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. 1993 Dec 15;90(24):11890–11894. doi: 10.1073/pnas.90.24.11890

Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells.

R A Brundage 1, G A Smith 1, A Camilli 1, J A Theriot 1, D A Portnoy 1
PMCID: PMC48090  PMID: 8265643

Abstract

Movement of Listeria monocytogenes within infected eukaryotic cells provides a simple model system to study the mechanism of actin-based motility in nonmuscle cells. The actA gene of L. monocytogenes is required to induce the polymerization of host actin filaments [Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1990) Cell 68, 521-531; Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wachter, M., Wuenscher, M. & Chakraborty, T. (1992) EMBO J. 11, 1981-1990]. In this study, an in-frame deletion mutation within the actA gene was constructed and introduced into the L. monocytogenes chromosome by allelic exchange. This mutation resulted in a decrease (3 orders of magnitude) in virulence for mice. In tissue culture cells, the actA mutant was absolutely defective for the nucleation of actin filaments and consequently was impaired in cell-to-cell spread. Antiserum raised to a synthetic peptide encompassing the proline-rich repeat (DFPPPPTDEEL) of ActA was used to characterize the expression of the ActA protein. The ActA protein derived from extracellular bacteria migrated as a 97-kDa polypeptide upon SDS/PAGE, whereas the protein from infected cells migrated as three distinct polypeptides, one that comigrated with the 97-kDa extracellular form and two slightly larger species. Treatment of infected cells with okadaic acid resulted in decreased amounts of all forms of ActA and the appearance of a larger species of ActA. Phosphatase treatment of ActA immunoprecipitated from intracellular bacteria resulted in conversion of the larger two species to the 97-kDa form. Labeling of infected cells with 32Pi followed by immunoprecipitation showed that the largest molecular form of ActA was phosphorylated. Taken together, these data indicate that ActA is phosphorylated during intracellular growth. The significance of the intracellular modification of ActA is not known, but we speculate that it may modulate the intracellular activity of ActA.

