Visualization of mycobacterial membrane dynamics in live cells

Article abstract of DOI:10.1021/jacs.6b12541

Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose-fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein-trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.

Full text of DOI:10.1021/jacs.6b12541

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Article
Visualization of mycobacterial membrane dynamics in live cells
Frances P. Rodriguez-Rivera, Xiaoxue Zhou, Julie A Theriot, and Carolyn R Bertozzi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b12541 • Publication Date (Web): 11 Jan 2017
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Visualization of mycobacterial membrane dynamics in live cells
†‡
§
§ #
‡#
∥
Frances P. RodriguezꢀRivera , Xiaoxue Zhou , Julie A. Theriot , Carolyn R. Bertozzi *
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† Department of Chemistry, University of California, Berkeley, CA 94720
‡ Department of Chemistry, Stanford University, Stanford, CA 94305
§ Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305
∥Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
# Howard Hughes Medical Institute
KEYWORDS mycobacteria, cell envelope, trehalose mycolate, metabolic labeling, live-cell imaging
ABSTRACT: Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many anꢀ
tibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organizaꢀ
tion and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane
dynamics that was enabled by trehaloseꢀfluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We
identified fluoresceinꢀtrehalose analogs that are metabolically incorporated into the trehalose mycolates of representative Mycobac-
terium, Corynebacterium, Nocardia and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids
by timeꢀlapse microscopy and fluorescence recovery after photobleaching (FRAP) experiments and found that mycomembrane
fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacꢀ
teria with ethambutol, a frontꢀline tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance
our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cockꢀ
tails.
Given its importance in protecting Mtb from drug action, the
cell envelope has been a focus of considerable structural work
INTRODUCTION
Tuberculosis (TB), the leading cause of death worldwide
from a single infectious agent, Mycobacterium tuberculosis
centered on isolating and identifying its various components
and visualizing layers by electron microscopy (EM).7 CryoEM
(Mtb), took 1.5 million lives in 2014 and remains a global
public health emergency.1 Mtb infects host macrophages and
studies have added information about the highly organized
vertical architecture of the cell envelope at high resolution and
survival within that hostile environment depends on an imꢀ
in a native state.9,10 Far less is known, however, about the
permeable cell envelope that protects bacilli from biological
stresses.2,3 Furthermore, the cell envelope has proven to be a
dynamics of the cell envelope and very few studies have foꢀ
cused on the mycomembrane, the major contributor to barrier
formidable physical barrier against many antibiotics that might
otherwise be efficacious against Mtb.4,5 For this reason, TB
function. This deficit likely reflects a lack of tools for probing
cell envelope metabolites in live cells, and with subcellular
must be treated with drug combinations that include at least
one compound that compromises cell wall integrity.6
resolution, a problem that chemists have tackled in recent
years with the development of new imaging methods.11 For
The mycobacterial cell envelope comprises inner membrane
example, early work using lipophilic fluorophore conjugates
and peptidoglycan layers that are similar to those of common
that nonspecifically intercalate into the mycomembrane sugꢀ
gested a heterogeneous cell surface landscape.12,13 Progress
Gramꢀnegative and –positive organisms, but then diverges
considerably in the molecular composition of its outer layers
toward defining the dynamic properties of Mtb’s cell enveꢀ
(Figure. 1A). Most prominent are mycolic acids that are covaꢀ
lope, however, requires more refined methods for imaging its
lently anchored to arabinogalactan chains.7 Up to an impresꢀ
specific cell envelope components.
sive 90 carbon atoms in length,8 these lipids constitute the
Recently, metabolic labeling has proven to be an effective
inner leaflet of the mycomembrane and form an interface with
strategy to image trehalose glycolipids in the mycomembrane
an outer leaflet composed of nonꢀcovalently associated glycoꢀ
of live mycobacteria.14–17 The approach exploits the promisꢀ
lipids, the most abundant being trehalose monomycolate
cuity of the antigen 85 complex (Ag85), a family of mycolꢀ
(TMM) and dimycolate (TDM) (Figure 1B). The result is a
yltransferases that convert two molecules of TMM to TDM
functional outer membrane that is unique to Mtb and other
members of the suborder Corynebacterineae.
and free trehalose (Figure 1A). In a screen of dozens of trehaꢀ
lose analogs, Backus et al. found that the backward reaction
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Metabolic labeling
Mycomembrane
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Outer leaflet
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Tre
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TDM
Inner leaflet
Ag85
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AG
Arabinogalactan
(AG)
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Peptidoglycan
TMM
Inner membrane
Cytosol
Legend
X
Peptigoglycan
Arabinose
Trehalose
metabolic
reporter
Trehalose
monomycolate (TMM)
Trehalose
dimycolate (TDM)
AG-mycolate
Mycobacterial
free lipids
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Species
M. smegmatis
M. tuberculosis
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OH
37ꢀ48
49ꢀ65
19, 21
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Trehalose
Trehalose monomycolate
(TMM)
Trehalose dimycolate
(TDM)
Figure 1. Mycobacterial cell envelope and structures of trehalose mycolates. A) Mycobacterial cell envelope components include
inner membrane, peptidoglycan, arabinogalactan, mycomembrane, and capsule. Antigen 85 mediates mycolylation of arabinogalacꢀ
tan from TMM donor. Two molecules of TMM are used to generate TDM, thereby releasing one molecule of trehalose. Antigen 85
is predicted to be active in the mycomembrane. Red dashed line depicts metabolic incorporation route for unnatural trehalose reꢀ
porters. B) Chemical structures for trehalose, trehalose monomycolate and trehalose dimycolate. Number of carbons (n1, n2) strictly
in linear chains of mycolates are shown for M. smegmatis and M. tuberculosis. Trehalose monomycolate (TMM), trehalose dimycoꢀ
late (TDM), trehalose (Tre), Antigen 85 (Ag85), arabinogalactan (AG).
can be used to deliver unnatural trehalose derivatives into
Mtb’s mycomembrane (Figure 1A), including a fluorescein
conjugate (FITCꢀTre, Figure 2A).14 This observation suggests
a means by which mycomembrane dynamics could be directly
interrogated by molecular imaging.
structure. Finally, we probed the effects of the frontꢀline TB
drug ethambutol on mycomembrane dynamics in live M.
smegmatis cells. We conclude that drugs targeting the Mtb
cell envelope influence mycomembrane fluidity and this paꢀ
rameter might therefore be considered when evaluating new
drug combinations.
