Chapter 7
Novel Single-Cell and High-Throughput Microscopy
Techniques to Monitor Dictyostelium
discoideum–Mycobacterium marinum Infection Dynamics
Manon Mottet, Cristina Bosmani, Nabil Hanna, Jahn Nitschke,
Louise H. Lefranc ¸ ois, and Thierry Soldati
Abstract
The Dictyostelium discoideum–Mycobacterium marinum host–pathogen system is a well-established and
powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three
microscopy methods that allow the precise identification and quantification of very diverse phenotypes
arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use
the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the
infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the
various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was
established to study the infection dynamics with high-throughput imaging of a large number of cells at the
different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline
allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single
D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered
assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of
infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and
quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can
adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity
conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning
disc confocal microscope.
Key words Dictyostelium discoideum, Mycobacterium marinum, Intracellular infection, Live microscopy, Single cell, Developmental cycle, High-throughput microscopy, Phenotypic heterogeneity
1 Introduction
Dictyostelium discoideum is a social amoeba that has emerged as a
powerful model system to study conserved mechanisms of host
defence involved in many human diseases caused by bacteria, as
well as mechanisms of bacterial pathogenicity. The main and highly
conserved cell-autonomous defence is constituted by the
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183
Novel Single-Cell and High-Throughput Microscopy
Techniques to Monitor Dictyostelium
discoideum–Mycobacterium marinum Infection Dynamics
Manon Mottet, Cristina Bosmani, Nabil Hanna, Jahn Nitschke,
Louise H. Lefranc ¸ ois, and Thierry Soldati
Abstract
The Dictyostelium discoideum–Mycobacterium marinum host–pathogen system is a well-established and
powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three
microscopy methods that allow the precise identification and quantification of very diverse phenotypes
arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use
the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the
infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the
various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was
established to study the infection dynamics with high-throughput imaging of a large number of cells at the
different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline
allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single
D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered
assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of
infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and
quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can
adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity
conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning
disc confocal microscope.
Key words Dictyostelium discoideum, Mycobacterium marinum, Intracellular infection, Live microscopy, Single cell, Developmental cycle, High-throughput microscopy, Phenotypic heterogeneity
1 Introduction
Dictyostelium discoideum is a social amoeba that has emerged as a
powerful model system to study conserved mechanisms of host
defence involved in many human diseases caused by bacteria, as
well as mechanisms of bacterial pathogenicity. The main and highly
conserved cell-autonomous defence is constituted by the
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
183