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. 1996 Jul 1;15(13):3315–3321.

Rho-dependent membrane folding causes Shigella entry into epithelial cells.

T Adam 1, M Giry 1, P Boquet 1, P Sansonetti 1
PMCID: PMC451894  PMID: 8670832

Abstract

The small GTPase rho is functionally involved in the formation of cytoskeletal structures like stress fibers or focal adhesion plaques. Shigella entry into HeLa cells induces a blossom-like membrane structure at the bacterial entry site. We show here that this membrane-folding process is rho-dependent. The three rho isoforms were recruited into bacterial entry sites with differential localization relative to the membrane structure. A rho-specific inhibitor abolished Shigella-induced membrane folding and impaired bacterial entry accordingly. S1-myosin labeling indicated that rho was involved in Shigella-induced actin polymerization but not actin nucleation in the bacterial invasion site. This provides a major link in the signalization cascade allowing entry of a bacterial pathogen into a eukaryotic cell.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  2. Adam T., Arpin M., Prévost M. C., Gounon P., Sansonetti P. J. Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells. J Cell Biol. 1995 Apr;129(2):367–381. doi: 10.1083/jcb.129.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aktories K., Rösener S., Blaschke U., Chhatwal G. S. Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. Eur J Biochem. 1988 Mar 1;172(2):445–450. doi: 10.1111/j.1432-1033.1988.tb13908.x. [DOI] [PubMed] [Google Scholar]
  4. Aktories K., Weller U., Chhatwal G. S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 1987 Feb 9;212(1):109–113. doi: 10.1016/0014-5793(87)81566-1. [DOI] [PubMed] [Google Scholar]
  5. Aullo P., Giry M., Olsnes S., Popoff M. R., Kocks C., Boquet P. A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly. EMBO J. 1993 Mar;12(3):921–931. doi: 10.1002/j.1460-2075.1993.tb05733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braun U., Habermann B., Just I., Aktories K., Vandekerckhove J. Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product. FEBS Lett. 1989 Jan 16;243(1):70–76. doi: 10.1016/0014-5793(89)81220-7. [DOI] [PubMed] [Google Scholar]
  7. Bromberg Y., Shani E., Joseph G., Gorzalczany Y., Sperling O., Pick E. The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages. J Biol Chem. 1994 Mar 11;269(10):7055–7058. [PubMed] [Google Scholar]
  8. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clerc P., Sansonetti P. J. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun. 1987 Nov;55(11):2681–2688. doi: 10.1128/iai.55.11.2681-2688.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Einberger H., Mertz R., Hofschneider P. H., Neubert W. J. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro. J Virol. 1990 Sep;64(9):4274–4280. doi: 10.1128/jvi.64.9.4274-4280.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galán J. E., Pace J., Hayman M. J. Involvement of the epidermal growth factor receptor in the invasion of cultured mammalian cells by Salmonella typhimurium. Nature. 1992 Jun 18;357(6379):588–589. doi: 10.1038/357588a0. [DOI] [PubMed] [Google Scholar]
  12. Giannini G., Rappuoli R., Ratti G. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 1984 May 25;12(10):4063–4069. doi: 10.1093/nar/12.10.4063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54. doi: 10.1146/annurev.cb.10.110194.000335. [DOI] [PubMed] [Google Scholar]
  14. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  15. Hori Y. Function of the post-translationally modified C-terminal region of rho p21. Kobe J Med Sci. 1992 Apr;38(2):79–92. [PubMed] [Google Scholar]
  16. Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones B. D., Paterson H. F., Hall A., Falkow S. Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10390–10394. doi: 10.1073/pnas.90.21.10390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Just I., Fritz G., Aktories K., Giry M., Popoff M. R., Boquet P., Hegenbarth S., von Eichel-Streiber C. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J Biol Chem. 1994 Apr 8;269(14):10706–10712. [PubMed] [Google Scholar]
