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. 1995 Nov;63(11):4470–4475. doi: 10.1128/iai.63.11.4470-4475.1995

Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10.

S H Huang 1, C Wass 1, Q Fu 1, N V Prasadarao 1, M Stins 1, K S Kim 1
PMCID: PMC173636  PMID: 7591087

Abstract

Most cases of neonatal Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. In an attempt to identify E. coli structures contributing to invasion into the central nervous system (CNS), TnphoA mutagenesis was performed with an invasive CSF isolate of E. coli K1 strain RS218 (O18:K1:H7), and TnphoA mutants were examined for their noninvasive capability in brain microvascular endothelial cells (BMEC). The noninvasive mutants exhibited the invasive ability of < 1% of the parent strain. One of the noninvasive mutants (10A-23) with a single TnphoA insertion and no changes in phenotypic characteristics was found to be significantly less invasive into the CNS in the newborn rat model of hematogenous E. coli meningitis. The TnphoA inserts with flanking sequences were cloned and sequenced. A novel open reading frame (8.2 kDa) was identified. Open reading frame analysis indicated that the 8.2-kDa protein (Ibe10) contained multiple transmembrane domains. ibe10 was cloned into an expression vector, pQE30, and the purified Ibe10 was shown to inhibit invasion of BMEC by strain RS218. These findings indicate that ibe10 is one of the E. coli genes involved in the invasion of BMEC in vitro and in vivo.

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Selected References

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  1. Bliska J. B., Galán J. E., Falkow S. Signal transduction in the mammalian cell during bacterial attachment and entry. Cell. 1993 Jun 4;73(5):903–920. doi: 10.1016/0092-8674(93)90270-z. [DOI] [PubMed] [Google Scholar]
  2. Coque J. J., Liras P., Martín J. F. Genes for a beta-lactamase, a penicillin-binding protein and a transmembrane protein are clustered with the cephamycin biosynthetic genes in Nocardia lactamdurans. EMBO J. 1993 Feb;12(2):631–639. doi: 10.1002/j.1460-2075.1993.tb05696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Crocker P. R., Mucklow S., Bouckson V., McWilliam A., Willis A. C., Gordon S., Milon G., Kelm S., Bradfield P. Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains. EMBO J. 1994 Oct 3;13(19):4490–4503. doi: 10.1002/j.1460-2075.1994.tb06771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cross A. S., Kim K. S., Wright D. C., Sadoff J. C., Gemski P. Role of lipopolysaccharide and capsule in the serum resistance of bacteremic strains of Escherichia coli. J Infect Dis. 1986 Sep;154(3):497–503. doi: 10.1093/infdis/154.3.497. [DOI] [PubMed] [Google Scholar]
  5. Deverson E. V., Gow I. R., Coadwell W. J., Monaco J. J., Butcher G. W., Howard J. C. MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters. Nature. 1990 Dec 20;348(6303):738–741. doi: 10.1038/348738a0. [DOI] [PubMed] [Google Scholar]
  6. Donnenberg M. S., Yu J., Kaper J. B. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol. 1993 Aug;175(15):4670–4680. doi: 10.1128/jb.175.15.4670-4680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duchêne M., Schweizer A., Lottspeich F., Krauss G., Marget M., Vogel K., von Specht B. U., Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol. 1988 Jan;170(1):155–162. doi: 10.1128/jb.170.1.155-162.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Falkow S., Isberg R. R., Portnoy D. A. The interaction of bacteria with mammalian cells. Annu Rev Cell Biol. 1992;8:333–363. doi: 10.1146/annurev.cb.08.110192.002001. [DOI] [PubMed] [Google Scholar]
  9. Gladstone I. M., Ehrenkranz R. A., Edberg S. C., Baltimore R. S. A ten-year review of neonatal sepsis and comparison with the previous fifty-year experience. Pediatr Infect Dis J. 1990 Nov;9(11):819–825. doi: 10.1097/00006454-199011000-00009. [DOI] [PubMed] [Google Scholar]
  10. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  12. Hsia R. C., Small P. L., Bavoil P. M. Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells. J Bacteriol. 1993 Aug;175(15):4817–4823. doi: 10.1128/jb.175.15.4817-4823.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang S. H., Tang A., Drisco B., Zhang S. Q., Seeger R., Li C., Jong A. Human dTMP kinase: gene expression and enzymatic activity coinciding with cell cycle progression and cell growth. DNA Cell Biol. 1994 May;13(5):461–471. doi: 10.1089/dna.1994.13.461. [DOI] [PubMed] [Google Scholar]
  14. Isberg R. R., Falkow S. A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature. 1985 Sep 19;317(6034):262–264. doi: 10.1038/317262a0. [DOI] [PubMed] [Google Scholar]
  15. Isberg R. R., Leong J. M. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell. 1990 Mar 9;60(5):861–871. doi: 10.1016/0092-8674(90)90099-z. [DOI] [PubMed] [Google Scholar]
  16. Isberg R. R., Voorhis D. L., Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769–778. doi: 10.1016/0092-8674(87)90335-7. [DOI] [PubMed] [Google Scholar]
  17. Kim K. S., Itabashi H., Gemski P., Sadoff J., Warren R. L., Cross A. S. The K1 capsule is the critical determinant in the development of Escherichia coli meningitis in the rat. J Clin Invest. 1992 Sep;90(3):897–905. doi: 10.1172/JCI115965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim K. S., Kang J. H., Cross A. S., Kaufman B., Zollinger W., Sadoff J. Functional activities of monoclonal antibodies to the O side chain of Escherichia coli lipopolysaccharides in vitro and in vivo. J Infect Dis. 1988 Jan;157(1):47–53. doi: 10.1093/infdis/157.1.47. [DOI] [PubMed] [Google Scholar]
  19. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Prasadarao N. V., Wass C. A., Hacker J., Jann K., Kim K. S. Adhesion of S-fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae. J Biol Chem. 1993 May 15;268(14):10356–10363. [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stins M. F., Prasadarao N. V., Ibric L., Wass C. A., Luckett P., Kim K. S. Binding characteristics of S fimbriated Escherichia coli to isolated brain microvascular endothelial cells. Am J Pathol. 1994 Nov;145(5):1228–1236. [PMC free article] [PubMed] [Google Scholar]
  25. Sunahara S., Muramoto K., Tenma K., Kamiya H. Amino acid sequence of two sea anemone toxins from Anthopleura fuscoviridis. Toxicon. 1987;25(2):211–219. doi: 10.1016/0041-0101(87)90243-1. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Unhanand M., Mustafa M. M., McCracken G. H., Jr, Nelson J. D. Gram-negative enteric bacillary meningitis: a twenty-one-year experience. J Pediatr. 1993 Jan;122(1):15–21. doi: 10.1016/s0022-3476(05)83480-8. [DOI] [PubMed] [Google Scholar]
  28. Weiser J. N., Gotschlich E. C. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun. 1991 Jul;59(7):2252–2258. doi: 10.1128/iai.59.7.2252-2258.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wilmes-Riesenberg M. R., Wanner B. L. TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization. J Bacteriol. 1992 Jul;174(14):4558–4575. doi: 10.1128/jb.174.14.4558-4575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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