Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

Article abstract of DOI:10.1002/cbic.201800687

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involv

Full text of DOI:10.1002/cbic.201800687

Accepted Article
Title: Engineering the Mycomembrane of Live Mycobacteria with an
Expanded Set of Trehalose Monomycolate Analogues
Authors: Taylor J Fiolek, Nicholas Banahene, Herbert W Kavunja,
Nathan J Holmes, Adrian K Rylski, Amol Arunrao Pohane,
Mary Sloan Siegrist, and Benjamin M Swarts
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ChemBioChem
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Engineering the Mycomembrane of Live Mycobacteria with an
Expanded Set of Trehalose Monomycolate Analogues
Taylor J. Fiolek,[a],† Nicholas Banahene,[a],† Herbert W. Kavunja, Nathan J. Holmes, Adrian K.
[a]
[a]
[a]
[b]
[b]
[a]
Rylski, Amol Arunrao Pohane, M. Sloan Siegrist, and Benjamin M. Swarts*
Abstract: Mycobacteria and related organisms in the
Corynebacterineae suborder are characterized by a distinctive outer
membrane referred to as the mycomembrane. Biosynthesis of the
mycomembrane occurs through an essential process called
mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of
mycolic acids from the mycoloyl donor trehalose monomycolate
Introduction
Bacteria in the Corynebacterineae suborder include species of
enormous medical, biotechnological, and environmental
importance, such as the global pathogen Mycobacterium
tuberculosis (Mtb)[ and the industrial fermentation workhorse
Corynebacterium glutamicum (Cglut).[ These bacteria have a
complex cell envelope featuring a distinctive outer membrane—
called the mycomembrane—which consists mainly of long-chain,
1,2]
3]
(TMM) to acceptor carbohydrates and, in some organisms, proteins.
We recently described an alkyne-modified TMM analogue (O-
AlkTMM-C7) which, in conjunction with click chemistry, acted as a
chemical reporter for mycoloylation in intact cells and allowed
metabolic labeling of mycoloylated components of the
mycomembrane. Here, we describe the synthesis and evaluation of a
toolbox of TMM-based reporters bearing alkyne, azide, trans-
cyclooctene, and fluorescent tags. These compounds gave further
insight into the substrate tolerance of mycoloyltransferases (e.g.,
Ag85s) in a cellular context and they provide significantly expanded
experimental versatility by allowing one- or two-step cell labeling, live
cell labeling, and rapid cell labeling via tetrazine ligation. Such
capabilities will facilitate research on mycomembrane composition,
biosynthesis, and dynamics. Moreover, because TMM is exclusively
metabolized by Corynebacterineae, the described probes may be
valuable for the specific detection and cell-surface engineering of
Mycobacterium tuberculosis and related pathogens. We also
performed experiments to establish the dependence of probe
incorporation on mycoloyltransferase activity, results from which
suggested that cellular labeling is a function not only of metabolic
incorporation (and likely removal) pathway(s), but also accessibility
across the envelope. Thus, whole-cell labeling experiments with TMM
reporters should be carefully designed and interpreted when envelope
permeability may be compromised. On the other hand, this property
of TMM reporters can potentially be exploited as a convenient way to
probe changes in envelope integrity and permeability, facilitating drug
development studies.
branched mycolic acids that are conjugated to carbohydrates and
other molecules (Figure 1A).[
4–7]
The mycomembrane is essential
for survival due to its roles in fundamental physiological processes
such as defense, nutrient acquisition, and cell–cell
communication, including host–pathogen interactions.[6,7] As a
result, there is significant interest in improving our understanding
of mycomembrane composition, biosynthesis, and function, as
this information is crucial for activities such as tuberculosis drug
and diagnostic development.[8] However, progress has been
hampered by the difficulty of analyzing mycomembrane
biosynthetic pathways and components using traditional
microbiological and biochemical techniques (e.g., radiolabeling
and fractionation), particularly in native settings.[9,10]
Metabolic reporters that exploit endogenous biosynthetic
pathways are valuable tools for studying cell envelope
components in intact bacterial cells.[11] Such efforts in
mycobacteria have benefited from the recent elucidation of key
steps in mycomembrane biosynthesis, which are mediated by the
non-mammalian disaccharide trehalose.[12] Trehalose shuttles
mycolic acids from the cytoplasm to the cell’s exterior to assemble
the mycomembrane (Figure 1A). Trehalose is first converted to
trehalose monomycolate (TMM),[ which, after transport across
the plasma membrane,[14] donates its mycoloyl group to acceptor
molecules.[15] This process, termed mycoloylation, is catalyzed by
a group of mycoloyltransferases, which in mycobacterial species
13]
[
15–
are collectively referred to as the antigen 85 (Ag85) complex.
