn5 insertions in this region and restored to these noninvasive mutants the ability to enter epithelial cells.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boileau C. R., d'Hauteville H. M., Sansonetti P. J. DNA hybridization technique to detect Shigella species and enteroinvasive escherichia coli. J Clin Microbiol. 1984 Nov;20(5):959–961. doi: 10.1128/jcm.20.5.959-961.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradley D. E., Taylor D. E., Cohen D. R. Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12. J Bacteriol. 1980 Sep;143(3):1466–1470. doi: 10.1128/jb.143.3.1466-1470.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DuPont H. L., Formal S. B., Hornick R. B., Snyder M. J., Libonati J. P., Sheahan D. G., LaBrec E. H., Kalas J. P. Pathogenesis of Escherichia coli diarrhea. N Engl J Med. 1971 Jul 1;285(1):1–9. doi: 10.1056/NEJM197107012850101. [DOI] [PubMed] [Google Scholar]
  5. Formal S. B., Hornick R. B. Invasive Escherichia coli. J Infect Dis. 1978 May;137(5):641–644. doi: 10.1093/infdis/137.5.641. [DOI] [PubMed] [Google Scholar]
  6. Hale T. L., Sansonetti P. J., Schad P. A., Austin S., Formal S. B. Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli. Infect Immun. 1983 Apr;40(1):340–350. doi: 10.1128/iai.40.1.340-350.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harris J. R., Wachsmuth I. K., Davis B. R., Cohen M. L. High-molecular-weight plasmid correlates with Escherichia coli enteroinvasiveness. Infect Immun. 1982 Sep;37(3):1295–1298. doi: 10.1128/iai.37.3.1295-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Maurelli A. T., Baudry B., d'Hauteville H., Hale T. L., Sansonetti P. J. Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect Immun. 1985 Jul;49(1):164–171. doi: 10.1128/iai.49.1.164-171.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Maurelli A. T., Blackmon B., Curtiss R., 3rd Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun. 1984 Jan;43(1):397–401. doi: 10.1128/iai.43.1.397-401.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maurelli A. T., Blackmon B., Curtiss R., 3rd Temperature-dependent expression of virulence genes in Shigella species. Infect Immun. 1984 Jan;43(1):195–201. doi: 10.1128/iai.43.1.195-201.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Robinson M. K., Bennett P. M., Falkow S., Dodd H. M. Isolation of a temperature-sensitive derivative of RP1. Plasmid. 1980 May;3(3):343–347. doi: 10.1016/0147-619x(80)90047-5. [DOI] [PubMed] [Google Scholar]
  12. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  13. Sakai T., Sasakawa C., Makino S., Kamata K., Yoshikawa M. Molecular cloning of a genetic determinant for Congo red binding ability which is essential for the virulence of Shigella flexneri. Infect Immun. 1986 Feb;51(2):476–482. doi: 10.1128/iai.51.2.476-482.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sakazaki R., Tamura K., Nakamura A. Further studies on enteropathogenic Escherichia coli associated with diarrhoeal diseases in children and adults. Jpn J Med Sci Biol. 1974 Feb;27(1):7–18. doi: 10.7883/yoken1952.27.7. [DOI] [PubMed] [Google Scholar]
  15. Sakazaki R., Tamura K., Saito M. Enteropathogenic Escherichia coli associated with diarrhea in children and adults. Jpn J Med Sci Biol. 1967 Oct;20(5):387–399. doi: 10.7883/yoken1952.20.387. [DOI] [PubMed] [Google Scholar]
  16. Sansonetti P. J., Kopecko D. J., Formal S. B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun. 1982 Mar;35(3):852–860. doi: 10.1128/iai.35.3.852-860.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sansonetti P. J., d'Hauteville H., Formal S. B., Toucas M. Plasmid-mediated invasiveness of "Shigella-like" Escherichia coli. Ann Microbiol (Paris) 1982 May-Jun;133(3):351–355. [PubMed] [Google Scholar]
  18. Sasakawa C., Kamata K., Sakai T., Murayama S. Y., Makino S., Yoshikawa M. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun. 1986 Feb;51(2):470–475. doi: 10.1128/iai.51.2.470-475.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Silva R. M., Toledo M. R., Trabulsi L. R. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980 May;11(5):441–444. doi: 10.1128/jcm.11.5.441-444.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Small P. L., Isberg R. R., Falkow S. Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells. Infect Immun. 1987 Jul;55(7):1674–1679. doi: 10.1128/iai.55.7.1674-1679.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. Toledo M. R., Reis M. H., Almeida R. G., Trabulsi L. R. Invasive strain of Escherichia coli belonging to O group 29. J Clin Microbiol. 1979 Feb;9(2):288–289. doi: 10.1128/jcm.9.2.288-289.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yakobson E. A., Guiney D. G., Jr Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5. J Bacteriol. 1984 Oct;160(1):451–453. doi: 10.1128/jb.160.1.451-453.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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