Here we identified fluoresceinꢀtrehalose analogs that are
recognized by mycolyltransferases of diverse actinobacterial
species, including Mycobacteria, Corynebacteria, Nocardia
and Rhodococcus genera. We used the new probes to deterꢀ
mine the subcellular distribution and dynamics of trehalose
mycolates within the mycomembrane of live cells. Using fluꢀ
orescence recovery after photobleaching (FRAP) experiments,
we found a striking disparity in mycomembrane mobilities
across species, which partially correlated with mycolic acid
RESULTS AND DISCUSSION
Design and synthesis of new fluorescein-trehalose conju-
gates. Our first goal was to develop a fluorescent trehalose
reagent that efficiently labels trehalose mycolates across many
actinobacterial species. A survey of previously reported trehaꢀ
lose analogs revealed them to be unsuitable for various reaꢀ
sons. We found that FITCꢀTre (Figure 2A) labeled Mycobac-
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Figure 2. Metabolic engineering of trehalose glycolipids with unnatural trehalose reporters. A) Previously reported unnatural trehaꢀ
lose reporters including FITCꢀTre, TreAz analogs and alkTMM analogs. B) Library of fluoresceinꢀtrehalose analogs (this work).
Fluorescein isothiocyanate (FITC), azidoꢀtrehalose (TreAz), fluoresceinꢀtrehalose (FlTre).
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teria and Nocardia species with poor efficiency, and did not
label glycolipids of Corynebacteria at a detectable level (vide
infra). We speculate that the anomeric methyl group (highꢀ
lighted in red, Figure 2A), a directing group used by Backus et
al. to form the α,αꢀ1,1ꢀglycosidic linkage, may compromise
the processing of FITCꢀTre by the Ag85 complex.14
compared to FITCꢀTre. Accordingly, we synthesized the panꢀ
el of regioisomeric fluoresceinꢀtrehalose conjugates (FlTre,
shown in Figure 2B), which all possess a native trehalose core
structure. The compounds were prepared from the correꢀ
sponding TreAz analogs by reduction to the amines followed
by reaction with fluorescein isothiocyanate (FITC) (details
provided in Supporting information). Notably, 2ꢀ, 3ꢀ and 4ꢀ
FlTre could, if recognized by Ag85, label both TMM and
TDM, whereas 6ꢀFlTre can only be metabolized to TMM.
Alternatively, we considered our own previously reported
azidoꢀtrehalose derivatives (TreAz, Figure 2A), which are
metabolically incorporated into trehalose glycolipids by severꢀ
al mycobacterial species and through both cytosolic and extraꢀ
cellular pathways.15 However, these analogs get transformed
to additional classes of trehalose metabolites beyond TMM
and TDM, which complicates data interpretation. Moreover,
the secondary reagents used to attach fluorescent probes (e.g.
cyclooctyneꢀfluorophore conjugates) have limited access to
the mycomembrane and engage in nonꢀspecific interactions.
Recently, Swarts and coworkers elegantly introduced alkyneꢀ
functionalized trehalose analogs (alkTMM, Figure 2A) into
the mycomembrane.17 This approach also requires the use of
secondary labeling reagents as well as Cu catalysis that may
be cytotoxic.
Fluorescein-trehalose analogs selectively label trehalose
mycolates. We tested the labeling activity of the FlTre analogs
as well as FITCꢀTre using live cultures of Mycobacterium
smegmatis and Corynebacterium glutamicum, organisms
whose cell envelope composition and structure are similar to
Mtb’s.18 All FlTre analogs outperformed FITCꢀTre in both
species as evaluated by flow cytometry (Figure 3A and B).
Surprisingly, FITCꢀTre was not metabolized at a detectable
level by C. glutamicum, while 2ꢀFlTre, which only differs
from FITCꢀTre by lacking an anomeric methyl group, labeled
strongly (Figure 3B). This observation illustrates that small
perturbations to probe structure can alter, or in this case, comꢀ
pletely ablate labeling. The brightest labeling was observed
with 6ꢀFlTre, while 2ꢀ, 3ꢀ and 4ꢀFlTre showed lower labeling
in both species.
In light of these issues, we returned to the notion of oneꢀstep
labeling with a trehaloseꢀfluorophore conjugate and focused
on developing reagents with improved metabolic efficiency
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morphology were observed under these conditions. In particuꢀ
lar, M. smegmatis showed polar labeling when treated for
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C. glutamicum
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shorter incubations suggesting incorporation into newly synꢀ
thesized cell envelope, where biosynthetic enzymes reside19,20
(Figure S1A). Treatment of bacteria that lack trehalose mycoꢀ
lates, such as canonical Gramꢀnegative Escherichia coli and
Gramꢀpositive Bacillus subtilis, with 6ꢀFlTre afforded no deꢀ
tectable labeling (Figure S1B).
To confirm that the FlTre isomers are biosynthetically conꢀ
verted to trehalose mycolates, we assessed whether exogenous
trehalose, a native substrate for mycolyltransferases, could
compete with reporter labeling. Bacterial cells that were coinꢀ
cubated with 2ꢀ, 3ꢀ, 4ꢀ or 6ꢀFlTre (100 ꢁM) and trehalose (0,
0.5, 5 mM) showed a doseꢀdependent decrease in metabolic
labeling (Figure S2). In addition, we found that ebselen, an
inhibitor of Ag85 activity in mycobacteria,21,22 decreased 2ꢀ, 3ꢀ
, 4ꢀ and 6ꢀFlTre labeling in both M. smegmatis and C. glu-
tamicum (Figure S2). Furthermore, partially purified trehalose
glycolipids from 2ꢀ or 6ꢀFlTreꢀlabeled C. glutamicum were
analyzed by mass spectrometry. For both analogs, we obꢀ
served ions corresponding to the expected fluoresceinꢀ
conjugated TMM analogs (Figure S3, Table S1). Finally, total
lipids extracted from 6ꢀFlTre labeled M. smegmatis cells
showed a single fluorescent band by TLC (Figure S3). Collecꢀ
tively, these data demonstrate that FlTre analogs are biosynꢀ
thetically converted to trehalose mycolates in live bacterial
cells.
