Abstract
Dictyostelium discoideum cells are “professional phagocytes,” as they ingest a large variety of bacteria, yeast, and inert particles. Several bacterial pathogens are able to survive intracellularly within specialized vacuoles of D. discoideum by interfering with host signaling pathways. To better understand the molecular mechanisms underlying these evolutionary conserved processes we have established a method for the isolation of pathogen-containing vacuoles (PCVs). The isolation protocol describes the infection of D. discoideum cells with the intracellular pathogen Legionella pneumophila, loading of the lysosomal compartment with colloidal iron, mechanical lysis of host cells, iodophenylnitrophenyltetrazolium (INT) heavy labeling of mitochondria, removal of nucleic acid by Benzonase treatment, separation of nuclei by low-speed centrifugation, and the magnetic removal of lysosomes. The subcellular fractionation in a discontinuous sucrose density OptiPrep gradient allows the separation of mitochondria and to prepare PCVs with high purity. The proteins isolated from PCVs have been successfully subjected to mass spectrometry and allowed to analyze pathogen-directed maturation processes of vacuoles. The method can also be applied for subsequent protein modification analyses and lipidome comparisons.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bozzaro S, Bucci C, Steinert M (2008) Phagocytosis and host-pathogen interactions in Dictyostelium with a look at macrophages. Int Rev Cell Mol Biol 271:253–300
Steinert M (2011) Pathogen-host interactions in Dictyostelium, Legionella, Mycobacterium and other pathogens. Semin Cell Dev Biol 22:70–76
Cosson P, Zulianello L, Join-Lambert O, Faurisson F, Gebbie L, Benghezal M, van Delden C, Curty LK, Kohler T (2002) Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. J Bacteriol 184:3027–3033
Alibaud L, Köhler T, Coudray A, Prigent-Combaret C, Bergeret E, Perrin J, Benghezal M, Reimmann C, Gauthier Y, van Delden C, Attree I, Fauvarque MO, Cosson P (2008) Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model. Cell Microbiol 10:729–740
Pukatzki S, Kessin RH, Mekalanos JJ (2002) The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc Natl Acad Sci U S A 99:3159–3164
Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci USA 103:1528–1533
Skriwan C, Fajardo M, Hägele S, Horn M, Wagner M, Michel R, Krohne G, Schleicher M, Hacker J, Steinert M (2002) Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum. Int J Med Microbiol 291:615–624
Solomon JM, Leung GS, Isberg RR (2003) Intracellular replication of Mycobacterium marinum within Dictyostelium discoideum: efficient replication in the absence of host coronin. Infect Immun 71:3578–3586
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
Solomon JM, Isberg RR (2000) Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbiol 8:478–480
Solomon JM, Rupper A, Cardelli JA, Isberg RR (2000) Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect Immun 68:2939–2947
Hasselbring BM, Patel MK, Schell MA (2011) Dictyostelium discoideum as a model system for identification of Burkholderia pseudomallei virulence factors. Infect Immun 79: 2079–2088
Horwitz MA (1983) The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med 158:2108–2126
Roy CR, Tilney LG (2002) The road less traveled: transport of Legionella to the endoplasmic reticulum. J Cell Biol 158:415–419
Desjardins M (1995) Biogenesis of phagolysosomes: the “kiss and run” hypothesis. Trends Cell Biol 5:183–186
Via LE, Deretic D, Ulmer RJ, Hibler NS, Huber LA, Deretic V (1997) Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem 272:13326–13331
Parish RW (1975) Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes. Eur J Biochem 58:523–531
Zhang C, Kuspa A (2009) Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection. PLoS One 4:e5706
Shevchuk O, Batzilla C, Hägele S, Kusch H, Engelmann S, Hecker M, Haas A, Heuner K, Glockner G, Steinert M (2009) Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium. Int J Med Microbiol 299:489–508
Rodriguez-Paris JM, Nolta KV, Steck TL (1993) Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation. J Biol Chem 268:9110–9116
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Shevchuk, O., Steinert, M. (2013). Isolation of Pathogen-Containing Vacuoles. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology, vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_23
Download citation
DOI: https://doi.org/10.1007/978-1-62703-302-2_23
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-301-5
Online ISBN: 978-1-62703-302-2
eBook Packages: Springer Protocols