1887

Abstract

The haploid amoeba is a versatile host system for studying cellular aspects of pathogenicity. Previous studies have shown that the internalization of leads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth of . Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of -infected host cells. In contrast to the control experiment with -containing phagosomes, both proteins decorated the replicative vacuole of during the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication of in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27111-0
2004-09-01
2019-11-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/9/mic1502825.html?itemId=/content/journal/micro/10.1099/mic.0.27111-0&mimeType=html&fmt=ahah

References

  1. Abu Kwaik, Y. ( 1996; ). The phagosome containing Legionella pneumophila within protozoan Hartmannella vermiformis is surrounded by the rough endoplasmatic reticulum. Appl Environ Microbiol 62, 2022–2028.
    [Google Scholar]
  2. Abu Kwaik, Y., Fields, B. S. & Engleberg, N. C. ( 1994; ). Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila. Infect Immun 62, 1860–1866.
    [Google Scholar]
  3. Abu Kwaik, Y. ( 1998; ). Fatal attraction of mammalian cells to Legionella. Mol Microbiol 30, 689–695.[CrossRef]
    [Google Scholar]
  4. Ahmed, S. A., Gogal, R. M. & Walsh, J. E. ( 1994; ). A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods 170, 211–224.[CrossRef]
    [Google Scholar]
  5. Berridge, M. J., Lipp, P. & Bootman, M. D. ( 2000; ). The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1, 11–21.
    [Google Scholar]
  6. Bers, D. M. ( 1982; ). A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am J Physiol 242, 404–408.
    [Google Scholar]
  7. Cardelli, J. ( 2001; ). Phagocytosis and macropinocytosis in Dictyostelium. Phosphoinositide-based processes, biochemically distinct. Traffic 2, 311–320.[CrossRef]
    [Google Scholar]
  8. Damiani, M. T. & Colombo, M. I. ( 2001; ). Involvement of heterotrimeric G protein in phagocytosis and recycling from the phagosomal compartment. Exp Cell Res 271, 189–199.[CrossRef]
    [Google Scholar]
  9. Dewitt, S. & Hallett, M. B. ( 2002; ). Cytosolic free Ca2+ changes and calpain activation are required for β integrin-accelerated phagocytosis by human neutrophils. J Cell Biol 159, 181–189.[CrossRef]
    [Google Scholar]
  10. Döring, V., Veretout, F., Albrecht, R., Mühlbauer, B., Schlatterer, C., Schleicher, M. & Noegel, A. A. ( 1995; ). The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca+2-regulated processes. J Cell Sci 108, 2065–2076.
    [Google Scholar]
  11. Eichinger, L., Lee, S. S. & Schleicher, M. ( 1999; ). Dictyostelium as a model system for studies of the actin cytoskeleton by molecular genetics. Microsc Res Tech 47, 124–134.[CrossRef]
    [Google Scholar]
  12. Garin, J., Diez, R., Kieffer, S., Dermine, J. F., Duyclos, S., Gagnon, E., Sadoul, R., Rondeau, C. & Desjardins, M. ( 2001; ). The phagosome proteome: insight into phagosome functions. J Cell Biol 152, 165–180.[CrossRef]
    [Google Scholar]
  13. Gloss, A., Rivero, F., Khaire, N., Müller, R., Loomis, W. F., Schleicher, M. & Noegel, A. A. ( 2003; ). Villidin, a novel WD-repeat and villidin-related protein from Dictyostelium, is associated with membranes and the cytoskeleton. Mol Biol Cell 14, 2716–2727.[CrossRef]
    [Google Scholar]
  14. Hägele, S., Köhler, R., Merkert, H., Schleicher, M., Hacker, J. & Steinert, M. ( 2000; ). Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2, 165–171.[CrossRef]
    [Google Scholar]
  15. Hammer, B. K., Tateda, E. S. & Swanson, M. S. ( 2002; ). A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila. Mol Microbiol 44, 107–118.[CrossRef]
    [Google Scholar]
  16. Haugwitz, M., Noegel, A. A., Karakesisoglou, J. & Schleicher, M. ( 1994; ). Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development. Cell 79, 303–314.[CrossRef]
    [Google Scholar]
  17. Hilbi, H., Segal, G. & Shuman, H. ( 2001; ). Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila. Mol Microbiol 42, 603–617.