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M. smegmatis
C
C. glutamicum
D
Fluorescein-trehalose analogs report on mycomembrane
dynamics in live bacteria. We next sought to investigate the
mobility of trehalose glycolipids in the mycomembrane during
the course of cell growth. To that end, we performed twoꢀ
color imaging experiments wherein we first labeled cells with
6ꢀFlTre to visualize trehalose glycolipids, then later marked
newly formed cell wall with a peptidoglycan (PG) reporter,
tetramethylrhodamine Dꢀlysine (TDL), which replaces Dꢀ
alanine residues in the stem peptides.23 M. smegmatis was
labeled with 6ꢀFlTre for several generations, washed to reꢀ
move excess reporter, and chased with TDL for 5 or 20 min
(Figure 4A). Fluorescence microscopy revealed that labeled
trehalose mycolates were excluded from newly biosynthesized
cell wall at the poles during growth, as denoted by minimal
overlap with polar labeling of PG. These results suggest that
trehalose glycolipids in the mycomembrane remain rather
immobile during growth in M. smegmatis. Conversely, C. glu-
tamicum showed complete redistribution of labeled glycolipids
after 6ꢀFlTre had been removed from the growth media for the
same time periods (Figure 4B). We were intrigued by the lack
of fluidity in the mycobacterial mycomembrane that did not
allow diffusion of labeled trehalose glycolipids to new cell
envelope regions. Indeed, low fluidity has been predicted
based on extremely low permeability of the mycomembrane to
lipophilic molecules24 as well as differential scanning calorimꢀ
etry (DSC) studies that revealed high temperature phase transiꢀ
tions in Mycobacteria.25–27 Our results provide direct experiꢀ
mental confirmation of this prediction in live cells.
Figure 3. Metabolic labeling of actinobacteria with FlTre anaꢀ
logs. Labeling profile of FlTre analogs compared to FITCꢀTre
in M. smegmatis (A) and C. glutamicum (B). Bacteria were
labeled with 100 ꢁM trehalose analogs or vehicle for at least 5
doubling times and analyzed by flow cytometry. Error bars
depict standard deviation of three replicate experiments. Reꢀ
sults are representative of at least two independent experiꢀ
ments. Mean fluorescence intensity (MFI). Fluorescence miꢀ
croscopy images of M. smegmatis (C) and C. glutamicum (D)
cells labeled with vehicle or 6ꢀFlTre. Scale bar, 2 ꢁm.
In addition, we evaluated the spatial distribution of the fluoꢀ
rescence labeling by microscopy. Gratifyingly, cell envelope
labeling was observed for both M. smegmatis and C. glutami-
cum after incubation with 100 ꢁM FlTre for several doubling
times (Figure 3, C and D, respectively). No defects in cell
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mobility phenomena were also observed when cells were laꢀ
A
M. smegmatis
1
beled with 2ꢀFlTre (Figure S5) or trehalose reporters modified
with different fluorophores at the same position (Figure S6,
and probe characterization is shown in Figure S4), suggesting
that the glycolipid dynamics we observed are not significantly
altered by probe structure. In addition, for a control experiꢀ
ment, we performed FRAP analysis on cells that were metaꢀ
bolically labeled with Dꢀamino acid reporters bearing the same
fluorophores. As expected, the fluorophores integrated into
PG were essentially immobile (Figure S7).
Merge
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TDL
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TDL
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B
C. glutamicum
Merge
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TDL
Merge
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TDL
Figure 4. FlTreꢀlabeled trehalose glycolipids show speciesꢀ
dependent mobility. A) Preꢀlabeled M. smegmatis glycolipids (6ꢀ
FlTre, green) are excluded from the poles as depicted by peptiꢀ
doglycan labeling (TDL, red). B) Preꢀlabeled C. glutamicum glyꢀ
colipids are highly mobile and redistribute to the entire cell after
6ꢀFlTre has been removed from the growth media. Scale bar, 2
ꢁm.
Mycolic acid-producing actinobacteria have a wide
range of mycomembrane fluidities that correlate with my-
colic acid chain structure. Fluorescence recovery after phoꢀ
tobleaching (FRAP)28 experiments have been previously used
to quantitate diffusion dynamics and subcellular organization
of membrane components in live bacteria with high temporal
and spatial resolution.29 We therefore applied the FRAP techꢀ
nique to directly elucidate the intrinsic mobility of labeled
trehalose glycolipids in mycobacteria. Photobleaching of polar
and midꢀcellular regions of M. smegmatis preꢀlabeled with 6ꢀ
FlTre revealed that trehalose glycolipids failed to move after
irradiation (Figure 5A), even when monitored up to 10 min.
However, under the same experimental conditions, C. glu-
tamicum glycolipids diffused through the photobleached area
in a few seconds (Figure 5B). The relatively high fluidity obꢀ
served for the corynebacterial mycomembrane is consistent
with previously reported lower temperature phase transitions
as measured by DSC.25 Quantitation of fluorescence recovery
traces and halfꢀtime measurements revealed similar recovery
dynamics regardless of the photobleached area of the cell
(pole or center) as shown in Figure 5C. Similar glycolipid
Figure 5. Mycobacterial trehalose glycolipids are relatively
immobile. A) FRAP experiment of 6ꢀFlTre labeled M. smeg-
matis (A) and C. glutamicum (B) after irradiation at the pole
(left panel) and center (right panel). Scale bar, 2 ꢁm. C) Fluoꢀ
rescence recovery curves after photobleaching for center and
pole regions in 6ꢀFlTre labeled cells across different bacterial
species with the corresponding number of carbon atoms in
mycolic acid chains. Lines represent the averaged signal of n ≥
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6 cells, where solid and dotted lines correspond to pole and
DSC studies.25–27,36 Taken together, our results correlate realꢀ
time mycomembrane fluidity to mycolic acid structure across
several actinobacteria.
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center regions, respectively. Comparison of fraction mobile
(D) and apparent diffusion coefficient (E) extracted from fitꢀ
ting FRAP curves for different actinobacterial species. Filled
and open circles correspond to pole and center regions, respecꢀ
tively. Number of cells evaluated with the corresponding mean
and standard deviation are shown for every species. pꢀvalues
between samples were calculated with a rank sum test. Fluoꢀ
rescence recovery after photobleaching (FRAP), M. smegmatis
(Ms), C. glutamicum (Cg), R. equi (Re), N. brasiliensis (Nb),
apparent diffusion coefficient (Dapp), not determined (n.d.)
Ethambutol treatment alters mycomembrane dynamics.
Impermeable membranes within the cell envelope can serve as
static barriers against antibiotics and biological stresses. Genes
involved in the biosynthesis of the bacterial cell envelope are
essential for growth and division and thus are important antiꢀ
biotic targets.7,37 Our imaging strategy could find broad appliꢀ
cations in characterizing mycobacterial cell wall changes in
live cells upon treatment with current frontꢀline TB drugs. For
example, ethambutol inhibits arabinosyl transferase EmbB that
installs arbaninose residues to growing arabinogalactan
chains,38,39 which results in reduced sites for mycolylation of
the inner leaflet of the mycomembrane. We hypothesized that
the fluidity of the mycomembrane could be altered by treatꢀ
ment with ethambutol as a result of changing the membrane’s
physical properties.
