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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
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

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

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

Notes

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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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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