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Selected References

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

  1. Bernardini M. L., Mounier J., d'Hauteville H., Coquis-Rondon M., Sansonetti P. J. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A. 1989 May;86(10):3867–3871. doi: 10.1073/pnas.86.10.3867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Camilli A., Tilney L. G., Portnoy D. A. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol. 1993 Apr;8(1):143–157. doi: 10.1111/j.1365-2958.1993.tb01211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cohen P., Holmes C. F., Tsukitani Y. Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci. 1990 Mar;15(3):98–102. doi: 10.1016/0968-0004(90)90192-e. [DOI] [PubMed] [Google Scholar]
  4. Dabiri G. A., Sanger J. M., Portnoy D. A., Southwick F. S. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6068–6072. doi: 10.1073/pnas.87.16.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeister-Wächter M., Wuenscher M., Chakraborty T. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 1992 May;11(5):1981–1990. doi: 10.1002/j.1460-2075.1992.tb05252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeister-Wächter M., Wuenscher M., Chakraborty T. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 1992 May;11(5):1981–1990. doi: 10.1002/j.1460-2075.1992.tb05252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell. 1991 Jun 28;65(7):1127–1141. doi: 10.1016/0092-8674(91)90009-n. [DOI] [PubMed] [Google Scholar]
  8. Gaillard J. L., Berche P., Mounier J., Richard S., Sansonetti P. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun. 1987 Nov;55(11):2822–2829. doi: 10.1128/iai.55.11.2822-2829.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geoffroy C., Raveneau J., Beretti J. L., Lecroisey A., Vazquez-Boland J. A., Alouf J. E., Berche P. Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes. Infect Immun. 1991 Jul;59(7):2382–2388. doi: 10.1128/iai.59.7.2382-2388.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldberg M. B., Bârzu O., Parsot C., Sansonetti P. J. Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. J Bacteriol. 1993 Apr;175(8):2189–2196. doi: 10.1128/jb.175.8.2189-2196.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldfine H., Johnston N. C., Knob C. Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes. J Bacteriol. 1993 Jul;175(14):4298–4306. doi: 10.1128/jb.175.14.4298-4306.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Headley V. L., Payne S. M. Differential protein expression by Shigella flexneri in intracellular and extracellular environments. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4179–4183. doi: 10.1073/pnas.87.11.4179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heinzen R. A., Hayes S. F., Peacock M. G., Hackstadt T. Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells. Infect Immun. 1993 May;61(5):1926–1935. doi: 10.1128/iai.61.5.1926-1935.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kadurugamuwa J. L., Rohde M., Wehland J., Timmis K. N. Intercellular spread of Shigella flexneri through a monolayer mediated by membranous protrusions and associated with reorganization of the cytoskeletal protein vinculin. Infect Immun. 1991 Oct;59(10):3463–3471. doi: 10.1128/iai.59.10.3463-3471.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaufmann S. H. Immunity to intracellular bacteria. Annu Rev Immunol. 1993;11:129–163. doi: 10.1146/annurev.iy.11.040193.001021. [DOI] [PubMed] [Google Scholar]
  16. Kocks C., Gouin E., Tabouret M., Berche P., Ohayon H., Cossart P. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell. 1992 Feb 7;68(3):521–531. doi: 10.1016/0092-8674(92)90188-i. [DOI] [PubMed] [Google Scholar]
  17. Kocks C., Hellio R., Gounon P., Ohayon H., Cossart P. Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly. J Cell Sci. 1993 Jul;105(Pt 3):699–710. doi: 10.1242/jcs.105.3.699. [DOI] [PubMed] [Google Scholar]
  18. Makino S., Sasakawa C., Kamata K., Kurata T., Yoshikawa M. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell. 1986 Aug 15;46(4):551–555. doi: 10.1016/0092-8674(86)90880-9. [DOI] [PubMed] [Google Scholar]
  19. Marquis H., Bouwer H. G., Hinrichs D. J., Portnoy D. A. Intracytoplasmic growth and virulence of Listeria monocytogenes auxotrophic mutants. Infect Immun. 1993 Sep;61(9):3756–3760. doi: 10.1128/iai.61.9.3756-3760.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mounier J., Ryter A., Coquis-Rondon M., Sansonetti P. J. Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2. Infect Immun. 1990 Apr;58(4):1048–1058. doi: 10.1128/iai.58.4.1048-1058.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Niebuhr K., Chakraborty T., Rohde M., Gazlig T., Jansen B., Köllner P., Wehland J. Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin "comets". Infect Immun. 1993 Jul;61(7):2793–2802. doi: 10.1128/iai.61.7.2793-2802.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Portnoy D. A., Chakraborty T., Goebel W., Cossart P. Molecular determinants of Listeria monocytogenes pathogenesis. Infect Immun. 1992 Apr;60(4):1263–1267. doi: 10.1128/iai.60.4.1263-1267.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Portnoy D. A., Jacks P. S., Hinrichs D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med. 1988 Apr 1;167(4):1459–1471. doi: 10.1084/jem.167.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Portnoy D. A., Tweten R. K., Kehoe M., Bielecki J. Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells. Infect Immun. 1992 Jul;60(7):2710–2717. doi: 10.1128/iai.60.7.2710-2717.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pál T., Newland J. W., Tall B. D., Formal S. B., Hale T. L. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect Immun. 1989 Feb;57(2):477–486. doi: 10.1128/iai.57.2.477-486.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger J. M., Sanger J. W., Southwick F. S. Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes. Infect Immun. 1992 Sep;60(9):3609–3619. doi: 10.1128/iai.60.9.3609-3619.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sawin K. E., Mitchison T. J., Wordeman L. G. Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J Cell Sci. 1992 Feb;101(Pt 2):303–313. doi: 10.1242/jcs.101.2.303. [DOI] [PubMed] [Google Scholar]
  28. Sun A. N., Camilli A., Portnoy D. A. Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect Immun. 1990 Nov;58(11):3770–3778. doi: 10.1128/iai.58.11.3770-3778.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tanaka M., Shibata H. Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin. Eur J Biochem. 1985 Sep 2;151(2):291–297. doi: 10.1111/j.1432-1033.1985.tb09099.x. [DOI] [PubMed] [Google Scholar]
  30. Teysseire N., Chiche-Portiche C., Raoult D. Intracellular movements of Rickettsia conorii and R. typhi based on actin polymerization. Res Microbiol. 1992 Nov-Dec;143(9):821–829. doi: 10.1016/0923-2508(92)90069-z. [DOI] [PubMed] [Google Scholar]
  31. Theriot J. A., Mitchison T. J. The nucleation-release model of actin filament dynamics in cell motility. Trends Cell Biol. 1992 Aug;2(8):219–222. doi: 10.1016/0962-8924(92)90298-2. [DOI] [PubMed] [Google Scholar]
  32. Theriot J. A., Mitchison T. J., Tilney L. G., Portnoy D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 1992 May 21;357(6375):257–260. doi: 10.1038/357257a0. [DOI] [PubMed] [Google Scholar]
  33. Tilney L. G., DeRosier D. J., Weber A., Tilney M. S. How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper. J Cell Biol. 1992 Jul;118(1):83–93. doi: 10.1083/jcb.118.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vazquez-Boland J. A., Kocks C., Dramsi S., Ohayon H., Geoffroy C., Mengaud J., Cossart P. Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun. 1992 Jan;60(1):219–230. doi: 10.1128/iai.60.1.219-230.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. d'Hauteville H., Sansonetti P. J. Phosphorylation of IcsA by cAMP-dependent protein kinase and its effect on intracellular spread of Shigella flexneri. Mol Microbiol. 1992 Apr;6(7):833–841. doi: 10.1111/j.1365-2958.1992.tb01534.x. [DOI] [PubMed] [Google Scholar]

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