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In order to get a more quantitative understanding of obꢀ
served diffusion phenomena, we modeled recovery data to
calculate the apparent diffusion coefficients for labeled trehaꢀ
lose mycolates (Figure S8). A diffusion coefficient of 0.06 ±
0.02 ꢁm2sꢀ1 (n = 20 cells) was obtained for 6ꢀFlTre labeled
glycolipids in C. glutamicum, while the lack of observable
diffusion in M. smegmatis limited our ability to model fluoresꢀ
cence recovery. For comparison, lipopolysaccharide (LPS), an
abundant glycolipid in the outer membrane of Gramꢀ negative
E. coli, diffuses slightly slower (0.020 ± 0.009 ꢁm2sꢀ1) as deꢀ
termined by an exogenous rhodamineꢀLPS conjugate.30 Howꢀ
ever, antibody binding of LPS to visualize glycolipid dynamꢀ
ics resulted in much slower diffusion dynamics (2.0 x 10ꢀ5
ꢁm2sꢀ1),31 suggesting potential perturbation by antibody detecꢀ
tion and underscoring the benefits of directly visualizable glyꢀ
colipids.
We evaluated trehalose mycolate’s mobility in 6ꢀFlTre preꢀ
labeled mycobacteria after ethambutol treatment. TDL labelꢀ
ing of PG marked cell wall biogenesis and also reported on
relative growth rates, an indicator of drug toxicity. M. smeg-
matis cells were preꢀlabeled with 6ꢀFlTre over several generaꢀ
tions and treated with different ethambutol doses for 3 h while
the reporter was still present. Cells were then chased with
TDL for 20 min, washed, fixed and imaged by microscopy.
As shown in Figure 6A, untreated cells showed exclusion of
preꢀlabeled glycolipids from new cell wall regions. However,
ethambutol treatment at doses as low as 0.5 ꢁg/mL led to reꢀ
distribution of labeled trehalose mycolates across the entire
cell surface, with accumulation of signal at the poles (Figure
6B). M. smegmatis has been reported to upregulate trehalose
mycolate biosynthesis after exposure to ethambutol,40,41 which
is consistent with higher metabolic labeling observed at the
poles during drug treatment. Overall, these results indicate that
ethambutol treatment enhances mycomembrane fluidity. Noꢀ
tably, we observed increased diffusion for subꢀminimal inhibiꢀ
tory concentration (MIC) doses (ethambutol’s MIC = 1.0
ꢁg/mL for M. smegmatis42). To confirm ethambutol’s effects
on mycomembrane dynamics, we examined ethambutol treatꢀ
ed cells by FRAP analysis, which revealed increased fraction
of mobile glycolipids in a drug doseꢀdependent manner (Figꢀ
ure 6 C and D, Figure S11). Our results demonstrate that myꢀ
comembrane fluidity can be altered with subꢀlethal antibiotic
concentrations, which could improve permeability and drug
accessibility in the context of coꢀtherapy. Modulation of myꢀ
comembrane integrity with sublethal concentrations of ethamꢀ
butol has been previously shown to reverse Mtb resistance to
clarithromycin,43 suggesting reduction of the barrier effectiveꢀ
ness of the mycomembrane. Moreover, increased susceptibilꢀ
ity to the betaꢀlactam cefepime has been observed during coꢀ
treatment with ethambutol.44 Together, these reports provide
precedent for potentiating approved therapeutics and repurposꢀ
ing high MIC drugs by coꢀtreatment with low concentrations
of ethambutol.6 Understanding how the permeability of the
bacterial cell envelope can be modulated could facilitate the
design or access of new therapeutic agents.
Our results, consistent with DSC studies, suggest a potential
correlation between trehalose mycolate structure and the emꢀ
pirically determined fluidity of the mycomembrane. Mycolic
acid chain lengths vary significantly between C. glutamicum
(22ꢀ38 carbon atoms)32 and M. smegmatis (60ꢀ81 carbon atꢀ
oms).33 Fascinated by the dramatic differences in trehalose
glycolipid mobility observed for M. smegmatis and C. glu-
tamicum, we sought to test the correlation of mycomembrane
fluidity and mycolic acid length by extending our analysis to
other bacterial species. Rhodococcus and Nocardia species
synthesize trehalose glycolipids with intermediate length myꢀ
colic acids, ranging from 30ꢀ54 and 46ꢀ60 carbon atoms, reꢀ
spectively.34,35 We thus hypothesized that Rhodococcus equi
and Nocardia brasiliensis would show intermediate glycolipid
mobilities relative to corynebacteria and mycobacteria. FlTre
analogs labeled both species as confirmed by flow cytometry
and fluorescence microscopy (Figure S9). FRAP mobility
studies revealed similar fluorescence recovery profiles for R.
equi, and C. glutamicum, while N. brasiliensis displayed an
intermediate recovery profile (Figure 5C). These results were
corroborated by evaluation of the fraction mobile of labeled
trehalose glycolipids, which showed striking differences beꢀ
tween all bacterial species (Figure 5D). Calculated apparent
diffusion coefficients revealed small differences between speꢀ
cies that were not statistically significant, suggesting subtle
effects on diffusion for minor variations in mycolic acid chain
length. The diffusion coefficient for M. smegmatis was not
calculated since recovery was not observed after 10 min (Figꢀ
ure 5A). However, functional groups such as ketones, methꢀ
oxy, and cyclopropyl groups in mycobacterial mycolic acids
significantly influence membrane fluidity as determined by
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A
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1. Ethambutol 3 h
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3. TDL 20 min
Ethambutol
10 ꢁg/mL
2 ꢁg/mL
0.5 ꢁg/mL
0 ꢁg/mL
0.8
0.6
0.4
0.2
0
4. Wash
5. Microscopy
6ꢀFlTre
M. smegmatis
Increasing mycoꢀ
membrane fluidity
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6FlTre
TDL
B
(ꢁg/mL)
0
50
100 150 200 250 300
Time (s)
0
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< 10ꢀ2
p
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p = 0.5
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(
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.