  19. Jähner D., Hunter T. The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts. Mol Cell Biol. 1991 Jul;11(7):3682–3690. doi: 10.1128/mcb.11.7.3682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kikuchi A., Yamamoto K., Fujita T., Takai Y. ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1. J Biol Chem. 1988 Nov 5;263(31):16303–16308. [PubMed] [Google Scholar]
  21. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  22. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem. 1993 Nov 25;268(33):24535–24538. [PubMed] [Google Scholar]
  24. Labigne-Roussel A. F., Lark D., Schoolnik G., Falkow S. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect Immun. 1984 Oct;46(1):251–259. doi: 10.1128/iai.46.1.251-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lang P., Guizani L., Vitté-Mony I., Stancou R., Dorseuil O., Gacon G., Bertoglio J. ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity. J Biol Chem. 1992 Jun 15;267(17):11677–11680. [PubMed] [Google Scholar]
  26. Leffers H., Nielsen M. S., Andersen A. H., Honoré B., Madsen P., Vandekerckhove J., Celis J. E. Identification of two human Rho GDP dissociation inhibitor proteins whose overexpression leads to disruption of the actin cytoskeleton. Exp Cell Res. 1993 Dec;209(2):165–174. doi: 10.1006/excr.1993.1298. [DOI] [PubMed] [Google Scholar]
  27. Narumiya S., Sekine A., Fujiwara M. Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. J Biol Chem. 1988 Nov 25;263(33):17255–17257. [PubMed] [Google Scholar]
  28. Nemoto Y., Namba T., Kozaki S., Narumiya S. Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli. J Biol Chem. 1991 Oct 15;266(29):19312–19319. [PubMed] [Google Scholar]
  29. Nishiyama T., Sasaki T., Takaishi K., Kato M., Yaku H., Araki K., Matsuura Y., Takai Y. rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells. Mol Cell Biol. 1994 Apr;14(4):2447–2456. doi: 10.1128/mcb.14.4.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  31. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  32. Olofsson B., Chardin P., Touchot N., Zahraoui A., Tavitian A. Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues. Oncogene. 1988 Aug;3(2):231–234. [PubMed] [Google Scholar]
  33. Paterson H. F., Self A. J., Garrett M. D., Just I., Aktories K., Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990 Sep;111(3):1001–1007. doi: 10.1083/jcb.111.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ridley A. J., Hall A. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J. 1994 Jun 1;13(11):2600–2610. doi: 10.1002/j.1460-2075.1994.tb06550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  36. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  37. Rubin E. J., Gill D. M., Boquet P., Popoff M. R. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol Cell Biol. 1988 Jan;8(1):418–426. doi: 10.1128/mcb.8.1.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sansonetti P. J. Genetic and molecular basis of epithelial cell invasion by Shigella species. Rev Infect Dis. 1991 Mar-Apr;13 (Suppl 4):S285–S292. doi: 10.1093/clinids/13.supplement_4.s285. [DOI] [PubMed] [Google Scholar]
  39. Sekine A., Fujiwara M., Narumiya S. Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem. 1989 May 25;264(15):8602–8605. [PubMed] [Google Scholar]
  40. Takaishi K., Sasaki T., Kato M., Yamochi W., Kuroda S., Nakamura T., Takeichi M., Takai Y. Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene. 1994 Jan;9(1):273–279. [PubMed] [Google Scholar]
  41. Tilney L. G., Portnoy D. A. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol. 1989 Oct;109(4 Pt 1):1597–1608. doi: 10.1083/jcb.109.4.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Uchida T., Pappenheimer A. M., Jr, Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J Biol Chem. 1973 Jun 10;248(11):3838–3844. [PubMed] [Google Scholar]
  43. Van Doren K., Hanahan D., Gluzman Y. Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA. J Virol. 1984 May;50(2):606–614. doi: 10.1128/jvi.50.2.606-614.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang J., King W. G., Dillon S., Hall A., Feig L., Rittenhouse S. E. Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. J Biol Chem. 1993 Oct 25;268(30):22251–22254. [PubMed] [Google Scholar]

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