1
7]
The major products of mycoloylation are arabinogalactan-
linked mycolate (AGM) and trehalose dimycolate (TDM), which
are the principal constituents of the inner- and outer-leaflets of the
mycomembrane, respectively.[ In some members of the
Corynebacterineae, including Cglut, mycomembrane proteins can
also be O-mycoloylated on serine residues,[18] although the scope
†
†
[
a]
T. J. Fiolek, N. Banahene, H. W. Kavunja, N. J. Holmes, A. K.
Rylski, Prof. B. M. Swarts
7]
Department of Chemistry & Biochemistry
Central Michigan University
Mount Pleasant, MI (USA)
E-mail: ben.swarts@cmich.edu
Homepage: http://people.cst.cmich.edu/swart1bm/swartslab/
and functional importance of this unique post-translational
modification remains largely unknown.
[b]
A. A. Pohane, Prof. M. S. Siegrist
Department of Microbiology
University of Massachusetts, Amherst
Amherst, MA (USA)
Trehalose metabolic pathways in mycobacteria have been
successfully exploited for the purpose of probe development.
Fluorescein- and azide-modified trehalose analogues can be
metabolically incorporated into trehalose-containing glycolipids in
the mycomembrane, i.e. TMM and/or TDM, allowing detection of
these species in cells either (i) directly, when the fluorophore is
E-mail: ben.swarts@cmich.edu
†These authors contributed equally to this work.
pre-attached to trehalose, or (ii) after click chemistry, wherein the
Supporting information for this article is given via a link at the end of
the document.
fluorophore is delivered in a second step.[
19,20]
These trehalose-
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Figure 1. (A) TMM is the mycoloyl donor used to construct the major mycomembrane components, including AGM, TDM, and O-mycoloylated proteins. O-AlkTMM-
C7 (2) is a previously developed TMM-mimicking reporter molecule that allows metabolic labeling and analysis of mycomembrane components in intact cells. (B)
Chemical structures and properties of the expanded set of TMM-based reporters (1–6) developed in this work.
based reporters have been used for various applications. For
instance, fluorescein-tagged trehalose analogues have been
used to image Mtb within macrophages and were recently used
to investigate mycomembrane fluidity in response to
antitubercular drugs.[21] In addition, 6-azido trehalose labeling,
deployed in conjunction with click chemistry, was used to confirm
the function of MmpL3 as the essential TMM flippase and to
characterize antitubercular compounds that inhibit it.[22] Fluorine-
modified trehalose analogues can also be metabolized by
mycobacteria, a finding which may pave the way for imaging
mycobacterial infections in vivo using positron emission
tomography (PET).[23,24] Recently, a trehalose analogue bearing a
solvatochromic moiety was used as a fluorogenic probe for the
rapid detection of Mtb in patient sputum samples.[25] The latter two
examples highlight the potential of trehalose-based metabolic
reporters for tuberculosis diagnostic applications.
Recently, our group introduced a new class of reporters,
which are based on TMM and provide complementary capabilities
for studying the mycomembrane.[26] The reporter we initially
developed, named O-AlkTMM-C7, mimics the structure of the
mycoloyl donor TMM, but in place of a native branched lipid we
added a linear, truncated (C7) chain containing a clickable
terminal alkyne tag (2, Figure 1B). When fed to mycobacterial or
corynebacterial cells, O-AlkTMM-C7 was processed by
mycoloyltransferases (e.g., Ag85), thus depositing its clickable
lipid chain onto mycoloyl acceptors and producing species such
as alkyne-labeled AGM and TDM. By combining O-AlkTMM-C7
labeling with Cu-catalyzed
azide–alkyne cycloaddition
(CuAAC[27,28])-mediated fluorophore delivery, various aspects of
mycomembrane composition and dynamics can be probed. For
instance, O-AlkTMM-C7 allowed the first direct visualization of
AGM biosynthesis during cell growth and division in M.
[26]
smegmatis (Msmeg). O-AlkTMM-C7 was also used to assist in
the functional characterization of
a
protein involved in
mycomembrane construction, namley the mycobacterial lipid
transporter LprG.[29] In another example, O-AlkTMM-C7 was
shown to label mycomembrane-resident O-mycoloylated proteins
in Cglut (via the pathway shown in Figure 1A), providing a
chemical biology approach to the discovery and characterization
of this distinctive class of proteins.[30] In the context of studying
this post-translational modification, the ability of O-AlkTMM-C7 to
exclusively label O-mycoloylated proteins provides a substantial
advantage over using alkynyl fatty acids, which label all types of
lipidated proteins in bacteria and thus lack selectivity for O-
mycoloylation.[31,32] Overall, O-AlkTMM-C7 is a powerful new tool
for studying various aspects of the mycomembrane. However, it
is limited in some contexts by its reliance on CuAAC—which is
often not suitable for use in living systems due to copper toxicity
or perturbations resulting from oxidative stress—as well as the
need to perform two steps to deliver a fluorophore (or other type
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of functional tag) to the cell surface. In this work, we sought to
further develop, characterize, and define the utility of the TMM
class of reporters for investigating the mycomembrane-containing
species of the Corynebacterineae suborder.