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(
9
n
n
n
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=
=
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5
)
8
5
)
)
)
Ethambutol concentration (ꢁg/mL)
Figure 6. FlTre metabolic labeling reports on cell wall dynamics during ethambutol (EMB) treatment. A) Scheme depicting experimental
workflow and labeling pattern expected for differential mycomembrane fluidity. M. smegmatis was preꢀlabeled with 6ꢀFlTre, then treated
with EMB at different concentrations for 3 h. Cells were washed to remove excess 6ꢀFlTre and chased with TDL in the absence of drug for
20 min. Spatial distribution of labeled glycolipids was evaluated by fluorescence microscopy after fixation (B). Representative cells are
shown in each panel. 6ꢀFlTre and TDL signals are in green and red channels, respectively, for merged images (left panel). FRAP recovery
traces (C) and fraction mobile (D) for cells treated with different concentrations of EMB. Fluorescence recovery trace lines represent the
averaged signal of n ≥ 5 cells. Number of cells evaluated with the corresponding mean and standard deviation are shown for all concentraꢀ
tions. pꢀvalues between samples were calculated with a rank sum test. Scale bar, 2 ꢁm.
found a relationship between diffusion kinetics and mycolic
acid chain structure. We also discovered that ethambutol, a
CONCLUSION
The complex and unusual cell envelope of mycobacteria has
captured the attention of biologists, chemists, and biophysiꢀ
cists alike for almost a century. Tightly packed mycolic acids
in the mycomembrane were first predicted by Minnikin to
explain the observed impermeability to lipophilic moleꢀ
cules.24,45 Nikaido and coworkers validated the prediction of a
quasiꢀcrystalline arrangement of mycolic acids extending perꢀ
pendicularly from the cell wall plane by Xꢀray absorption
studies.46 As well, they elegantly studied the mobility of spinꢀ
labeled fatty acids inserted into mycomembranes from M.
chelonae cell walls.47 Additionally, DSC studies provided an
empirical foundation for our understanding of membrane fluꢀ
idity in terms of phase transition temperatures across bacterial
cell walls.25 This pioneering series of studies provided insights
into mycomembrane dynamics in the context of isolated cell
walls preparations, but could not be extended to studies in live
cells.
frontꢀline TB drug, enhances mycomembrane fluidity at subꢀ
lethal doses, an effect that may underlie its synergism with
other TB drugs. Consistent with this postulate is a recent reꢀ
port that mycobacteriophage SWU1 gp39 affects M. smegmaꢀ
tis’ cell envelope permeability and thereby potentiates the
efficacy of multiple antibiotics.48 These observations argue
that more highꢀthroughput screens against Mtb should be perꢀ
formed in the presence of a mycomembraneꢀcompromising
agent such as ethambutol.44,49–52
Furthermore, the imaging method we developed here could
be adapted to visualize trehalose glycolipid dynamics in the
context of host cell infection, as trehalose is metabolically
orthogonal to eukaryotic hosts. Consequent insights could
provide avenues for development of new drug combinations
for the treatment of TB.
ASSOCIATED CONTENT
Supporting Information
The targeted metabolic labeling strategy used herein enaꢀ
bled direct interrogation of trehalose glycolipids in live cells,
in their native context, with minimal perturbation. With this
approach, we quantitated trehalose glycolipid mobilities and
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Page 8 of 23
(19) Carel, C.; Nukdee, K.; Cantaloube, S.; Bonne, M.; Diagne,
C. T.; Laval, F.; Daffé, M.; Zerbib, D. PLOS ONE 2014, 9 (5),
e97148.
(20) Meniche, X.; Otten, R.; Siegrist, M. S.; Baer, C. E.; Murꢀ
phy, K. C.; Bertozzi, C. R.; Sassetti, C. M. Proc. Natl. Acad. Sci.
2014, 111 (31), E3243.
Experimental procedures, characterization data, supporting figꢀ
ures, and schemes. This material is available free of charge on the
ACS Publications website.
1
2
3
4
Supporting Information (PDF)
5
6
7
8
(21) Favrot, L.; Grzegorzewicz, A. E.; Lajiness, D. H.; Marvin,
R. K.; Boucau, J.; Isailovic, D.; Jackson, M.; Ronning, D. R. Nat.
Commun. 2013, 4, 2748.
(22) Favrot, L.; Lajiness, D. H.; Ronning, D. R. J. Biol. Chem.
2014, 289 (36), 25031.
(23) Kuru, E.; Hughes, H. V.; Brown, P. J.; Hall, E.; Tekkam,
S.; Cava, F.; de Pedro, M. A.; Brun, Y. V.; VanNieuwenhze, M. S.
Angew. Chem. Int. Ed. 2012, 51 (50), 12519.
(24) Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64
(1), 29.
(25) Liu, J.; Barry, C. E.; Besra, G. S.; Nikaido, H. J. Biol.
Chem. 1996, 271 (47), 29545.
(26) George, K. M.; Yuan, Y.; Sherman, D. R.; Barry, C. E. J.
Biol. Chem. 1995, 270 (45), 27292.
(27) Yuan, Y.; Crane, D. C.; Musser, J. M.; Sreevatsan, S.; Barꢀ
ry, C. E. J. Biol. Chem. 1997, 272 (15), 10041.
(28) IshikawaꢀAnkerhold, H. C.; Ankerhold, R.; Drummen, G.
P. C. Molecules 2012, 17 (4), 4047.
(29) Ghosh, A. S.; Young, K. D. J. Bacteriol. 2005, 187 (6),
1913.
(30) Schindler, M.; Osborn, M. J.; Koppel, D. E. Nature 1980,
285 (5762), 261.
(31) Muhlradt, P.F., Menzel J., Golecki, J.R., Speth, V. Eur. J. Biꢀ
ochem. 1973, 35, 471.
(32) Yang, Y.; Shi, F.; Tao, G.; Wang, X. J. Microbiol. 2012, 50
(2), 235.
(33) Baba, T.; Kaneda, K.; Kusunose, E.; Kusunose, M.; Yano,
I. Lipids 1988, 23 (12), 1132.
(34) Nishiuchi, Y.; Baba, T.; Yano, I. J. Microbiol. Methods
2000, 40 (1), 1.
(35) Nishiuchi, Y.; Baba, T.; Hotta, H. H.; Yano, I. J. Microbiol.
Methods 1999, 37 (2), 111.
(36) Barkan, D.; Liu, Z.; Sacchettini, J. C.; Glickman, M. S.
Chem. Biol. 2009, 16 (5), 499.
(37) Hett, E. C.; Rubin, E. J. Microbiol. Mol. Biol. Rev. 2008,
72 (1), 126.
(38) Takayama, K.; Armstrong, E. L.; Kunugi, K. A.; Kilburn, J.
O. Antimicrob. Agents Chemother. 1979, 16 (2), 240.
(39) Telenti, A.; Philipp, W. J.; Sreevatsan, S.; Bernasconi, C.;
Stockbauer, K. E.; Wieles, B.; Musser, J. M.; Jacobs, W. R. Nat. Med.
1997, 3 (5), 567.
(40) Kilburn, J. O.; Takayama, K. Antimicrob. Agents
Chemother. 1981, 20 (3), 401.
(41) Mikusová, K.; Slayden, R. A.; Besra, G. S.; Brennan, P. J.
Antimicrob. Agents Chemother. 1995, 39 (11), 2484.
(42) Li, X.ꢀZ.; Zhang, L.; Nikaido, H. Antimicrob. Agents
Chemother. 2004, 48 (7), 2415.
(43) BosneꢀDavid, S.; Barros, V.; Verde, S. C.; Portugal, C.;
David, H. L. J. Antimicrob. Chemother. 2000, 46 (3), 391.
