Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment
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 wordsAcanthamoeba castellanii Legionella Single cell Infection Microcolony Agarose Embedment Flow chamber Fluorescent protein
Green fluorescent protein
Intracellular multiplication/defective organelle trafficking
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.
- 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