Chapter 17
Exploiting Fluorescent Proteins to Understand
Mycobacterium tuberculosis Biology
David Giacalone, Lu Huang, and Shumin Tan
Abstract
The utility of fluorescent proteins in bacterial research has long been appreciated, with extensive use in the
Mycobacterium tuberculosis field. In more recent years, a new generation of fluorescent tools has been
developed for use in M. tuberculosis research. These new fluorescent reporters exploit the immense genetic
and transcriptional knowledge now available, and enable the use of the bacteria as direct reporters of the
local environment during infection, as well as provide insight into bacterial replication status in situ. Here
we describe methods for the construction of such fluorescent reporter M. tuberculosis strains, and their use
in combination with confocal microscopy and flow cytometry approaches for single bacterium-level analyses
of M. tuberculosis physiology and M. tuberculosis-host interactions.
Key words Mycobacterium tuberculosis, Fluorescent reporters, Host-pathogen interactions, Confocal
microscopy, Flow cytometry
1 Introduction
Fluorescent proteins are powerful tools that have been widely used
in biological research, illuminating a myriad of processes, including
bacterial-host interactions [1–4]. In the Mycobacterium tuberculosis
field, fluorescent proteins have been utilized to visualize the bacteria during infection and to delineate the location of proteins within
the bacterium [5–9]. These tools have provided insight into various
aspects of M. tuberculosis biology, and enabled studies ranging from
in vitro drug screens to analysis of cell wall synthesis and bacterial
division dynamics [10–16]. Beyond enabling visualization of the
bacteria or localization of a particular protein of interest during
in vitro growth or infection however, a new generation of fluorescent reporters have now been developed that provide insight into
the physiology or local environment experienced by M. tuberculosis
at a single bacillus level. The environmental reporters take advantage of well-characterized transcriptional changes that occur upon
M. tuberculosis exposure to a given signal, with a fluorescent protein
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
365
Exploiting Fluorescent Proteins to Understand
Mycobacterium tuberculosis Biology
David Giacalone, Lu Huang, and Shumin Tan
Abstract
The utility of fluorescent proteins in bacterial research has long been appreciated, with extensive use in the
Mycobacterium tuberculosis field. In more recent years, a new generation of fluorescent tools has been
developed for use in M. tuberculosis research. These new fluorescent reporters exploit the immense genetic
and transcriptional knowledge now available, and enable the use of the bacteria as direct reporters of the
local environment during infection, as well as provide insight into bacterial replication status in situ. Here
we describe methods for the construction of such fluorescent reporter M. tuberculosis strains, and their use
in combination with confocal microscopy and flow cytometry approaches for single bacterium-level analyses
of M. tuberculosis physiology and M. tuberculosis-host interactions.
Key words Mycobacterium tuberculosis, Fluorescent reporters, Host-pathogen interactions, Confocal
microscopy, Flow cytometry
1 Introduction
Fluorescent proteins are powerful tools that have been widely used
in biological research, illuminating a myriad of processes, including
bacterial-host interactions [1–4]. In the Mycobacterium tuberculosis
field, fluorescent proteins have been utilized to visualize the bacteria during infection and to delineate the location of proteins within
the bacterium [5–9]. These tools have provided insight into various
aspects of M. tuberculosis biology, and enabled studies ranging from
in vitro drug screens to analysis of cell wall synthesis and bacterial
division dynamics [10–16]. Beyond enabling visualization of the
bacteria or localization of a particular protein of interest during
in vitro growth or infection however, a new generation of fluorescent reporters have now been developed that provide insight into
the physiology or local environment experienced by M. tuberculosis
at a single bacillus level. The environmental reporters take advantage of well-characterized transcriptional changes that occur upon
M. tuberculosis exposure to a given signal, with a fluorescent protein
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
365