(44) Abate, G.; Hoffner, S. E. Diagn. Microbiol. Infect. Dis.
1997, 28 (3), 119.
(45) Minnikin, D. E. Lipids: complex lipids, their chemistry, biꢀ
osynthesis and roles. The Biology of the Mycobacteria: Vol. 1.44.
Physiology, Identification and Classification; eds Ratledge C, Stanꢀ
ford J; New York: Academic Press, 1982, 95.
(46) Nikaido, H.; Kim, S.ꢀH.; Rosenberg, E. Y. Mol. Microbiol.
1993, 8 (6), 1025.
AUTHOR INFORMATION
Corresponding Author
* bertozzi@stanford.edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
We thank Douglas Fox for critical reading of the manuscript and
Anthony Iavarone for mass spectra acquisition, instrumentation
located in QB3/Chemistry Mass Spectrometry Facility at the Uniꢀ
versity of California, Berkeley. We thank Jason Bell for initial
help with the FRAP experiments and Aaron Straight for microꢀ
scope access. F.P.RꢀR. was supported by Ford Foundation Preꢀ
doctoral Fellowship and University of California Chancellor’s
Fellowship. X.Z. was supported by a Stanford University Interdisꢀ
ciplinary Graduate Fellowship. This work was supported by Naꢀ
tional Institutes of Health grants GM058867 (to C.R.B.),
AI051622 (to C.R.B.), AI036929 (to J.A.T.), and the Stanford
Center for Systems Biology grant P50ꢀGM107615 (to J.A.T.).
REFERENCES
(1) World Health Organization. Global Tuberculosis Report 2015,
20 th ed.; World Health Organization: Geneva, 2015.
(2)
(3)
Russell, D. G. Nat. Rev. Mol. Cell Biol. 2001, 2 (8), 569.
Philips, J. A.; Ernst, J. D. Annu. Rev. Pathol. Mech. Dis.
2012, 7 (1), 353.
(4)
(1), 11.
(5)
529.
(6)
Jarlier, V.; Nikaido, H. FEMS Microbiol. Lett. 1994, 123
Zhang, Y. Annu. Rev. Pharmacol. Toxicol. 2005, 45 (1),
Zumla, A.; Nahid, P.; Cole, S. T. Nat. Rev. Drug Discov.
2013, 12 (5), 388.
(7)
Jankute, M.; Cox, J. A. G.; Harrison, J.; Besra, G. S. Annu.
Rev. Microbiol. 2015, 69 (1), 405.
(8)
Marrakchi, H.; Lanéelle, M.ꢀA.; Daffé, M. Chem. Biol.
2014, 21 (1), 67.
(9)
Hoffmann, C.; Leis, A.; Niederweis, M.; Plitzko, J. M.;
Engelhardt, H. Proc. Natl. Acad. Sci. 2008, 105 (10), 3963.
(10) Zuber, B.; Chami, M.; Houssin, C.; Dubochet, J.; Griffiths,
G.; Daffé, M. J. Bacteriol. 2008, 190 (16), 5672.
(11) Siegrist, M. S.; Swarts, B. M.; Fox, D. M.; Lim, S. A.;
Bertozzi, C. R. FEMS Microbiol. Rev. 2015, 39 (2), 184.
(12) Christensen, H.; Garton, N. J.; Horobin, R. W.; Minnikin,
D. E.; Barer, M. R. Mol. Microbiol. 1999, 31 (5), 1561.
(13) Maloney, E. A.; Lun, S.; Stankowska, D.; Guo, H.; Raꢀ
jagopalan, M.; Bishai, W.; Madiraju, M. V. Front. Microbiol. 2011, 2,
19.
(14) Backus, K. M.; Boshoff, H. I.; Barry, C. S.; Boutureira, O.;
Patel, M. K.; D’Hooge, F.; Lee, S. S.; Via, L. E.; Tahlan, K.; Iii, C. E.
B.; Davis, B. G. Nat. Chem. Biol. 2011, 7 (4), 228.
(15) Swarts, B. M.; Holsclaw, C. M.; Jewett, J. C.; Alber, M.;
Fox, D. M.; Siegrist, M. S.; Leary, J. A.; Kalscheuer, R.; Bertozzi, C.
R. J. Am. Chem. Soc. 2012, 134 (39), 16123.
(16) Urbanek, B. L.; Wing, D. C.; Haislop, K. S.; Hamel, C. J.;
Kalscheuer, R.; Woodruff, P. J.; Swarts, B. M. Chem. Bio. Chem
2014, 15 (14), 2066.
(17) Foley, H. N.; Stewart, J. A.; Kavunja, H. W.; Rundell, S.
R.; Swarts, B. M. Angew. Chem. Int. Ed. 2016, 55 (6), 2053.
(18) Dover, L. G.; CerdeñoꢀTárraga, A. M.; Pallen, M. J.;
Parkhill, J.; Besra, G. S. FEMS Microbiol. Rev. 2004, 28 (2), 225.
(47) Liu, J.; Rosenberg, E. Y.; Nikaido, H. Proc. Natl. Acad.
Sci. U. S. A. 1995, 92 (24), 11254.
(48) Li, Q.; Zhou, M.; Fan, X.; Yan, J.; Li, W.; Xie, J. Sci. Rep.
2016, 6.
(49) Farha, M. A.; Brown, E. D. Ann. N. Y. Acad. Sci. 2015,
1354 (1), 54.
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Page 9 of 23
Journal of the American Chemical Society
(50) RamónꢀGarcía, S.; Ng, C.; Anderson, H.; Chao, J. D.;
Zheng, X.; Pfeifer, T.; AvꢀGay, Y.; Roberge, M.; Thompson, C. J.
Antimicrob. Agents Chemother. 2011, 55 (8), 3861.
(51) RamónꢀGarcía, S.; González del Río, R.; Villarejo, A. S.;
Sweet, G. D.; Cunningham, F.; Barros, D.; Ballell, L.; Mendozaꢀ
Losana, A.; FerrerꢀBazaga, S.; Thompson, C. J. Sci. Rep. 2016, 6,
34293.
(52) Rens, C.; Laval, F.; Daffé, M.; Denis, O.; Frita, R.; Bauꢀ
lard, A.; Wattiez, R.; Lefèvre, P.; Fontaine, V. Antimicrob. Agents
Chemother. 2016, AAC.00872.