6 in 62% and 85% yield, respectively. Conveniently, intermediate
8 allows modification of the TMM scaffold with virtually any type
of amine-reactive chemical cargo, which in the future will provide
ready access to TMM reporters tailored to specific applications.
Results and Discussion
Design and Synthesis of an Expanded Set of TMM reporters
The objective of this study was to develop and characterize an
expanded toolbox of TMM-based reporters that are designed to
provide broad experimental flexibility for studying the
mycomembrane (Figure 1B). First, we designed O-AlkTMM
reporters with varying chain lengths, from C5–C11 (1–3), to
determine whether this property had any effect on metabolic
incorporation efficiency. Second, we designed the azide-modified
TMM reporter O-AzTMM-C10 (4) to allow non-toxic Cu-free click
chemistry [i.e., strain-promoted azide–alkyne cycloaddition
SPAAC[33,34])] on living cells. O-AzTMM-C10 was also viewed as
Scheme 1. (A) Syntheses of alkyne- and azide-modified TMM reporters 1–4. a)
(
TMSCl, Et
DCC, DMAP, CH
given). (B) Syntheses of TCO- and fluorescein-modified TMM reporters 5 and 6.
e) Pd/C, H , CH OH; f) NHS-TCO, Et N, N,N’-dimethylformamide (DMF); g)
FITC, Et N, DMF; h) reaction of 8 with commercially available amine-reactive
3
N; b) K
2
CO
3
, CH
3
OH (92% over two steps, ref. 36); c) carboxylic acid,
a versatile synthetic intermediate, since it can be readily
elaborated to other TMM analogues via either (i) click chemistry
with alkyne-modified reagents or (ii) azide reduction to the
corresponding amine, followed by reaction with amine-reactive
reagents. Third, we designed the trans-cyclooctene-modified
TMM reporter O-TCO-TMM (5) to allow secondary labeling using
the tetrazine ligation, which is exceptionally fast and can be
performed on live cells.[35,36] Fourth, because reporters 1–5 all
require two-step labeling, we designed the fluorescein-modified
TMM reporter O-FITC-TMM (6) to allow one-step labeling of live
cells. The diversity of TMM modifications explored in this work
also allowed for an in-depth evaluation of the tolerance of
mycoloylation machinery for varying size and polarity of the tag.
To synthesize compounds 1–4, we used Kulkarni’s
approach to accessing unsymmetrical trehalose esters (Scheme
; d) Dowex H+ resin, CH
2
Cl
2
3
OH (yields over two steps are
2
3
3
3
reagents will provide access to custom TMM reporters.
Effect of Acyl Chain Length on Incorporation Efficiency
With compounds 1–6 in hand, our first objective was to evaluate
the O-AlkTMM series (1–3) to determine whether changes in
chain length had an impact on labeling efficiency. Native mycolic
acids are α-branched and β-hydroxylated, and they contain
anywhere from 22–100 total carbons, depending on the species.[
The shortened linear chain of our original TMM reporter, O-
AlkTMM-C7, was a fairly drastic simplification of the native
mycolate, but the compound was still efficiently incorporated into
the mycomembrane.
7]
1
A).[37] First, trehalose was subjected to per-O-trimethylsilylation,
then both 6-O-trimethylsilyl groups were selectively removed
using CO /CH OH to give diol 7. This -symmetric
K
2
3
3
C
2
In the present comparison, we predicted to observe that
longer-chain reporters would have favorable incorporation. To
test this hypothesis, Msmeg was cultured in 0–50 µM of each
reporter, then subjected to CuAAC reaction with an azido-488
fluorophore and analyzed by flow cytometry (Figure 2A). All three
compounds labeled Msmeg efficiently, which is consistent with
the remarkable substrate plasticity that has been reported for
mycoloyltransferases.[19,26] Interestingly, at lower probe
concentrations (5 and 10 µM), we observed a clear trend in
Msmeg in which the intermediate-length O-AlkTMM-C7 (2) gave
optimal labeling. One explanation for this observation is that the
C7 reporter has an optimal balance of penetration across the
mycomembrane and processing by mycoloyltransferases. It is
also possible that the smaller C7 reporter labels more terminal AG
residues than larger reporters due to a steric advantage.