    [Google Scholar]
  18. Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K., Ohkura, Y. & Ueno, K. ( 1996; ). A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 19, 1518–1520.[CrossRef]
    [Google Scholar]
  19. Jacob, T., Escallier, J. C., Sanguedolce, M. V. C., Chicheportiche, C., Bongran Capo, C. & Mege, J. L. ( 1994; ). Legionella pneumophila inhibits superoxide generation in human monocytes via the down-modulation of alpha and beta protein kinase C isotypes. J Leukocyte Biol 55, 310–312.
    [Google Scholar]
  20. Jaconi, M. E., Lew, D. P., Carpentier, J. L., Magnusson, K. E., Sjogren, M. & Stendahl, O. ( 1990; ). Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils. J Cell Biol 110, 1555–1564.[CrossRef]
    [Google Scholar]
  21. Kagan, J. C. & Roy, C. R. ( 2002; ). Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol 4, 945–954.[CrossRef]
    [Google Scholar]
  22. Khurana, B., Khurana, T., Khaire, N. & Noegel, A. A. ( 2002; ). Functions of LIM proteins in cell polarity and chemotactic motility. EMBO J 21, 5331–5342.[CrossRef]
    [Google Scholar]
  23. Köhler, R., Bubert, A., Goebel, W., Steinert, M., Hacker, J. & Bubert, B. ( 2000; ). Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila. Mol Gen Genet 262, 1060–1069.[CrossRef]
    [Google Scholar]
  24. Konzok, A., Weber, I., Simmeth, E., Hacker, U., Maniak, M. & Müller-Taubenberger, A. ( 1999; ). Daip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility. J Cell Biol 146, 453–464.[CrossRef]
    [Google Scholar]
  25. Malik, Z. A., Denning, G. M. & Kusner, D. J. ( 2000; ). Inhibition of Ca2+ signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages. J Exp Med 191, 287–302.[CrossRef]
    [Google Scholar]
  26. Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J. & Gerisch, G. ( 1995; ). Coronin involved in phagocytosis: dynamics of particle induced relocalization visualized by a green fluorescent protein tag. Cell 83, 915–924.[CrossRef]
    [Google Scholar]
  27. May, R. C. & Machesky, L. M. ( 2001; ). Phagocytosis and the actin cytoskeleton. J Cell Sci 114, 1061–1077.
    [Google Scholar]
  28. Müller, A., Hacker, J. & Brand, B. C. ( 1996; ). Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. Infect Immun 64, 4900–4906.
    [Google Scholar]
  29. Müller-Taubenberger, A., Lupas, A. N., Li, H., Ecke, M., Simmeth, E. & Gerisch, G. ( 2001; ). Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J 23, 6772–6782.
    [Google Scholar]
  30. Noegel, A. A. & Schleicher, M. ( 2000; ). The actin cytoskeleton of Dictyostelium: a story told by mutants. J Cell Sci 113, 759–766.
    [Google Scholar]
  31. Otto, G. P., Wu, M. Y., Clarke, M., Lu, H., Anderson, O. R., Hilbi, H., Shuman, H. A. & Kessin, R. H. ( 2004; ). Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum. Mol Microbiol 51, 63–72.
    [Google Scholar]
  32. Payne, N. R. & Horwitz, M. A. ( 1987; ). Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med 166, 1377–1389.[CrossRef]
    [Google Scholar]
  33. Peracino, B., Borleis, J., Jin, T. & 7 other authors ( 1998; ). G protein β subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton. J Cell Biol 141, 1529–1537.[CrossRef]
    [Google Scholar]
  34. Prassler, J., Murr, A., Stocker, S., Faix, J., Murphy, J. & Marriott, G. ( 1998; ). DdLim is a cytoskeleton-associated protein involved in protusion of lamellipodia in Dictyostelium. Mol Biol Cell 9, 545–559.[CrossRef]
    [Google Scholar]
  35. Rivero, F., Köppel, B., Peracino, B., Bozzaro, S., Siegert, F., Weijer, C. J., Schleicher, M., Albrecht, R. & Noegel, A. A. ( 1996; ). The cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress and ensure cell size, cell shape and motility and contribute to phagocytosis and development. J Cell Sci 109, 2679–2691.
