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. 1995 Dec;63(12):4721–4728. doi: 10.1128/iai.63.12.4721-4728.1995

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

J P Nataro 1, J Seriwatana 1, A Fasano 1, D R Maneval 1, L D Guers 1, F Noriega 1, F Dubovsky 1, M M Levine 1, J G Morris Jr 1
PMCID: PMC173677  PMID: 7591128

Abstract

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.

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Selected References

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  1. Allaoui A., Sansonetti P. J., Parsot C. MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins. Mol Microbiol. 1993 Jan;7(1):59–68. doi: 10.1111/j.1365-2958.1993.tb01097.x. [DOI] [PubMed] [Google Scholar]
  2. Andrews G. P., Hromockyj A. E., Coker C., Maurelli A. T. Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens. Infect Immun. 1991 Jun;59(6):1997–2005. doi: 10.1128/iai.59.6.1997-2005.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Fasano A., Kay B. A., Russell R. G., Maneval D. R., Jr, Levin M. M. Enterotoxin and cytotoxin production by enteroinvasive Escherichia coli. Infect Immun. 1990 Nov;58(11):3717–3723. doi: 10.1128/iai.58.11.3717-3723.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fasano A., Noriega F. R., Maneval D. R., Jr, Chanasongcram S., Russell R., Guandalini S., Levine M. M. Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro. J Clin Invest. 1995 Jun;95(6):2853–2861. doi: 10.1172/JCI117991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Goldberg M. B., Sansonetti P. J. Shigella subversion of the cellular cytoskeleton: a strategy for epithelial colonization. Infect Immun. 1993 Dec;61(12):4941–4946. doi: 10.1128/iai.61.12.4941-4946.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hale T. L. Genetic basis of virulence in Shigella species. Microbiol Rev. 1991 Jun;55(2):206–224. doi: 10.1128/mr.55.2.206-224.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  10. Kinsey M. D., Formal S. B., Dammin G. J., Giannella R. A. Fluid and electrolyte transport in rhesus monkeys challenged intracecally with Shigella flexneri 2a. Infect Immun. 1976 Aug;14(2):368–371. doi: 10.1128/iai.14.2.368-371.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kotloff K. L., Herrington D. A., Hale T. L., Newland J. W., Van De Verg L., Cogan J. P., Snoy P. J., Sadoff J. C., Formal S. B., Levine M. M. Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen. Infect Immun. 1992 Jun;60(6):2218–2224. doi: 10.1128/iai.60.6.2218-2224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Levine M. M., Bergquist E. J., Nalin D. R., Waterman D. H., Hornick R. B., Young C. R., Sotman S. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet. 1978 May 27;1(8074):1119–1122. doi: 10.1016/s0140-6736(78)90299-4. [DOI] [PubMed] [Google Scholar]
  13. Lin K. H., Cheng S. Y. An efficient method to purify active eukaryotic proteins from the inclusion bodies in Escherichia coli. Biotechniques. 1991 Dec;11(6):748, 750, 752-3. [PubMed] [Google Scholar]
  14. Marston F. A., Hartley D. L. Solubilization of protein aggregates. Methods Enzymol. 1990;182:264–276. doi: 10.1016/0076-6879(90)82022-t. [DOI] [PubMed] [Google Scholar]
  15. Rout W. R., Formal S. B., Giannella R. A., Dammin G. J. Pathophysiology of Shigella diarrhea in the rhesus monkey: intestinal transport, morphological, and bacteriological studies. Gastroenterology. 1975 Feb;68(2):270–278. [PubMed] [Google Scholar]
  16. Sansonetti P. J., Hale T. L., Dammin G. J., Kapfer C., Collins H. H., Jr, Formal S. B. Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri. Infect Immun. 1983 Mar;39(3):1392–1402. doi: 10.1128/iai.39.3.1392-1402.1983. [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. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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