intermediate was desymmetrized in the subsequent step, which
entailed 6-O-monoesterification with an appropriate alkyne- or
azide-modified carboxylic acid in the presence of N,N‘-
dicyclohexylcarbodiimide
(DCC)
and
catalytic
4-
(
dimethylamino)pyridine (DMAP). The intermediate was then
desilylated in the presence of Dowex H+ resin, delivering the
desired products. The yields for compounds 1–4 over the two-step
monoesterification–deprotection sequence ranged from 34–44%,
consistent with prior results from Kulkarni[37] and our lab.[26]
To access trans-cyclooctene- and fluorescein-modified
TMM analogues 5 and 6, we sought to capitalize on the
commercial availability of amine-reactive versions of these tags
(
Scheme 1B). Therefore, we converted O-AzTMM-C10 (4) into
the corresponding amine 8 by Pd-catalyzed hydrogenation, which
proceeded in nearly quantitative yield (99%). Next, we reacted
amine 8 with either the N-hydroxysuccinimidyl carbonate of trans-
cyclooctene (NHS-TCO) to generate O-TCO-TMM (5) or
fluorescein isothiocyanate (FITC) to generate O-FITC-TMM (6).
Purification by C18 reverse-phase chromatography yielded 5 and
It will be interesting in the future to compare reporters 1–3
to versions that more closely resemble the native TMM. For
example, compounds with and without α-branching and β-
hydroxylation, spanning a broader range of chain length, and
containing other mycolic acid modifications would be beneficial to
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Figure 2. (A) Dependence of metabolic labeling on TMM reporter acyl chain length. Msmeg was incubated with 0–50 µM of 1, 2, or 3 (or left untreated) for 4 h, fixed
with para-formaldehyde, reacted with Az488 by CuAAC, and analyzed by flow cytometry. (B) Concentration-dependent metabolic labeling of Msmeg and Cglut
using azide-, trans-cyclooctene, and fluorophore-modified TMM reporters 4, 5, and 6. Msmeg or Cglut was incubated with 4, 5, or 6 (or left untreated) for 4 h, then
in a second step the live cells were reacted with DBCO-488 (for 4), tetrazine-Cy3 (for 5), or left untreated (for 6), then fixed and analyzed by flow cytometry. Error
bars denote the standard deviation of three replicate experiments. Y axis on each plot is mean fluorescence intensity (MFI) measured in arbitrary units (a.u.).
evaluate. To permit such a comparison, strategies for the
synthesis of native mycolic acids (e.g., ref. [38] and references
therein) will need to be adapted to accomodate alkyne or azide
modifications. These studies are ongoing in our lab. In the
meantime, there are criteria to be considered for selection of TMM
reporter chain length for a given application. The smaller C5 and
C7 reporters are conveniently water-soluble and possibly have
better porin-mediated uptake across the mycomembrane,
whereas larger C11 reporters need co-solvent (e.g., DMSO) to
solubilize and may not cross the mycomembrane as efficiently.
Metabolic incorporation of the C7 reporter was also significantly
higher than either the C5 or C11 compounds. On the other hand,
the TMM reporters with longer chains that more closely resemble
native mycolates are also more likely to faithfully replicate the
biophysical properties of native TMM (and labeled TDM, AGM, or
O-mycoloylated protein products) within a cellular environment.
These considerations could prove particularly relevant in the
development of mycobacteria-specific detection probes.
labeling experiments, all three reporters efficiently labeled both
Msmeg and Cglut as compared to the control cells. Labeling
saturation was observed for both O- AzTMM-C10 (4) and O-TCO-
TMM (5) at approximately 25 µM concentration, whereas for O-
FITC-TMM saturation occurred at higher concentrations (≥ 100
µM), presumably because the larger size of the fluorophore
impeded access to mycoloyltransferases or curbed incorporation
by mycoloyltransferases. Interestingly, the reporters with the
larger TCO and FITC tags, 5 and 6, labeled Cglut much more
efficiently than Msmeg. This could be due to increased uptake of
the larger reporters across the mycomembrane of Cglut, which
has shorter mycolic acids and higher fluidity than Msmeg.[21]
Notably, we did not observe growth inhibition or reduced
viability upon treatment of Msmeg with compounds 1–6 at
concentrations up to 100 µM over 16 h, demonstrating their
apparent non-perturbing nature under these conditions (Figure
S1). Interestingly, at 250 µM concentration, partial growth
inhibition was observed for O-AlkTMM-C11 (3), O-AzTMM-C10
(
4), and O-TCO-TMM (5), all of which have longer acyl chains.
The azido TMM analogue had the most significant impairment of
Msmeg growth, which likely explains why this compound showed
reduced labeling of Msmeg at higher concentrations (Figure 2B).
Further investigation of the inhibitory properties of TMM
analogues is a topic of future interest.
Labeling of Live Bacteria with TMM Reporters
Due to the complications of using copper in vivo, alkyne-labeled
cells (e.g., O-AlkTMM-labeled bacteria) are typically chemically
fixed prior to carrying out CuAAC reactions on the cell surface.
Fixation prevents studies in live cells, whether in broth culture or
more complex settings such as macrophage or animal infection
models. To address this issue, the new TMM reporters 4–6 were
designed to enable use in living cells, which we investigated next.