    [Google Scholar]
  36. Roy, C. R. ( 2002; ). Exploitation of the endoplasmic reticulum by bacterial pathogens. Trends Microbiol 10, 418–424.[CrossRef]
    [Google Scholar]
  37. Roy, C. R. & Tilney, L. G. ( 2002; ). The road less traveled: transport of Legionella to the endoplasmic reticulum. J Cell Biol 158, 415–419.[CrossRef]
    [Google Scholar]
  38. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory.
  39. Seastone, D. J., Zhang, L., Buczynski, G., Rebstein, P., Weeks, G., Spiegelman, G. & Cardelli, J. ( 1999; ). The small Mr Ra-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum. Mol Biol Cell 10, 393–406.[CrossRef]
    [Google Scholar]
  40. Segal, G., Russo, J. J. & Shuman, H. A. ( 1999; ). Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila. Mol Microbiol 34, 799–809.[CrossRef]
    [Google Scholar]
  41. Shelling, J. G. & Sykes, B. D. ( 1985; ). 1H nuclear magnetic resonance study of the two calcium-binding sites of porcine intestinal calcium-binding protein. J Biol Chem 260, 8342–8347.
    [Google Scholar]
  42. Skriwan, C., Fajardo, M., Hägele, S. & 7 other authors ( 2002; ). Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum. Int J Med Microbiol 291, 615–624.[CrossRef]
    [Google Scholar]
  43. Solomon, J. M. & Isberg, R. R. ( 2000; ). Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbiol 10, 478–480.
    [Google Scholar]
  44. Solomon, J. M., Rupper, A., Cardelli, J. A. & Isberg, R. R. ( 2000; ). Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect Immun 68, 2939–2947.[CrossRef]
    [Google Scholar]
  45. Steinert, M., Hentschel, U. & Hacker, J. ( 2002; ). Legionella pneumophila: an aquatic microbe goes astray. FEMS Microbiol Rev 743, 1–14.
    [Google Scholar]
  46. Swanson, M. S. & Hammer, B. K. ( 2000; ). Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54, 567–613.[CrossRef]
    [Google Scholar]
  47. Swanson, M. S. & Isberg, R. R. ( 1995; ). Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun 63, 3609–3620.
    [Google Scholar]
  48. Vardar, D., Chisti, A., Frank, B. S., Luna, E., Noegel, A. A., Oh, S. W., Schleicher, M. & McKnight, J. C. ( 2002; ). Villin-type headpiece domains show a wide range of F-actin binding affinities. Cell Motil Cytoskel 52, 9–21.[CrossRef]
    [Google Scholar]
  49. Venkataraman, C., Gao, L. Y., Bondada, S. & Abu Kwaik, Y. ( 1998; ). Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila. J Exp Med 188, 505–514.[CrossRef]
    [Google Scholar]
  50. Wintermeyer, E., Ludwig, B., Steinert, M., Schmidt, B., Fischer, G. & Hacker, J. ( 1995; ). Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect Immun 63, 4576–4583.
    [Google Scholar]
  51. Yamamoto, Y., Klein, T. W., Shinomia, B., Nakano, M. & Friedman, H. ( 1992; ). Infection of macrophages with Legionella pneumophila induces phosphorylation of a 76-kilodalton protein. Infect Immun 60, 3452–3455.
    [Google Scholar]
  52. Yumura, S., Mori, H. & Fukui, Y. ( 1984; ). Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence. J Cell Biol 99, 894–899.[CrossRef]
    [Google Scholar]
  53. Zink, S. D., Pederson, L., Cianciotto, N. P. & Abu-Kwaik, Y. ( 2002; ). The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages. Infect Immun 70, 1657–1663.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27111-0
Loading
/content/journal/micro/10.1099/mic.0.27111-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error