microbicidal machinery that arms the phagosome in both
D. discoideum and human phagocytes [1]. This includes a number
of genes and pathways recently reviewed by our group
[2]. D. discoideum has a haploid genome that is fully sequenced
[3] and annotated (www.dictybase.org). Mutants can be generated
by homologous recombination, random restriction enzymemediated integration (REMI) mutagenesis, as well as adaptations
of CRISPR/Cas9 [4, 5] and Cre-Lox strategies of genome editing
[6]. Moreover, D. discoideum is easily tractable for biochemistry
and live imaging methods. This organism is a professional phagocyte that feeds on bacteria via the highly conserved process of
phagocytosis. In addition, D. discoideum has evolved a range of
mechanisms to distinguish between nutrients and pathogens in
order to defend itself. D. discoideum lives as a vegetative unicellular
organism when the conditions are favourable. However, if the
nutrients become scarce, the amoeba initiates an aggregation
phase where thousands of cells assemble to form a multicellular
organism, a process called the developmental cycle. Here, we
describe techniques to monitor infection of D. discoideum by Mycobacterium marinum.
M. marinum is the causative agent of a tuberculosis-like disease
in fish and frogs. It is a genetically close relative of Mycobacterium
tuberculosis with which it shares more than 85% nucleotide similarity [7]. Moreover, both species have very similar pathogenicity
mechanisms [8, 9], including intracellular localization, and manipulation of the host vesicular trafficking. M. tuberculosis, responsible
for tuberculosis, killed 1.2 million people in 2018 (WHO Global
Tuberculosis Report, 2019). This intracellular pathogen is able to
manipulate the phagosomal pathway by preventing the maturation
of the compartment, thus residing in the phagosome during infection. Indeed, M. tuberculosis can establish a “permissive niche” in
which it proliferates by preventing the delivery of the v-ATPase and
lysosomal enzymes [10–13].
This chapter covers three techniques used to precisely identify
and quantify diverse phenotypes arising during infection of
D. discoideum with M. marinum. The first method allows to
study the infection at the single cell level. Most of our knowledge
of infected cells has been acquired by studying cell populations,
which are intrinsically heterogeneous. Such studies use techniques
that mask cell individuality and therefore might hide the potential
impacts of small subpopulations on the infection course
[14, 15]. For this reason, single-cell analysis needs to be performed
to investigate bacteria–amoeba interactions. To map and quantitate
precisely the fates of both the host and the pathogen, a high spatial
and temporal resolution is also needed. Importantly, identifying the
causality links between the various fates observed requires a reconstruction of the integrated history of the infection course. To
achieve this, we use the InfectChip, a microfluidic device recently
184
Manon Mottet et al.
D. discoideum and human phagocytes [1]. This includes a number
of genes and pathways recently reviewed by our group
[2]. D. discoideum has a haploid genome that is fully sequenced
[3] and annotated (www.dictybase.org). Mutants can be generated
by homologous recombination, random restriction enzymemediated integration (REMI) mutagenesis, as well as adaptations
of CRISPR/Cas9 [4, 5] and Cre-Lox strategies of genome editing
[6]. Moreover, D. discoideum is easily tractable for biochemistry
and live imaging methods. This organism is a professional phagocyte that feeds on bacteria via the highly conserved process of
phagocytosis. In addition, D. discoideum has evolved a range of
mechanisms to distinguish between nutrients and pathogens in
order to defend itself. D. discoideum lives as a vegetative unicellular
organism when the conditions are favourable. However, if the
nutrients become scarce, the amoeba initiates an aggregation
phase where thousands of cells assemble to form a multicellular
organism, a process called the developmental cycle. Here, we
describe techniques to monitor infection of D. discoideum by Mycobacterium marinum.
M. marinum is the causative agent of a tuberculosis-like disease
in fish and frogs. It is a genetically close relative of Mycobacterium
tuberculosis with which it shares more than 85% nucleotide similarity [7]. Moreover, both species have very similar pathogenicity
mechanisms [8, 9], including intracellular localization, and manipulation of the host vesicular trafficking. M. tuberculosis, responsible
for tuberculosis, killed 1.2 million people in 2018 (WHO Global
Tuberculosis Report, 2019). This intracellular pathogen is able to
manipulate the phagosomal pathway by preventing the maturation
of the compartment, thus residing in the phagosome during infection. Indeed, M. tuberculosis can establish a “permissive niche” in
which it proliferates by preventing the delivery of the v-ATPase and
lysosomal enzymes [10–13].
This chapter covers three techniques used to precisely identify
and quantify diverse phenotypes arising during infection of
D. discoideum with M. marinum. The first method allows to
study the infection at the single cell level. Most of our knowledge
of infected cells has been acquired by studying cell populations,
which are intrinsically heterogeneous. Such studies use techniques
that mask cell individuality and therefore might hide the potential
impacts of small subpopulations on the infection course
[14, 15]. For this reason, single-cell analysis needs to be performed
to investigate bacteria–amoeba interactions. To map and quantitate
precisely the fates of both the host and the pathogen, a high spatial
and temporal resolution is also needed. Importantly, identifying the
causality links between the various fates observed requires a reconstruction of the integrated history of the infection course. To
achieve this, we use the InfectChip, a microfluidic device recently
184
Manon Mottet et al.