Abstract
The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hägele S, Köhler R, Merkert H, Schleicher M et al (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
Farbrother P, Wagner C, Na J, Tunggal B et al (2006) Dictyostelium transcriptional host cell response upon infection with Legionella. Cell Microbiol 8:438–456
Clarke M (2010) Recent insights into host-pathogen interactions from Dictyostelium. Cell Microbiol 12:283–291
Bozzaro S, Eichinger L (2011) The professional phagocyte Dictyostelium discoideum as a model host for bacterial pathogens. Curr Drug Targets 12:942–954
Steinert M (2011) Pathogen-host interactions in Dictyostelium, Legionella, Mycobacterium and other pathogens. Semin Cell Dev Biol 22:70–76
Hempstead AD, Isberg RR (2013) Host signal transduction and protein kinases implicated in Legionella infection. Curr Top Microbiol Immunol 376:249–269
Peracino B, Buracco S, Bozzaro S (2013) The Nramp (Slc11) proteins regulate development, resistance to pathogenic bacteria and iron homeostasis in Dictyostelium discoideum. J Cell Sci 126:301–311
Steiner B, Weber S, Hilbi H (2018) Formation of the Legionella-containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics. Int J Med Microbiol 308(1):49–57
Koller B, Schramm C, Siebert S, Triebel J et al (2016) Dictyostelium discoideum as a novel host system to study the interaction between phagocytes and yeasts. Front Microbiol 7:1665
Hillmann F, Novohradská S, Mattern DJ, Forberger T et al (2015) Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation. Environ Microbiol 17:2858–2869
Steenbergen JN, Nosanchuk JD, Malliaris SD, Casadevall A (2003) Cryptococcus neoformans virulence is enhanced after growth in the genetically malleable host Dictyostelium discoideum. Infect Immun 71:4862–4872
Lima WC, Lelong E, Cosson P (2011) What can Dictyostelium bring to the study of Pseudomonas infections? Semin Cell Dev Biol 22:77–81
Lima WC, Balestrino D, Forestier C, Cosson P (2014) Two distinct sensing pathways allow recognition of Klebsiella pneumoniae by Dictyostelium amoebae. Cell Microbiol 16:311–323
Sillo A, Matthias J, Konertz R, Bozzaro S et al (2011) Salmonella typhimurium is pathogenic for Dictyostelium cells and subverts the starvation response. Cell Microbiol 13:1793–1811
Barisch C, Soldati T (2017) Mycobacterium marinum degrades both triacylglycerols and phospholipids from its Dictyostelium host to synthesise its own triacylglycerols and generate lipid inclusions. PLoS Pathog 13:e1006095
Brenz Y, Winther-Larsen HC, Hagedorn M (2017) Expanding Francisella models: pairing up the soil amoeba Dictyostelium with aquatic Francisella. Int J Med Microbiol
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
Maniak M (2011) Dictyostelium as a model for human lysosomal and trafficking diseases. Semin Cell Dev Biol 22:114–119
Lu H, Clarke M (2005) Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007
Fajardo M, Schleicher M, Noegel A, Bozzaro S et al (2004) Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum. Microbiology 150:2825–2835
Li Z, Dugan AS, Bloomfield G, Skelton J et al (2009) The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA. Cell Host Microbe 6:253–267
Shevchuk O, Batzilla C, Hägele S, Kusch H et al (2009) Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium. Int J Med Microbiol 299:489–508
Urwyler S, Nyfeler Y, Ragaz C, Lee H et al (2009) Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases. Traffic 10:76–87
Weber SS, Ragaz C, Hilbi H (2009) The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell Microbiol 11:442–460
Weber SS, Ragaz C, Reus K, Nyfeler Y et al (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2:e46
Brombacher E, Urwyler S, Ragaz C, Weber SS et al (2009) Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila. J Biol Chem 284:4846–4856
Personnic N, Bärlocher K, Finsel I, Hilbi H (2016) Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol 24:450–462
Simon S, Hilbi H (2015) Subversion of cell-autonomous immunity and cell migration by Legionella pneumophila effectors. Front Immunol 6:447
Fey P, Dodson RJ, Basu S, Chisholm RL (2013) One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012. Methods Mol Biol 983:59–92
Bozzaro S, Peracino B, Eichinger L (2013) Dictyostelium host response to Legionella infection: strategies and assays. Methods Mol Biol 954:417–438
Müller-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 92:45–53
Swart AL, Harrison CF, Eichinger L, Steinert M et al (2018) Acanthamoeba and Dictyostelium as cellular models for Legionella infection. Front Cell Infect Microbiol 8:61
Mesquita A, Elena C-M, Dominguez E, Sandra M-B et al (2016) Autophagy in Dictyostelium: mechanisms, regulation and disease in a simple biomedical model. Autophagy:1–17
Sherwood R, Roy CR (2016) Autophagy evasion and endoplasmic reticulum subversion: the yin and yang of Legionella intracellular infection. Annu Rev Microbiol 70:413–433
