M. tuberculosis blocks innate immune defense of the host to establish its intracellular replicative niche. However, the host machinery
and bacterial effectors that control the spatio-temporal vacuolar
maturation and intracellular survival are not fully characterized
and need further investigation [5].
With the alarming emergence of multi-drug resistant strains, it
has become a race against time to discover new anti-tuberculosis
drugs. Antibacterial compounds identified during the last decades
were mainly revealed by whole cell phenotypic screens. To study
mycobacterial survival within host cells, in vitro models usually rely
on the enumeration of colony-forming units (CFU), which is
labor-intensive and time-consuming. Moreover, these assays do
not take into account individual host cell behavior from the sample.
In this context, the identification of small molecule inhibitors,
siRNA, or molecular genetic interventions interfering with the
pathological cycle of the bacteria would help to elaborate new
therapeutic strategies.
Fluorescence microscopy-based approaches and user-friendly
interfaces of image analysis provide the possibility to visualize and
quantify biological events at different time points post-infection,
making them particularly well-suited for host-pathogen interaction
studies [6]. In this chapter, taking into account that dynamics of
M. tuberculosis infection can vary depending on the virulence of the
strain and the type of host cells, we detailed miniaturized and
automated assays allowing the screening of large libraries of compounds, genetic modulators of host pathways and their mutants.
Moreover, we also described high-content fluorescent microcopy
assays and their analysis to follow successive biological events implicated in the intracellular establishment of M. tuberculosis.
1.1 Experiment
Outline
We provide here multiple fluorescence-based high-content and
high-throughput phenotypic assays to monitor the intracellular
trafficking and replication of M. tuberculosis. These methods allow
the quantitative and qualitative investigation of the impact of various genetic or chemical modulators on M. tuberculosis-infected host
cells (Fig. 1) [2, 7–12].
The following protocols describe details of the preparation of
various types of professional phagocytes as well as a type II pulmonary epithelial cell model to monitor M. tuberculosis interactions
with the host at the single cell level, thereby adding a considerable
value over the CFU-based methods that fail to take into account
individual host cell parameters. The high-throughput screening
(HTS) assays presented here are based on the adaptation of cellular
imaging to automation, which consist of acquiring automated
images from a large set of samples. The acquisition device used in
this method is an automated confocal fluorescence microscope
recording several fields and different focal planes for each well of
optical multi-well plates, such as 96-well, 384-well, etc. Due to the
650
Nathalie Deboosere et al.
and bacterial effectors that control the spatio-temporal vacuolar
maturation and intracellular survival are not fully characterized
and need further investigation [5].
With the alarming emergence of multi-drug resistant strains, it
has become a race against time to discover new anti-tuberculosis
drugs. Antibacterial compounds identified during the last decades
were mainly revealed by whole cell phenotypic screens. To study
mycobacterial survival within host cells, in vitro models usually rely
on the enumeration of colony-forming units (CFU), which is
labor-intensive and time-consuming. Moreover, these assays do
not take into account individual host cell behavior from the sample.
In this context, the identification of small molecule inhibitors,
siRNA, or molecular genetic interventions interfering with the
pathological cycle of the bacteria would help to elaborate new
therapeutic strategies.
Fluorescence microscopy-based approaches and user-friendly
interfaces of image analysis provide the possibility to visualize and
quantify biological events at different time points post-infection,
making them particularly well-suited for host-pathogen interaction
studies [6]. In this chapter, taking into account that dynamics of
M. tuberculosis infection can vary depending on the virulence of the
strain and the type of host cells, we detailed miniaturized and
automated assays allowing the screening of large libraries of compounds, genetic modulators of host pathways and their mutants.
Moreover, we also described high-content fluorescent microcopy
assays and their analysis to follow successive biological events implicated in the intracellular establishment of M. tuberculosis.
1.1 Experiment
Outline
We provide here multiple fluorescence-based high-content and
high-throughput phenotypic assays to monitor the intracellular
trafficking and replication of M. tuberculosis. These methods allow
the quantitative and qualitative investigation of the impact of various genetic or chemical modulators on M. tuberculosis-infected host
cells (Fig. 1) [2, 7–12].
The following protocols describe details of the preparation of
various types of professional phagocytes as well as a type II pulmonary epithelial cell model to monitor M. tuberculosis interactions
with the host at the single cell level, thereby adding a considerable
value over the CFU-based methods that fail to take into account
individual host cell parameters. The high-throughput screening
(HTS) assays presented here are based on the adaptation of cellular
imaging to automation, which consist of acquiring automated
images from a large set of samples. The acquisition device used in
this method is an automated confocal fluorescence microscope
recording several fields and different focal planes for each well of
optical multi-well plates, such as 96-well, 384-well, etc. Due to the
650
Nathalie Deboosere et al.