hand, when the acyl chain is natively ester-linked, as in ester-linked
6-heptynoyl-modified TMM (O-AlkTMM-C7), the TMM probe
can either accept or donate mycoloyl groups via Ag85, thus it labels
all mycolates, including TDM, AGM, and O-mycoloylated proteins
[22, 23]. Additional ester-linked TMM-mimicking probes have
been developed to expand experimental versatility, including
fluorophore-modified TMM (O-FITC-TMM), azide-modified
TMM (O-AzTMM-C10), and trans-cyclooctene-modified TMM
(O-TCO-TMM) [24]. O-FITC-TMM enables one-step fluorescence labeling of mycolates [24]. N-AlkTMM-C7, O-AlkTMMC7, and O-AzTMM enable “click” chemistry-mediated two-step
labeling, with all three being CuAAC-capable and the latter also
being SPAAC-compatible [22–24]. Finally, O-TCO-TMM enables
copper-free tetrazine ligation-mediated two-step labeling, which is
extremely fast and sensitive [24, 25].
The toolbox of trehalose-based probes offers a range of complementary options to perform metabolic labeling of mycobacteria.
The selection of which probe and, if employing two-step labeling,
which chemistry and secondary reagent, depends mainly on the
objective of the experiment. In general, some key considerations
include: (1) which metabolic labeling route and target mycomembrane component is desired, because, as just described, different
trehalose- and TMM-mimicking probes exploit different incorporation mechanisms and label different mycolate structures;
(2) what technique will be used for end-point analysis (e.g., flow
cytometry, microscopy, SDS-PAGE, LC–MS/MS); (3) whether a
one-step or two-step labeling approach offers the desired balance of
incorporation efficiency, experimental simplicity, and experimental
versatility; (4) whether the analysis step must occur on live cells, in
which case in vivo-compatible bioorthogonal reaction conditions
should be used for the two-step approach; (5) whether the probe
has been shown to be effective for labeling in the species desired at
concentrations that do not impact growth (most probes mentioned
have been tested and work in M. smegmatis, M. tuberculosis, and
C. glutamicum); (6) whether the probe and/or secondary reagent
are accessible, i.e., readily synthesized or commercially available (see
Note 2). For full consideration of selection criteria, readers are
referred to the extensive reviews on bacterial cell surface labeling
by Siegrist et al. [26] and on bioorthogonal reactions by Patterson
et al. [27].
Collectively, trehalose-based metabolic labeling probes enable a
broad range of applications. Some representative examples reported
so far include studying the dynamics mycomembrane biosynthesis
and remodeling [18, 22, 28–32], characterizing inhibitors of cell
envelope biosynthesis [29, 33], labeling and identifying O-mycoloylated proteins [23], specifically detecting mycobacteria in complex samples [16], developing in vivo PET probes for imaging
mycobacterial infections [34, 35], delivering therapeutic cargo
388
Nicholas Banahene and Benjamin M. Swarts
6-heptynoyl-modified TMM (O-AlkTMM-C7), the TMM probe
can either accept or donate mycoloyl groups via Ag85, thus it labels
all mycolates, including TDM, AGM, and O-mycoloylated proteins
[22, 23]. Additional ester-linked TMM-mimicking probes have
been developed to expand experimental versatility, including
fluorophore-modified TMM (O-FITC-TMM), azide-modified
TMM (O-AzTMM-C10), and trans-cyclooctene-modified TMM
(O-TCO-TMM) [24]. O-FITC-TMM enables one-step fluorescence labeling of mycolates [24]. N-AlkTMM-C7, O-AlkTMMC7, and O-AzTMM enable “click” chemistry-mediated two-step
labeling, with all three being CuAAC-capable and the latter also
being SPAAC-compatible [22–24]. Finally, O-TCO-TMM enables
copper-free tetrazine ligation-mediated two-step labeling, which is
extremely fast and sensitive [24, 25].
The toolbox of trehalose-based probes offers a range of complementary options to perform metabolic labeling of mycobacteria.
The selection of which probe and, if employing two-step labeling,
which chemistry and secondary reagent, depends mainly on the
objective of the experiment. In general, some key considerations
include: (1) which metabolic labeling route and target mycomembrane component is desired, because, as just described, different
trehalose- and TMM-mimicking probes exploit different incorporation mechanisms and label different mycolate structures;
(2) what technique will be used for end-point analysis (e.g., flow
cytometry, microscopy, SDS-PAGE, LC–MS/MS); (3) whether a
one-step or two-step labeling approach offers the desired balance of
incorporation efficiency, experimental simplicity, and experimental
versatility; (4) whether the analysis step must occur on live cells, in
which case in vivo-compatible bioorthogonal reaction conditions
should be used for the two-step approach; (5) whether the probe
has been shown to be effective for labeling in the species desired at
concentrations that do not impact growth (most probes mentioned
have been tested and work in M. smegmatis, M. tuberculosis, and
C. glutamicum); (6) whether the probe and/or secondary reagent
are accessible, i.e., readily synthesized or commercially available (see
Note 2). For full consideration of selection criteria, readers are
referred to the extensive reviews on bacterial cell surface labeling
by Siegrist et al. [26] and on bioorthogonal reactions by Patterson
et al. [27].
Collectively, trehalose-based metabolic labeling probes enable a
broad range of applications. Some representative examples reported
so far include studying the dynamics mycomembrane biosynthesis
and remodeling [18, 22, 28–32], characterizing inhibitors of cell
envelope biosynthesis [29, 33], labeling and identifying O-mycoloylated proteins [23], specifically detecting mycobacteria in complex samples [16], developing in vivo PET probes for imaging
mycobacterial infections [34, 35], delivering therapeutic cargo
388
Nicholas Banahene and Benjamin M. Swarts