Otto GP, Wu MY, Clarke M, Lu H et al (2004) Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum. Mol Microbiol 51:63–72
Tung SM, Unal C, Ley A, Peña C et al (2010) Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila. Cell Microbiol 12:765–780
Xiong Q, Ãœnal C, Matthias J, Steinert M et al (2015) The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions. Open Biol 5:150008
Choy A, Dancourt J, Mugo B, O’Connor TJ et al (2012) The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338:1072–1076
Rolando M, Escoll P, Nora T, Botti J et al (2016) Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy. Proc Natl Acad Sci U S A 113:1901–1906
Weber SS, Ragaz C, Hilbi H (2009) Pathogen trafficking pathways and host phosphoinositide metabolism. Mol Microbiol 71:1341–1352
Haneburger I, Hilbi H (2013) Phosphoinositide lipids and the Legionella pathogen vacuole. Curr Top Microbiol Immunol 376:155–173
Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443:651–657
Peracino B, Balest A, Bozzaro S (2010) Phosphoinositides differentially regulate bacterial uptake and Nramp1-induced resistance to Legionella infection in Dictyostelium. J Cell Sci 123:4039–4051
Riyahi TY, Frese F, Steinert M, Omosigho NN et al (2011) RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense. PLoS One 6:e27311
Raper KB (1951) Isolation, cultivation, and conservation of simple slime molds. Q Rev Biol 26:169–190
Gerisch G (1960) Zellfunktionen und Zellfunktionswechsel in der Entwicklung vonDictyostelium discoideum: Zellagglutination und Induktion der Fruchtkörperpolarität. Wilhelm Roux Arch Entwickl Mech Org 152:632–654
Fey P, Kowal AS, Gaudet P, Pilcher KE et al (2007) Protocols for growth and development of Dictyostelium discoideum. Nat Protoc 2:1307–1316
Froquet R, Lelong E, Marchetti A, Cosson P (2008) Dictyostelium discoideum: a model host to measure bacterial virulence. Nat Protoc 4:25–30
Sussman M (1966) Biochemical and genetic methods in the study of cellular slime mold development. In: Prescott D (ed) Methods in cell physiology. Academic, Cambridge, MA, pp 397–409
Franke J, Kessin R (1977) A defined minimal medium for axenic strains of Dictyostelium discoideum. Proc Natl Acad Sci U S A 74:2157–2161
Watts DJ, Ashworth JM (1970) Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J 119:171–174
Loomis WF (1971) Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp Cell Res 64:484–486
Maniak M (2001) Fluid-phase uptake and transit in axenic Dictyostelium cells. Biochim Biophys Acta 1525:197–204
Buracco S, Peracino B, Andreini C, Bozzaro S (2018) Differential effects of iron, but not zinc or copper, affects Dictyostelium discoideum cell growth and resistance to Legionella pneumophila. Front Cell Infect Microbiol 7:536
Sussman M (1987) Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. Methods Cell Biol 28:9–29
Laine J, Roxby N, Coukell MB (1975) A simple method for storing cellular slime mold amoebae. Can J Microbiol 21:959–962
Hilbi H, Segal G, Shuman HA (2001) Icm/dot-dependent upregulation of phagocytosis by Legionella pneumophila. Mol Microbiol 42:603–617
Hilbi H, Kortholt A (2017) Role of the small GTPase Rap1 in signal transduction, cell dynamics and bacterial infection. Small GTPases:1–7
Isaac DT, Laguna RK, Valtz N, Isberg RR (2015) MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth. Proc Natl Acad Sci U S A 112:E5208–E5217
Shevchuk O, Pägelow D, Rasch J, Döhrmann S et al (2014) Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection. Int J Med Microbiol 304:1169–1181
Peracino B, Wagner C, Balest A, Balbo A et al (2006) Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection. Traffic 7:22–38
Schmölders J, Manske C, Otto A, Hoffmann C et al (2017) Comparative proteomics of purified pathogen vacuoles correlates intracellular replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1). Mol Cell Proteomics 16:622–641
Tiaden ANN, Kessler A, Hilbi H (2013) Analysis of Legionella infection by flow cytometry. Methods Mol Biol 954:233–249
Eichinger L, Rivero F (2006) Methods in molecular biology—Dictyostelium discoideum protocols. Humana Press, Totowa, NJ
Weber S, Hilbi H (2014) Live cell imaging of phosphoinositide dynamics during Legionella infection. Methods Mol Biol 1197:153–167
Acknowledgments
This work was supported by the Compagnia San Paolo (12-CSP-C03-065) (SB), the Deutsche Forschungsgemeinschaft (TP01, SFB 670, Innate Immunity), and Köln Fortune (LE).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bozzaro, S., Buracco, S., Peracino, B., Eichinger, L. (2019). Dictyostelium Host Response to Legionella Infection: Strategies and Assays. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9048-1_23
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9047-4
Online ISBN: 978-1-4939-9048-1
eBook Packages: Springer Protocols