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Mycobacterial cell envelope
Capsule
1
2
3
Mycomembrane
4
5
6
Outer leaflet
7
8
Live Mycobacterium
9
Inner leaflet
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Mycomembrane fluidity
Correlates with mycolic acid structure
Altered by front-line TB drugs
Implications for TB-drug development
Cytosol
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Capsule
A
Metabolic labeling
1
2
Mycomembrane
X
3
Outer leaflet
4
5
X
6
7
Tre
8
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TDM
Inner leaflet
Ag85
AG
Arabinogalactan
(AG)
X
Peptidoglycan
TMM
Inner membrane
Cytosol
Legend
X
Peptigoglycan
Arabinose
Trehalose
metabolic
reporter
Trehalose
monomycolate (TMM)
Trehalose
dimycolate (TDM)
AG-mycolate
Mycobacterial
free lipids
OH
OR
O
OR
O
OH
B
44
O
O
HO
HO
HO
HO
HO
R =
45
46
47
48
49
50
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HO
OH
O
n1
OH
O
OH
O
n2
OH
OH
OH
OH
OR
O
O
O
OH
HO
HO
HO
Species
M. smegmatis
M. tuberculosis
n1
n2
OH
OH
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OH
37-48
49-65
18, 22
20, 22
Trehalose
Trehalose monomycolate
(TMM)
Trehalose dimycolate
(TDM)

OH
OH
Journal of the American Chemical Society
OH
Page 12 of 23
A
O
O
O
HO
HO
HO
HO
HO
HO
CH3
O
1
2
3
4
OH
OH
OH
O
O
O
OH
OH
X
R4
H
N
H
5
O
O
HO2C
N
O
6
R3
OH
O
R1
HO
OH
R2
OH
7
8
S
9
O
O-alkTMM X = O
N-alkTMM X = NH
(Ref. 17)
FITC-Tre
(Ref. 14)
TreAz (Ref. 15)
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2-TreAz R1 = N3, R2 = R3 = R4 = OH
3-TreAz R2 = N3, R1 = R3 = R4 = OH
4-TreAz R3 = N3, R1 = R2 = R4 = OH
6-TreAz R4 = N3, R1 = R2 = R3 = OH
OH
HO
O
O
B
17
OH
OH
OH
OH
18
19
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
20
21
22
23
24
25
26
27
28
29
HO
CO2H
OH
OH
OH
OH
O
O
O
O
OH
OH
OH
OH
OH
R
S
NH
O
O
O
O
R
R
OH
HO
HO
HO
NH
OH
OH
OH
R
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3-FlTre
4-FlTre
6-FlTre
R = Fl

Page 13 of 23
A
Journal of the American Chemical Society
B
M. smegmatis
C. glutamicum
8,000
80,000
60,000
40,000
20,000
0
1
2
3
4
5
6
7
8
6,000
4,000
2,000
0
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M. smegmatis
C
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D
C. glutamicum
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M. smegmatis
A
Merge
6FlTre
TDL
Merge
6FlTre
TDL
1
2
3
4
5
6
7
8
9
10
11
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B
C. glutamicum
25
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43
Merge
6FlTre
TDL
Merge
6FlTre
TDL
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Page 15 of 23 M. smegJmouartnisal of the American Chemical SocietCy . glutamicum
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Time (s)
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0
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18
19
20
21
22
23
24
25
26
27
28
20
30
6
8
60
10
20
30
300
600
29
C
30
C. glutamicum
(22-38 carbon atoms)
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
1.0
0.8
0.6
0.4
0.2
0
R. equi
(30-54 carbon atoms)
N. brasiliensis
(46-60 carbon atoms)
M. smegmatis
(60-81 carbon atoms)
0
10 20 30 40 50 60
Time (s)
50
D
51
E
p < 10-4
p < 10-3
p = 0.06
p = 0.4
52
53
54
55
56
57
p = 0.02
p = 0.1
0.2
0.1
0
1.0
58 0.8
59
60
0.6
0.4
0.2
0
n.d.
Cg
Re
Nb
Ms
Cg
Re
Nb
Ms
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1.0
A
C
1.ꢀEthambutol 3 h
2.ꢀWash
3.ꢀTDL 20 min
Ethambutol
10 µg/mL
2 µg/mL
0.5 µg/mL
0 µg/mL
1
2
3
4
5
6
7
8
0.8
0.6
4.ꢀWash
5.ꢀMicroscopy
6-FlTre
M. smegmatis
0.4
Increasing myco-
membrane fluidity
9
0.2
10
11
12
B
13
EMB
Merge
6FlTre
TDL
Merge
6FlTre
TDL
0
(µg/mL)
0
50
100 150 200 250 300
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Time (s)
0
1.0
0.8
0.6
0.4
0.2
0
D
p < 10-2
p = 0.17
p = 0.5
0.5
2
0
0.5
2
10
0.18
0.37
0.46
0.40
±
= 8)
±
= 5)
±
= 8)
±
0.03
0.10
0.07
0.09
(
n
= 5)
10
(
n
(
(
n
n
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Ethambutol concentration (µg/mL)

Journal of the AmericanMChyecmoicbaal Scotceireityal cell envelope
Capsule
Page 17 of 23
1
2
3
Mycomembrane
4
5
6
Outer leaflet
7
8
Live Mycobacterium
9
10
11
12
Inner leaflet
Mycomembrane fluidity
13
14
!ꢀ Correlates with mycolic acid structure
15
16
17
!ꢀ Altered by front-line TB drugs
18
19
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20
21
Implications for TB-drug development
Cytosol

Journal of the American Chemical Society
Page 18 of 23
1
2
3
Capsule
A
Metabolic labeling
4
5
6
Mycomembrane
X
Outer leaflet
X
7
8
Tre
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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36
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38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TDM
Inner leaflet
Ag85
AG
Arabinogalactan
(AG)
X
Peptidoglycan
TMM
Inner membrane
Cytosol
Legend
X
Peptigoglycan
Arabinose
Trehalose
metabolic
reporter
Trehalose
monomycolate (TMM)
Trehalose
dimycolate (TDM)
AG-mycolate
Mycobacterial
free lipids
OH
OR
O
OR
O
OH
B
O
O
HO
HO
HO
HO
HO
R =
HO
OH
O
n1
OH
O
OH
O
n2
OH
OH
OH
OH
OR
O
O
O
OH
HO
HO
HO
Species
M. smegmatis
M. tuberculosis
n1
n2
OH
OH
OH
37-48
49-65
18, 22
20, 22
Trehalose
Trehalose monomycolate
(TMM)
Trehalose dimycolate
(TDM)
Figure 1. Mycobacterial cell envelope and structures of trehalose mycolates. A) Mycobacterial cell envelope
components include inner membrane, peptidoglycan, arabinogalactan, mycomembrane, and capsule. Antigen
85 mediates mycolylation of arabinogalactan from TMM donor. Two molecules of TMM are used to generate
TDM, thereby releasing one molecule of trehalose. Antigen 85 is predicted to be active in the mycomembrane.
