Live Imaging of Mycobacterium marinum Infection in Dictyostelium discoideum

  • Caroline Barisch
  • Ana T. López-Jiménez
  • Thierry SoldatiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)


The Dictyostelium discoideumMycobacterium marinum host–pathogen system is a recently established and powerful model system for mycobacterial infection. In this chapter, two simple protocols for live imaging of Dictyostelium discoideum infection are described. The first method is used to monitor the dynamics of recruitment of GFP-tagged Dictyostelium discoideum proteins at single time-points corresponding to the main stages of the infection (1.5–72 h post infection). The second method focuses at the early stages of the establishment of an infection (0–3 h post infection). In addition, several procedures to improve the imaging of the bacterium-containing compartment are described. Basic bacterial parameters such as bacterial growth and the recruitment of host proteins to the bacterium-containing compartment can be easily and precisely quantified using macros for ImageJ. These methods can be adapted to monitoring mycobacteria infection in other systems using mammalian cells.

Key words

Dictyostelium discoideum Mycobacteria Intracellular infection Live microscopy Fluorescence 



The TS laboratory is supported by multiple grants from the Swiss National Science Foundation.


  1. 1.
    Muller-Taubenberger A, Kortholt A, Eichinger L (2013) Simple system–substantial share: the use of Dictyostelium in cell biology and molecular medicine. Eur J Cell Biol 92(2):45–53CrossRefPubMedGoogle Scholar
  2. 2.
    Faix J, Linkner J, Nordholz B, Platt JL, Liao XH, Kimmel AR (2013) The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci. Methods Mol Biol 983:249–267CrossRefPubMedGoogle Scholar
  3. 3.
    Boulais J, Trost M, Landry CR, Dieckmann R, Levy ED, Soldati T, Michnick SW, Thibault P, Desjardins M (2010) Molecular characterization of the evolution of phagosomes. Mol Syst Biol 6:423CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Clarke M, Maddera L, Engel U, Gerisch G (2010) Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging. PLoS One 5(1):e8585CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Carnell M, Zech T, Calaminus SD, Ura S, Hagedorn M, Johnston SA, May RC, Soldati T, Machesky LM, Insall RH (2011) Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis. J Cell Biol 193(5):831–839CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Muller-Taubenberger A, Ishikawa-Ankerhold HC (2013) Fluorescent reporters and methods to analyze fluorescent signals. Methods Mol Biol 983:93–112CrossRefPubMedGoogle Scholar
  7. 7.
    Jia K, Thomas C, Akbar M, Sun Q, Adams-Huet B, Gilpin C, Levine B (2009) Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance. Proc Natl Acad Sci U S A 106(34):14564–14569CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Alibaud L, Kohler 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(3):729–740CrossRefPubMedGoogle Scholar
  9. 9.
    Fuchs BB, Mylonakis E (2006) Using non-mammalian hosts to study fungal virulence and host defense. Curr Opin Microbiol 9(4):346–351CrossRefPubMedGoogle Scholar
  10. 10.
    Miyata ST, Kitaoka M, Brooks TM, McAuley SB, Pukatzki S (2011) Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun 79(7):2941–2949CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hoffmann C, Harrison CF, Hilbi H (2013) The natural alternative: protozoa as cellular models for Legionella infection. Cell Microbiol 16:15–26CrossRefGoogle Scholar
  12. 12.
    Harenberg J, Giese C, Hagedorn A, Traeger I, Fenyvesi T (2007) Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time. Semin Thromb Hemost 33(5):503–507CrossRefPubMedGoogle Scholar
  13. 13.
    Hagedorn M, Rohde KH, Russell DG, Soldati T (2009) Infection by tubercular mycobacteria is spread by nonlytic ejection from their amoeba hosts. Science 323(5922):1729–1733CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Weber S, Wagner M, Hilbi H (2013) Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection. mBio 5(1):e00839-13CrossRefGoogle Scholar
  15. 15.
    Tonjum T, Welty DB, Jantzen E, Small PL (1998) Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis. J Clin Microbiol 36(4):918–925PubMedPubMedCentralGoogle Scholar
  16. 16.
    Tobin DM, Ramakrishnan L (2008) Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis. Cell Microbiol 10(5):1027–1039CrossRefPubMedGoogle Scholar
  17. 17.
    Cosma CL, Sherman DR, Ramakrishnan L (2003) The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol 57:641–676CrossRefPubMedGoogle Scholar
  18. 18.
    MacGurn JA, Cox JS (2007) A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect Immun 75(6):2668–2678CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Deretic V, Singh S, Master S, Kyei G, Davis A, Naylor J, de Haro S, Harris J, Delgado M, Roberts E, Vergne I (2007) Phosphoinositides in phagolysosome and autophagosome biogenesis. Biochem Soc Symp 74:141–148CrossRefGoogle Scholar
  20. 20.
    Seto S, Tsujimura K, Koide Y (2011) Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes. Traffic 12(4):407–420CrossRefPubMedGoogle Scholar
  21. 21.
    Bloomfield G, Tanaka Y, Skelton J, Ivens A, Kay RR (2008) Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum. Genome Biol 9(4):R75CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    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(6):3578–3586CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Collins TJ (2007) ImageJ for microscopy. Biotechniques 43(1 Suppl):25–30CrossRefPubMedGoogle Scholar
  24. 24.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefPubMedGoogle Scholar
  25. 25.
    Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T (2010) Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS One 5(3):e9823CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Santi I, Dhar N, Bousbaine D, Wakamoto Y, McKinney JD (2013) Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria. Nat Commun 4:2470PubMedGoogle Scholar
  27. 27.
    King JS, Veltman DM, Insall RH (2011) The induction of autophagy by mechanical stress. Autophagy 7(12):1490–1499CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Garrod DR, Born GV (1971) Effect of temperature on the mutual adhesion of preaggregation cells of the slime mould, Dictyostelium discoideum. J Cell Sci 8(3):751–765PubMedGoogle Scholar
  29. 29.
    Glynn PJ (1981) A quantitative study of the phagocytosis of Escherichia coli by myxamoebae of the slime mould Dictyostelium discoideum. Cytobios 30(119):153–166PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Caroline Barisch
    • 1
  • Ana T. López-Jiménez
    • 1
  • Thierry Soldati
    • 1
    Email author
  1. 1.Département de Biochimie, Faculté des SciencesUniversité de Genève, Sciences IIGenève-4Switzerland

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