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. 1988 Sep;85(18):6682–6686. doi: 10.1073/pnas.85.18.6682

Cultured mammalian cells attach to the invasin protein of Yersinia pseudotuberculosis.

R R Isberg 1, J M Leong 1
PMCID: PMC282041  PMID: 3413117

Abstract

The expression of invasin, the product of the Yersinia pseudotuberculosis inv gene, allows enteric bacteria to enter cultured mammalian cells. The ability of invasin to bind animal cells and the potential significance of this interaction in the entry process were investigated. It was found that HEp-2 cells could attach to surfaces coated with bacterial membranes containing invasin. By fractionating bacterial membrane proteins on NaDodSO4/polyacrylamide gels and transferring the protein to filters, we demonstrated that the cell-binding component of the membranes comigrated with invasin. Mutations that changed the electrophoretic mobility of the protein also caused a corresponding shift in the migration of the cell-binding activity, showing that the comigrating protein was indeed invasin. Monoclonal antibodies directed against invasin that blocked invasin-HEp-2 cell interaction also inhibited bacteria from penetrating HEp-2 cells, indicating that interaction of this protein with animal cells is critical for cellular penetration.

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Selected References

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  1. Bell M. L., Engvall E. The specific detection of collagenous proteins after electrophoresis using enzyme-conjugated collagen-binding fibronectin fragments. Anal Biochem. 1982 Jul 1;123(2):329–335. doi: 10.1016/0003-2697(82)90454-7. [DOI] [PubMed] [Google Scholar]
  2. Bovallius A., Nilsson G. Ingestion and survival of Y. pseudotuberculosis in HeLa cells. Can J Microbiol. 1975 Dec;21(12):1997–2007. doi: 10.1139/m75-287. [DOI] [PubMed] [Google Scholar]
  3. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carter P. B. Pathogenecity of Yersinia enterocolitica for mice. Infect Immun. 1975 Jan;11(1):164–170. doi: 10.1128/iai.11.1.164-170.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Civin C. I., Banquerigo M. L. Cloning of murine hybridoma cells in ultra-low gelation temperature agarose. Methods Enzymol. 1986;121:322–327. doi: 10.1016/0076-6879(86)21030-7. [DOI] [PubMed] [Google Scholar]
  6. Devenish J. A., Schiemann D. A. HeLa cell infection by Yersinia enterocolitica: evidence for lack of intracellular multiplication and development of a new procedure for quantitative expression of infectivity. Infect Immun. 1981 Apr;32(1):48–55. doi: 10.1128/iai.32.1.48-55.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Falkow S., Small P., Isberg R., Hayes S. F., Corwin D. A molecular strategy for the study of bacterial invasion. Rev Infect Dis. 1987 Sep-Oct;9 (Suppl 5):S450–S455. doi: 10.1093/clinids/9.supplement_5.s450. [DOI] [PubMed] [Google Scholar]
  8. Fields P. I., Swanson R. V., Haidaris C. G., Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5189–5193. doi: 10.1073/pnas.83.14.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Formal S. B., Hale T. L., Sansonetti P. J. Invasive enteric pathogens. Rev Infect Dis. 1983 Sep-Oct;5 (Suppl 4):S702–S707. doi: 10.1093/clinids/5.supplement_4.s702. [DOI] [PubMed] [Google Scholar]
  10. Gemski P., Lazere J. R., Casey T., Wohlhieter J. A. Presence of a virulence-associated plasmid in Yersinia pseudotuberculosis. Infect Immun. 1980 Jun;28(3):1044–1047. doi: 10.1128/iai.28.3.1044-1047.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hayman E. G., Engvall E., A'Hearn E., Barnes D., Pierschbacher M., Ruoslahti E. Cell attachment on replicas of SDS polyacrylamide gels reveals two adhesive plasma proteins. J Cell Biol. 1982 Oct;95(1):20–23. doi: 10.1083/jcb.95.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Isberg R. R., Swain A., Falkow S. Analysis of expression and thermoregulation of the Yersinia pseudotuberculosis inv gene with hybrid proteins. Infect Immun. 1988 Aug;56(8):2133–2138. doi: 10.1128/iai.56.8.2133-2138.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Kumamoto C. A., Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. doi: 10.1128/jb.154.1.253-260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. 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]
  18. Landegren U. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol Methods. 1984 Mar 16;67(2):379–388. doi: 10.1016/0022-1759(84)90477-0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Pierschbacher M., Hayman E. G., Ruoslahti E. Synthetic peptide with cell attachment activity of fibronectin. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1224–1227. doi: 10.1073/pnas.80.5.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ruoslahti E., Hayman E. G., Pierschbacher M., Engvall E. Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol. 1982;82(Pt A):803–831. doi: 10.1016/0076-6879(82)82103-4. [DOI] [PubMed] [Google Scholar]
  23. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  24. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. doi: 10.1128/iai.51.2.461-469.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Smit J., Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol. 1975 Nov;124(2):942–958. doi: 10.1128/jb.124.2.942-958.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taggart R. T., Samloff I. M. Stable antibody-producing murine hybridomas. Science. 1983 Mar 11;219(4589):1228–1230. doi: 10.1126/science.6402815. [DOI] [PubMed] [Google Scholar]
  30. Takeuchi A., Sprinz H. Electron-Microscope Studies of Experimental Salmonella Infection in the Preconditioned Guinea Pig: II. Response of the Intestinal Mucosa to the Invasion by Salmonella typhimurium. Am J Pathol. 1967 Jul;51(1):137–161. [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Underwood P. A., Kelly J. F., Harman D. F., MacMillan H. M. Use of protein A to remove immunoglobulins from serum in hybridoma culture media. J Immunol Methods. 1983 May 27;60(1-2):33–45. doi: 10.1016/0022-1759(83)90332-0. [DOI] [PubMed] [Google Scholar]

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