Each reporter was evaluated over the same concentration range
Thus, in contrast to the original reporter O-AlkTMM-C7, the
new TMM reporters 4–6 provide the ability to probe incorporation
processes in live bacteria. Of course, O-FITC-TMM (6), or related
fluorophore-modified TMM reporters (which can be readily
accessed from 8 as shown in Scheme 1B), should be used when
a one-step live-cell labeling workflow is desired. For example,
one-step labeling may be preferred for its simplicity (e.g., fewer
reagents and washes) or when there is concern about whether
the secondary fluorophore has sufficient access to the tagged
biomolecule. Two-step labeling can be desirable for many
reasons, including generally higher metabolic incorporation
efficiency and the versatility to deliver virtually any type of
secondary reagent to the tagged biomolecule without having to
synthesize and evaluate a new TMM analogue.[11]
(
0–250 µM) in Msmeg and Cglut (Figure 2B). O-AzTMM-C10 (4)
labeling was followed by SPAAC reaction on live cells with a
dibenzocycloocytne (DBCO)-488 fluorophore; O-TCO-TMM (5)
labeling was followed by tetrazine ligation on live cells with a
tetrazine-Cy3 fluorophore; since O-FITC-TMM (6) directly delivers
the fluorophore during incorporation, no secondary step was
required for this reporter. Cellular fluorescence was quantified by
flow cytometry. Consistent with prior results from O-AlkTMM
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Comparison of SPAAC and Tetrazine Ligation
Specificity of TMM Reporters for the Mycomembrane
The TMM reporters that allow two-step live-cell labeling workflows, Only species in the Corynebacterineae suborder possess the
O-AzTMM-C10 (4) and O-TCO-TMM (5), are both useful in these
scenarios, although we were interested in characterizing these
reporters further to guide probe selection in future applications.
mycomembrane and its associated biosynthetic pathways. We
previously showed that O-AlkTMM-C7 (2) efficiently labeled
Msmeg and Cglut but not canonical Gram-positive or Gram-
negative bacteria, thus demonstrating that TMM-based reporters
3
6
TCO–tetrazine ligations, with rate constants ranging from 10 –10
–
1
–1
[26]
M s , are the fastest bioorthogonal reactions, making them 3–6
are specific for members of the Corynebacterineae suborder.
orders of magnitude faster than SPAAC reactions employing
To assess the specificity of the new TMM reporters 4–6 for
mycomembrane-containing bacteria, we performed labeling
experiments essentially as described above in Msmeg, Cglut,
Bacillus subtilis (Bsub, a model Gram-positive organism), and
Escherichia coli (E. coli, a model Gram-negative organism).
Cellular fluorescence was analyzed by flow cytometry and
fluorescence microscopy (Figure 4). These data indicated that the
three new TMM reporters exhibited high specificity for members
of the Corynebacterineae, with robust labeling of Msmeg and
Cglut but no labeling of Bsub or E. coli (Figure 4A). In addition,
–
1 –1 [39]
DBCO, which have rate constants of approximately 0.3 M s .
Thus, we predicted that O-TCO-TMM (5), in combination with the
tetrazine ligation, would give optimal cell-surface labeling.
To compare the efficiency of cell-surface labeling using
SPAAC or the tetrazine ligation, Msmeg was treated with either 4
or
5 and then reacted with DBCO-Cy3 or tetrazine-Cy3,
respectively, for reaction times ranging from 1–30 min. The signal-
to-noise (S/N) ratios for each condition, which were determined
by flow cytometry, are shown in Figure 3A (signal-to-noise values
provide a clearer comparison than mean fluorescence intensities). the microscopy images in Figure 4B showed that treatment with
O-TCO-TMM-labeled cells could be detected by tetrazine ligation
with a S/N ratio of nearly 300:1 at only 1 min (Figures 3A and 3B).
O-AzTMM-C10-labeled cells could also detected in 1 min by
SPAAC, although the S/N ratio was much lower at approximately
compounds 4–6 resulted in cell-surface fluorescence
concentrated at the poles and septa of bacteria, which is
consistent with a metabolic route of incorporation, the polar
growth mode of Corynebacterineae, and with prior imaging
studies employing O-AlkTMM-C7 (2).[23][40]
10:1. Overall, O-TCO-TMM, in conjunction with the tetrazine
ligation, is considered to be an excellent reporter for rapid two-
step labeling in living systems, and it should prove useful in
situations where very low concentrations of reagents are needed
Out of the new TMM reporters 4–6, O-FITC-TMM (6)
represents the most significant structural departure from the
original reporter O-AlkTMM-C7 (2). We considered the possibility
that the fluorescein group could promote non-specific association
of 6 with the cell surface rather than metabolic incorporation. To
test whether the trehalose ester moiety of O-FITC-TMM was in
fact responsible for targeting it to the cell as designed, we also
synthesized its corresponding FITC-modified carboxylic acid
lacking the ester-linked trehalose (compound 9; see SI for
synthesis) (Figure 5A). In a head-to-head comparison, O-FITC-
TMM labeled Msmeg efficiently, while compound 9 gave no
labeling above the untreated control (Figure 5A), supporting the
proposition that O-FITC-TMM incorporation is dependent on
trehalose metabolism.
