Chapter 29
High-Content Analysis Monitoring Intracellular Trafficking
and Replication of Mycobacterium tuberculosis Inside Host
Cells
Nathalie Deboosere, Ime ` ne Belhaouane, Arnaud Machelart, Eik Hoffmann,
Alexandre Vandeputte, and Priscille Brodin
Abstract
Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as
phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of
tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully
understood and require further investigations. Therefore, increasing the knowledge of this process will help
to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically
tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances
in imaging techniques and images-based analysis allow the rapid quantification of biological objects in
complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to
track the bacillus within host cell settings.
Key words Mycobacterium tuberculosis, High-content/high-throughput screening, Drug discovery,
siRNA library, Phenotypic assays, Automated confocal microscopy, Image-based analysis
1 Introduction
There is a strong interplay between M. tuberculosis and the host cell
at various levels, including uptake, intracellular trafficking, and
metabolic reprogramming [1]. Once internalized, mycobacteria
prevent and control the acidification of the M. tuberculosis-containing vacuole (pH at around 6.3–6.5), thereby inducing an intraphagosomal environment favoring its survival [2]. These organelles
were long considered sealed, but it was recently shown that the
bacilli induce vacuolar rupture, thereby allowing direct interactions
of bacterial components with host cytosolic proteins [3, 4]. Thus,
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_29, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Nathalie Deboosere and Ime `ne Belhaouane contributed equally.
649
High-Content Analysis Monitoring Intracellular Trafficking
and Replication of Mycobacterium tuberculosis Inside Host
Cells
Nathalie Deboosere, Ime ` ne Belhaouane, Arnaud Machelart, Eik Hoffmann,
Alexandre Vandeputte, and Priscille Brodin
Abstract
Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as
phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of
tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully
understood and require further investigations. Therefore, increasing the knowledge of this process will help
to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically
tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances
in imaging techniques and images-based analysis allow the rapid quantification of biological objects in
complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to
track the bacillus within host cell settings.
Key words Mycobacterium tuberculosis, High-content/high-throughput screening, Drug discovery,
siRNA library, Phenotypic assays, Automated confocal microscopy, Image-based analysis
1 Introduction
There is a strong interplay between M. tuberculosis and the host cell
at various levels, including uptake, intracellular trafficking, and
metabolic reprogramming [1]. Once internalized, mycobacteria
prevent and control the acidification of the M. tuberculosis-containing vacuole (pH at around 6.3–6.5), thereby inducing an intraphagosomal environment favoring its survival [2]. These organelles
were long considered sealed, but it was recently shown that the
bacilli induce vacuolar rupture, thereby allowing direct interactions
of bacterial components with host cytosolic proteins [3, 4]. Thus,
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_29, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Nathalie Deboosere and Ime `ne Belhaouane contributed equally.
649