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

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Legionella

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1921))

Abstract

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.

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Abbreviations

ACES:

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

GFP:

Green fluorescent protein

Icm/Dot:

Intracellular multiplication/defective organelle trafficking

LCV:

Legionella-containing vacuole

MES:

2-N-morpholino-ethanesulfonic acid

MOI:

Multiplicity of infection

T4SS:

Type IV secretion system

References

  1. Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23:274–298

    Article  CAS  Google Scholar 

  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 72:2885–2895

    Article  CAS  Google Scholar 

  3. Declerck P (2010) Biofilms: the environmental playground of Legionella pneumophila. Environ Microbiol 12:557–566

    Article  CAS  Google Scholar 

  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 41:3159–3167

    Article  CAS  Google Scholar 

  5. Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71:20–28

    Article  CAS  Google Scholar 

  6. Steinert M, Heuner K (2005) Dictyostelium as host model for pathogenesis. Cell Microbiol 7:307–314

    Article  CAS  Google Scholar 

  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 17:935–950

    Article  CAS  Google Scholar 

  8. Hilbi H, Haas A (2012) Secretive bacterial pathogens and the secretory pathway. Traffic 13:1187–1197

    Article  CAS  Google Scholar 

  9. Xu L, Luo ZQ (2013) Cell biology of infection by Legionella pneumophila. Microbes Infect 15:157–167

    Article  CAS  Google Scholar 

  10. Personnic N, Bärlocher K, Finsel I, Hilbi H (2016) Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol 24:450–462

    Article  CAS  Google Scholar 

  11. Molofsky AB, Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53:29–40

    Article  CAS  Google Scholar 

  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 102:9924–9929

    Article  CAS  Google Scholar 

  13. Dalebroux ZD, Edwards RL, Swanson MS (2009) SpoT governs Legionella pneumophila differentiation in host macrophages. Mol Microbiol 71:640–658

    Article  CAS  Google Scholar 

  14. Edwards RL, Dalebroux ZD, Swanson MS (2009) Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence. Mol Microbiol 71:1190–1204

    Article  Google Scholar 

  15. Cosson P, Soldati T (2008) Eat, kill or die: when amoeba meets bacteria. Curr Opin Microbiol 11:271–276

    Article  CAS  Google Scholar 

  16. Siddiqui R, Khan NA (2012) Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5:6

    Article  Google Scholar 

  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 60:399–405

    Article  CAS  Google Scholar 

  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 97:39–47

    Article  CAS  Google Scholar 

  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 303:1358–1361

    Article  CAS  Google Scholar 

  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 64:2240–2246

    CAS  PubMed  PubMed Central  Google Scholar 

  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 54:203–211

    Article  CAS  Google Scholar 

  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 31:149–151

    CAS  PubMed  PubMed Central  Google Scholar 

  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 18:1817–1836

    Article  CAS  Google Scholar 

  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 9:2903–2920

    Article  CAS  Google Scholar 

  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 99:778–793

    Article  CAS  Google Scholar 

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Acknowledgments

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.

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Authors and Affiliations

  1. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Nicolas Personnic, Bianca Striednig & Hubert Hilbi

Authors
  1. Nicolas Personnic
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  2. Bianca Striednig
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  3. Hubert Hilbi
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Corresponding author

Correspondence to Nicolas Personnic .

Editor information

Editors and Affiliations

  1. Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France

    Carmen Buchrieser

  2. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Hubert Hilbi

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Personnic, N., Striednig, B., Hilbi, H. (2019). Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment. 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_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9048-1_12

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  • 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

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