(
e.g., in animal infection models). Also, O-TCO-TMM did not
exhibit decreased labeling when used at higher concentrations,
whereas O-AzTMM-C10 did (Figure 2B). However, O-TCO-TMM
does have minor trade-offs, including: (i) the TCO group can
isomerize to the unreactive cis isomer over time; and (ii) at
present, the availability of tetrazine reagents is still somewhat
limited compared to cyclooctyne reagents.
As noted above and depicted in Figure 1A, TMM reporters
have the potential to label both major mycomembrane
components, including inner-leaflet AGM and outer-leaflet
TDM.[41] We previously determined that O-AlkTMM-C7 mainly
incorporated into the AGM-containing cellular fraction from
Msmeg and less so into the TDM-containing extractable lipids
fraction, which is consistent with AGM being the most abundant
mycolate-containing envelope component.[26,30] Here, we
performed the same cellular fractionation experiments following
treatment of Msmeg with 4–6. In agreement with the O-AlkTMM-
C7 results, all three new reporters showed the majority of
fluorescence localized to the AGM-containing fraction (Figure 5B).
It was interesting to note that O-FITC-TMM (6) appeared to give
a smaller increase in fluorescence versus the control compound
(9) in this fractionation experiment as compared to the cellular
labeling experiment, although the reason for this is unclear
Figure 3. (A) Cell-surface reaction kinetics comparison between O-AzTMM-C10
(4) with SPAAC and O-TCO-TMM (5) with tetrazine ligation. Msmeg was
cultured in 4 or 5 (25 µM each) or left untreated, then reacted with DBCO-Cy3
or tetrazine-Cy3 (20 µM each) for varying times and analyzed by flow cytometry.
Signal-to-noise ratios were determined by dividing the MFI value for the
reporter-treated sample by the MFI value for the vehicle-treated control exposed
to the same bioorthogonal reaction conditions. Error bars denote the standard
deviation of three replicate experiments. (B) Fluorescence imaging of O-TCO-
TMM-labeled Msmeg after 1 min tetrazine reaction. Scale bar (5 µm) applies to
all images. Left, transmitted light images; right, fluorescence images.
(
Figure 5A).
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Figure 4. Specificity of TMM reporters 4–6 for mycomembrane-containing members of the Corynebacterineae suborder. Msmeg, Cglut, Bsub, or E. coli were
incubated with 4 (25 µM), 5 (25 µM), 6 (100 µM), or left untreated for 4 h, then the cells were reacted with DBCO-488 (for 4), tetrazine-Cy3 (for 5), or left untreated
(for 6), and analyzed by (A) flow cytometry and (B) fluorescence microscopy. In (A), error bars denote the standard deviation of three replicate experiments. In (B),
scale bar (5 µm) at lower right applies to all images; grayscale images are transmitted light images.
Next, we sought to test whether incorporation of the new
TMM reporters 4–6 into mycomembrane-containing bacteria was
dependent on mycoloyltransferase activity. This is a challenging
task because all members of the Corynebacterineae have
multiple mycoloyltransferase isoforms, which are collectively
essential for viability and exhibit high functional redundancy.[15]
Not only does this make the knockout of all mycoloyltransferases
impossible due to lethality, but experiments involving partial
blockage of mycoloyltransferase activity (e.g, by chemical
inhibition or partial knockout) are complicated due to isoform
redundancy and the resulting perturbation of cell envelope
structure and permeability,[42] which presumably can impact probe
uptake and thus labeling efficiency. We attempted to use the
Ag85C inhibitor ebselen[26,43] and previously reported Msmeg
mutants partially lacking mycoloyltransferase activity (e.g.,
Msmeg ag85a::Tn[44] and the triple knockout Msmeg
ΔMSMEG_6396–99[25]), but the results were inconclusive. For
example, we observed increases in cellular labeling for all of the
TMM reporters in both Msmeg mutants, perhaps suggesting that
partial mycoloyltransferase disruption leads to increased reporter
uptake, followed by envelope incorporation via the remaining
mycoloyltransferases.
