driven by the promoter of a M. tuberculosis gene known to respond
to the cue of interest. Examples of such reporters include those that
respond to acidic pH, high chloride concentrations ([Cl
À
]), low
potassium concentrations, iron, hypoxia, or nitrosative stress
[6, 17–20].
Reporters that inform on M. tuberculosis physiology include
those that exploit intrinsic or engineered properties of fluorescent
proteins, enabling determination of aspects such as intrabacterial
pH and mycothiol redox potential [21, 22]. A unique fluorescent
reporter that provides information on M. tuberculosis physiology is
the single-strand binding protein (SSB)-GFP reporter, which is a
translational fusion of single-strand binding protein from
M. tuberculosis to GFP, driven by the native ssb promoter
[7, 23]. This results in the presence of green foci during periods
of active DNA replication, and serves as a proxy for analysis of
M. tuberculosis growth status in situ [7, 23].
These new fluorescent reporters have marked utility in vitro,
enabling for example identification of novel small molecule compounds that inhibit the DosRST or PhoPR two-component system
of M. tuberculosis [24, 25], and revealing novel regulators that
modulate the bacterial response to environmental signals [19]. In
addition, there is immense power in their use in whole animal
infection studies, in combination with approaches such as microscopy and flow cytometry. In particular, they enable analysis of
multiple aspects of M. tuberculosis-host interactions at the single
bacterium level while retaining the context of host cell type and/or
intact host tissue architecture [1, 6, 7, 23], especially critical given
the burgeoning appreciation for the presence and impact of heterogeneity across varied facets of M. tuberculosis infection biology
[21, 26–32].
In this chapter, we describe protocols for the construction and
characterization of this newer generation of fluorescent reporter
M. tuberculosis strains, and focus on their utilization in a murine
model of M. tuberculosis infection, in combination with confocal
microscopy and flow cytometry methods.
2 Materials
2.1 Construction
of Single Fluorescent
Reporter
M. tuberculosis Strains
1. M. tuberculosis genomic DNA (obtained via phenolchloroform-based extraction method or other equivalent
method).
2. QIAquick gel extraction kit (Qiagen, Germany).
3. Appropriate cloning vector. Used in particular here is pGFP-N
(containing GFPmut2 with an Esat6 terminator) [6].
4. Restriction enzymes (e.g., BamHI, HindIII, XbaI) and other
routine reagents needed for molecular cloning (e.g., Fast-link
366
David Giacalone et al.
to the cue of interest. Examples of such reporters include those that
respond to acidic pH, high chloride concentrations ([Cl
À
]), low
potassium concentrations, iron, hypoxia, or nitrosative stress
[6, 17–20].
Reporters that inform on M. tuberculosis physiology include
those that exploit intrinsic or engineered properties of fluorescent
proteins, enabling determination of aspects such as intrabacterial
pH and mycothiol redox potential [21, 22]. A unique fluorescent
reporter that provides information on M. tuberculosis physiology is
the single-strand binding protein (SSB)-GFP reporter, which is a
translational fusion of single-strand binding protein from
M. tuberculosis to GFP, driven by the native ssb promoter
[7, 23]. This results in the presence of green foci during periods
of active DNA replication, and serves as a proxy for analysis of
M. tuberculosis growth status in situ [7, 23].
These new fluorescent reporters have marked utility in vitro,
enabling for example identification of novel small molecule compounds that inhibit the DosRST or PhoPR two-component system
of M. tuberculosis [24, 25], and revealing novel regulators that
modulate the bacterial response to environmental signals [19]. In
addition, there is immense power in their use in whole animal
infection studies, in combination with approaches such as microscopy and flow cytometry. In particular, they enable analysis of
multiple aspects of M. tuberculosis-host interactions at the single
bacterium level while retaining the context of host cell type and/or
intact host tissue architecture [1, 6, 7, 23], especially critical given
the burgeoning appreciation for the presence and impact of heterogeneity across varied facets of M. tuberculosis infection biology
[21, 26–32].
In this chapter, we describe protocols for the construction and
characterization of this newer generation of fluorescent reporter
M. tuberculosis strains, and focus on their utilization in a murine
model of M. tuberculosis infection, in combination with confocal
microscopy and flow cytometry methods.
2 Materials
2.1 Construction
of Single Fluorescent
Reporter
M. tuberculosis Strains
1. M. tuberculosis genomic DNA (obtained via phenolchloroform-based extraction method or other equivalent
method).
2. QIAquick gel extraction kit (Qiagen, Germany).
3. Appropriate cloning vector. Used in particular here is pGFP-N
(containing GFPmut2 with an Esat6 terminator) [6].
4. Restriction enzymes (e.g., BamHI, HindIII, XbaI) and other
routine reagents needed for molecular cloning (e.g., Fast-link
366
David Giacalone et al.