Legionella pp 191-204 | Cite as

Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment

  • Nicolas PersonnicEmail author
  • Bianca Striednig
  • Hubert Hilbi
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)


Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.

Key words

Acanthamoeba castellanii Legionella Single cell Infection Microcolony Agarose Embedment Flow chamber Fluorescent protein 



N-(2-acetamido)-2-aminoethanesulfonic acid


Green fluorescent protein


Intracellular multiplication/defective organelle trafficking


Legionella-containing vacuole


2-N-morpholino-ethanesulfonic acid


Multiplicity of infection


Type IV secretion system



This work was supported by a Swiss National Science Foundation (SNF) Ambizione Fellowship (PZ00P3_161492) awarded to N.P. and an SNF project grant (31003A_153200) awarded to H.H.


  1. 1.
    Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23:274–298CrossRefGoogle Scholar
  2. 2.
    Mampel J, Spirig T, Weber SS, Haagensen JAJ, Molin S, Hilbi H (2006) Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl Environ Microbiol 72:2885–2895CrossRefGoogle Scholar
  3. 3.
    Declerck P (2010) Biofilms: the environmental playground of Legionella pneumophila. Environ Microbiol 12:557–566CrossRefGoogle Scholar
  4. 4.
    Declerck P, Behets J, van Hoef V, Ollevier F (2007) Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments. Water Res 41:3159–3167CrossRefGoogle Scholar
  5. 5.
    Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71:20–28CrossRefGoogle Scholar
  6. 6.
    Steinert M, Heuner K (2005) Dictyostelium as host model for pathogenesis. Cell Microbiol 7:307–314CrossRefGoogle Scholar
  7. 7.
    Finsel I, Hilbi H (2015) Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones. Cell Microbiol 17:935–950CrossRefGoogle Scholar
  8. 8.
    Hilbi H, Haas A (2012) Secretive bacterial pathogens and the secretory pathway. Traffic 13:1187–1197CrossRefGoogle Scholar
  9. 9.
    Xu L, Luo ZQ (2013) Cell biology of infection by Legionella pneumophila. Microbes Infect 15:157–167CrossRefGoogle Scholar
  10. 10.
    Personnic N, Bärlocher K, Finsel I, Hilbi H (2016) Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol 24:450–462CrossRefGoogle Scholar
  11. 11.
    Molofsky AB, Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53:29–40CrossRefGoogle Scholar
  12. 12.
    Sauer JD, Bachman MA, Swanson MS (2005) The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proc Natl Acad Sci U S A 102:9924–9929CrossRefGoogle Scholar
  13. 13.
    Dalebroux ZD, Edwards RL, Swanson MS (2009) SpoT governs Legionella pneumophila differentiation in host macrophages. Mol Microbiol 71:640–658CrossRefGoogle Scholar
  14. 14.
    Edwards RL, Dalebroux ZD, Swanson MS (2009) Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence. Mol Microbiol 71:1190–1204CrossRefGoogle Scholar
  15. 15.
    Cosson P, Soldati T (2008) Eat, kill or die: when amoeba meets bacteria. Curr Opin Microbiol 11:271–276CrossRefGoogle Scholar
  16. 16.
    Siddiqui R, Khan NA (2012) Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5:6CrossRefGoogle Scholar
  17. 17.
    Trabelsi H, Dendana F, Sellami A, Sellami H, Cheikhrouhou F, Neji S, Makni F, Ayadi A (2012) Pathogenic free-living amoebae: epidemiology and clinical review. Pathol Biol 60:399–405CrossRefGoogle Scholar
  18. 18.
    Morales VM, Backman A, Bagdasarian M (1991) A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39–47CrossRefGoogle Scholar
  19. 19.
    Chen J, de Felipe KS, Clarke M, Lu H, Anderson OR, Segal G, Shuman HA (2004) Legionella effectors that promote nonlytic release from protozoa. Science 303:1358–1361CrossRefGoogle Scholar
  20. 20.
    Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64:2240–2246PubMedPubMedCentralGoogle Scholar
  21. 21.
    Blokpoel MC, O’Toole R, Smeulders MJ, Williams HD (2003) Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression. J Microbiol Meth 54:203–211CrossRefGoogle Scholar
  22. 22.
    Rogers JE, Jones GW, Engleberg NC (1993) Growth and phenotypic characterization of Legionella species on semisolid media made with washed agar. J Clin Microbiol 31:149–151PubMedPubMedCentralGoogle Scholar
  23. 23.
    Steiner B, Swart AL, Welin A, Weber S, Personnic N, Kaech A, Freyre C, Ziegler U, Klemm RW, Hilbi H (2017) ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila. EMBO Rep 18:1817–1836CrossRefGoogle Scholar
  24. 24.
    Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R, Buchrieser C, Hilbi H (2007) The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol 9:2903–2920CrossRefGoogle Scholar
  25. 25.
    Schell U, Simon S, Sahr T, Hager D, Albers MF, Kessler A, Fahrnbauer F, Trauner D, Hedberg C, Buchrieser C, Hilbi H (2016) The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila. Mol Microbiol 99:778–793CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nicolas Personnic
    • 1
    Email author
  • Bianca Striednig
    • 1
  • Hubert Hilbi
    • 1
  1. 1.Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland

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