Given the complexities posed by the chemical inhibition and
Msmeg mutant experiments, we conceived of an alternative
approach to determine whether cellular incorporation of TMM-
based reporters is mycoloyltransferase-dependent. Cglut
encodes six mycoloyltransferases, only one of which, CgMytC,
transfers mycoloyl groups from TMM onto polypeptides to
generate O-mycoloylated proteins (see Figure 1A).[45] Our group
previously showed that O-AlkTMM-C7 could be used to
metabolically label O-mycoloylated proteins in Cglut with alkyne
tags, enabling their subsequent visualization using CuAAC
followed by SDS-PAGE with in-gel fluorescence analysis.[30] We
reasoned that using this experimental platform with a Cglut
mutant lacking CgMytC would allow us to assess whether TMM
reporters operate in a mycoloyltransferase-dependent manner,
because (i) CgMytC is not essential; and (ii) this system
eliminates the issue of mycoloyltransferase redundancy—again,
available evidence suggests that only CgMytC adds O-mycolates
to proteins in Cglut. Thus, we expected it would provide a facile
readout for specific probe incorporation.
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the new TMM reporters 4–6. After washing the cells, aliquots for
each condition were collected, secondarily labeled with a
fluorophore using the appropriate conditions (if necessary), and
analyzed by flow cytometry. As depicted in Figure 6A, cellular
fluorescence analysis was consistent with our aforementioned
Msmeg mutant results—the Δcg0413 mutant showed increased
fluorescence for all of the probes compared to wild-type Cglut. As
suggested above for the Msmeg mutants, we speculate that this
increased labeling in the CgMytC mutant was due to
a
combination of increased cellular permeability and the activity of
the remaining mycoloyltransferases in the mutant. Next, we lysed
the remaining TMM reporter-treated cells to collect proteins. The
lysates were subjected to appropriate secondary fluorophore
labeling (if necessary), then resolved by SDS-PAGE and
analyzed by in-gel fluorescence (Figure 6B). O-AlkTMM-C7 and
O-AzTMM-C10 showed robust 488 fluorescence for numerous
proteins in the 10–75 kDa range, consistent with our prior work
with O-AlkTMM-C7,[30] and labeling was completely abolished in
the Δcg0413 mutant. The similar probe O-AlkTMM-C11 gave
results identical to those obtained for O-AlkTMM-C7 and O-
AzTMM-C10 (data not shown). Likewise, the tetrazine ligation-
capable reporter O-TCO-TMM exhibited CgMytC-dependent
protein labeling, with a major band in the 10–15 kDa region
(
similar to the O-AlkTMM and O-AzTMM compounds) and lower
fluorescence associated with larger proteins, perhaps because
this larger probe selectively labels smaller proteins. These data
suggest that the O-AlkTMM series, O-AzTMM-C10, and O-TCO-
TMM specifically incorporate into mycolate-containing envelope
components via mycoloyltransferase activity, as hypothesized.
O-FITC-TMM also labeled Cglut proteins (Figure 6B), but
this signal was present in both wild-type Cglut and the Δcg0413
mutant, implying a lack of CgMytC dependence. Our other data
(
e.g., Figures 4, 5, and 6A) demonstrate consistent behavior
between O-FITC-TMM and the other TMM reporters, so it is
possible that O-FITC-TMM is not a good substrate for CgMytC but
it is nonetheless efficiently processed by other glycolipid-
synthesizing mycoloyltransferases. Regardless, based on the
Cglut protein labeling results (Figure 6B), it cannot be ruled out
that O-FITC-TMM incorporates into mycomembrane-containing
bacteria by a mycoloyltransferase-independent route. Taken
together, the new TMM reporters described in this study generally
behaved like the previously reported O-AlkTMM-C7 compound
with respect to their mycomembrane specificity. However,
because we could not confirm that O-FITC-TMM labeled bacteria
via mycoloyltransferase activity, at present this compound should
be limited to applications where mycobacteria-specific surface
labeling is desired but the labeling route is inconsequential. It will
be of interest to further explore the ramifications of adding large
fluorophores (or other sizeable chemical cargo) to the TMM
scaffold in follow-on studies. For instance, it was recently shown
that D-amino acid-based reporters of peptidoglycan synthesis
incorporate via different labeling routes depending on the nature
of the detectable tag.[40]
Figure 5. (A) Cellular labeling comparison of O-FITC-TMM (6) and its trehalose-
deficient variant 9. Msmeg was incubated with 6 or 9 (100 µM), or left untreated,
then analyzed by flow cytometry. Error bars denote the standard deviation of
three replicate experiments. (B) Labeling distribution of compounds 4–6 in
different cellular fractions. Msmeg was incubated with 4 (25 µM), 5 (25 µM), 6
(100 µM), 9 (100 µM), or left untreated for 4 h, then reacted with an appropriate
fluorophore (if necessary), fractionated into an AGM-containing fraction and
extractable lipids, and analyzed using a fluorescence plate reader. Error bars
denote the standard deviation of three replicate experiments.
To test this CgMytC deletion approach in Cglut, we
regenerated the CgMytC-deficient mutant Cglut Δcg0413
reported in the literature[45] by homologous recombination and
confirmed the absence of the gene by both PCR and sequencing
(
Table S1 and Figure S2). Cglut wild type and the Δcg0413
mutant were incubated in the presence of O-AlkTMM-C7 (2) and
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Figure 6. Mycoloyltransferase dependence of labeling Cglut cells (A) and Cglut O-mycoloylated proteins (B) with TMM reporters. Cglut wild type or Δcg0413 mutant
were incubated overnight with 100 µM 2, 4, 5, 6 (or 9), or left untreated, then aliquots of cells were removed and reacted with Az488 (for 2), Alk488 (for 4), tetrazine-
Cy3 (for 5), or left untreated (for 6 and its corresponding control compound 9), and analyzed by flow cytometry. Error bars denote the standard deviation of three
replicate experiments. From the remaining cells, lysates were collected and reacted with the same secondary reagents (if needed), then analyzed by SDS-PAGE.
For each probe evaluated, lanes 1 and 2 are from Cglut wild type and lanes 3 and 4 are from Cglut Δcg0413; the Coomassie-stained gel is shown on the left, and
in-gel fluorescence is shown on the right.
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Conclusions
inactivation should be designed and interepreted cautiously.
Irrespective of this issue, TMM-based reporters have numerous
capabilities with respect to mycobacteria research, including
visualization of envelope dynamics, characterization of
mycomembrane proteins, specific detection of mycobacterial cells,
and versatile cell-surface engineering of live mycobacteria.
In summary, we reported herein the efficient chemical
synthesis and evaluation of a collection of TMM-based metabolic
reporters that will facilitate research on the mycomembrane. The
chemical tags appended to the TMM analogues‘ acyl chains allow
labeling of mycomembrane-containing bacteria through two-step
strategies employing the major bioorthogonal reactions (CuAAC,
SPAAC, or tetrazine ligation) or one-step strategies employing Experimental Section
TMM reporters bearing pre-attached fluorophores. In addition to
providing improved flexibility in experimental design versus the
original O-AlkTMM-C7 reporter, the new reporters can be
deployed in living mycobacteria. Of particular interest, this is the
first method reported for modifying the mycobacterial cell surface
using the rapid and highly efficient TCO–tetrazine ligation.
The Supporting Information contains supplementary results, experimental
procedures, and NMR spectra of synthetic compounds.
Acknowledgements
In addition, this study further exemplified the high
promiscuity of cellular mycoloylation machinery for unnatural
TMM analogues. This feature of mycobacteria, along with the
versatile synthetic intermediates (i.e., 4 and 8) and high specificity
of TMM-based reporters for mycobacteria reported herein, shows
that TMM analogues can be used to develop novel strategies for
targeting mycobacteria with various types of chemical payload.
Indeed, the TMM scaffold offers opportunities for the development
of new mycomembrane-targeting probes. For instance, the
Kiessling group recently expanded on the TMM reporter concept
with a FRET-based TMM analogue, called QTF, which fluoresced
upon Ag85-mediated separation of its trehalose-linked quencher
and “lipid“-linked fluorophore.[46] QTF does not label
mycomembrane components, rather it allows direct visualization
of mycoloyltransferase activity, a novel and attractive feature that
complements the TMM reporters developed by our group so far.
B.M.S. was supported by an NSF CAREER Award (1654408), a
Cottrell College Scholar Award from the Research Corporation for
Science Advancement (22525), and a Henry Dreyfus Teacher-
Scholar Award from The Camille & Henry Dreyfus Foundation
(
TH-17-034). M.S.S was supported by the National Institutes of
Health (NIH DP2 AI138238). We thank R. J. Hood for assistance
with NMR spectroscopy and A. N. Ilacqua for assistance with flow
cytometry.
Keywords: Corynebacterineae • mycobacteria •
mycomembrane • mycolic acid • trehalose • chemical reporter •
bioorthogonal chemistry • imaging
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Taylor J. Fiolek, Nicholas Banahene,
Herbert W. Kavunja, Nathan J. Holmes,
Adrian K. Rylski, Amol A. Pohane, M.
Sloan Siegrist, and Benjamin M. Swarts*
Page No. – Page No.
Engineering the Mycomembrane of
Live Mycobacteria with an Expanded
Set of Trehalose Monomycolate
Analogues
Modifying mycobacteria via mycoloylation: A suite of trehalose monomycolate
(
TMM)-based metabolic reporters provides versatility and specificity for analyzing
and engineering the outer membrane of living mycobacteria.
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†
†
[
a]
T. J. Fiolek, N. Banahene, H. W. Kavunja, N. J. Holmes,
Rylski, Prof. B. M. Swarts
Department of Chemistry & Biochemistry
Central Michigan University
Mount Pleasant, MI (USA)
This article is protected by copyright. All rights reserved.
E-mail: ben.swarts@cmich.edu