Red dashed line depicts metabolic incorporation route for unnatural trehalose reporters. B) Chemical structures
for trehalose, trehalose monomycolate and trehalose dimycolate. Number of carbons (n1, n2) strictly in linear
chains of mycolates are shown for M. smegmatis and M. tuberculosis. Trehalose monomycolate (TMM),
trehalose dimycolate (TDM), trehalose (Tre), Antigen 85 (Ag85), arabinogalactan (AG).
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1
2
3
4
5
6
7
8
OH
OH
O
OH
A
O
O
HO
HO
HO
HO
HO
HO
CH3
O
OH
OH
OH
O
O
O
OH
OH
X
R4
R3
H
N
H
O
O
HO2C
N
O
OH
O
R1
HO
OH
R2
OH
9
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O-alkTMM X = O
N-alkTMM X = NH
(Ref. 17)
FITC-Tre
(Ref. 14)
TreAz (Ref. 15)
2-TreAz R1 = N3, R2 = R3 = R4 = OH
3-TreAz R2 = N3, R1 = R3 = R4 = OH
4-TreAz R3 = N3, R1 = R2 = R4 = OH
6-TreAz R4 = N3, R1 = R2 = R3 = OH
OH
HO
O
O
B
OH
OH
OH
OH
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
CO2H
OH
OH
OH
OH
O
O
O
O
OH
OH
OH
OH
OH
R
S
NH
O
O
O
O
R
R
OH
HO
HO
HO
NH
OH
OH
OH
R
2-FlTre
3-FlTre
4-FlTre
6-FlTre
R = Fl
Figure 2. Metabolic engineering of trehalose glycolipids with unnatural trehalose reporters. A) Previously
reported unnatural trehalose reporters including FITC-Tre, TreAz analogs and alkTMM analogs. B) Library of
fluorescein-trehalose analogs (this work). Fluorescein isothiocyanate (FITC), azido-trehalose (TreAz),
fluorescein-trehalose (FlTre).
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Page 20 of 23
1
2
3
A
B
M. smegmatis
C. glutamicum
4
5
6
7
8
9
8,000
6,000
4,000
2,000
0
80,000
60,000
40,000
20,000
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
M. smegmatis
C
C. glutamicum
D
Figure 3. Metabolic labeling of actinobacteria with FlTre analogs. Labeling profile of FlTre analogs compared
to FITC-Tre in M. smegmatis (A) and C. glutamicum (B). Bacteria were labeled with 100 µM trehalose analogs
or vehicle for several doubling times. Error bars depict standard deviation of three replicate experiments.
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Journal of the American Chemical Society
Results are representative of at least two independent experiments. Mean fluorescence intensity (MFI). (C)
Fluorescence microscopy images of M. smegmatis and C. glutamicum cells labeled with vehicle or 6-FlTre.
Scale bar, 2 µm.
1
2
3
4
5
6
7
8
9
A
M. smegmatis
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Merge
6FlTre
TDL
Merge
6FlTre
TDL
B
C. glutamicum
Merge
6FlTre
TDL
Merge
6FlTre
TDL
Figure 4. FlTre-labeled trehalose glycolipids show species-dependent mobility. A) Pre-labeled M. smegmatis
glycolipids (6-FlTre, green) are excluded from the poles as depicted by peptidoglycan labeling (TDL, red). B)
Pre-labeled C. glutamicum glycolipids are highly mobile and redistribute to the entire cell after 6-FlTre has
been removed from the growth media. Scale bar, 2 µm.
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Page 22 of 23
A
B
1
2
M. smegmatis
C. glutamicum
3
4
5
6
Time (s)
0
2
0
7
8
9
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
20
30
6
8
60
10
20
30
300
600
C
C. glutamicum
(22-38 carbon atoms)
1.0
0.8
0.6
0.4
0.2
0
R. equi
(30-54 carbon atoms)
N. brasiliensis
(46-60 carbon atoms)
M. smegmatis
(60-81 carbon atoms)
0
10 20 30 40 50 60
Time (s)
D
E
p < 10-4
p < 10-3
p = 0.06
p = 0.4
p = 0.02
p = 0.1
0.2
0.1
0
1.0
0.8
0.6
0.4
0.2
0
n.d.
Cg
0.91
Re
Nb
Ms
0.18
Cg
Re
Nb
Ms
0.96
0.69
0.06
0.09
0.10
(
(
(
(
(
n
±
±
±
±
±
±
±
0.05
(
(
n
n
n
n
n
0.06
= 39)
0.10
= 16)
0.10
= 12)
n
0.03
0.02
= 26)
0.04
= 12)
= 8)
= 6)
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Figure 5. Mycobacterial trehalose glycolipids are relatively immobile. A) FRAP experiment of 6-FlTre labeled
M. smegmatis (A) and C. glutamicum (B) after irradiation at the pole (left panel) and center (right panel). Scale
bar, 2 µm. C) Fluorescence recovery curves after photobleaching for center and pole regions in 6-FlTre labeled
cells across different bacterial species with the corresponding number of carbon atoms in mycolic acid chains.
Lines represent the averaged signal of n ≥ 6 cells, where solid and dotted lines correspond to pole and center
regions, respectively. Comparison of fraction mobile (D) and apparent diffusion coefficient (E) extracted from
fitting FRAP curves for different actinobacterial species. Filled and open circles correspond to pole and center
regions, respectively. Number of cells evaluated and standard deviation are shown for every species. p-values
between samples were calculated with a rank sum test. Fluorescence recovery after photobleaching (FRAP),
M. smegmatis (Ms), C. glutamicum (Cg), R. equi (Re), N. brasiliensis (Nb), apparent diffusion coefficient (Dapp),
not determined (n.d.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.0
A
C
1.ꢀEthambutol 3 h
2.ꢀWash
Ethambutol
10 µg/mL
2 µg/mL
0.5 µg/mL
0 µg/mL
0.8
0.6
0.4
0.2
0
3.ꢀTDL 20 min
4.ꢀWash
5.ꢀMicroscopy
6-FlTre
M. smegmatis
Increasing myco-
membrane fluidity
EMB
(µg/mL)
Merge
6FlTre
TDL
Merge
6FlTre
TDL
B
0
50
100 150 200 250 300
Time (s)
0
1.0
0.8
0.6
0.4
0.2
0
D
p < 10-2
p = 0.17
p = 0.5
0.5
2
0
0.5
2
10
0.18
0.37
0.46
0.40
±
= 8)
±
0.10
= 5)
±
= 8)
±
0.03
0.07
0.09
(
n
= 5)
10
(
n
(
(
n
n
Ethambutol concentration (µg/mL)
Figure 6. FlTre metabolic labeling reports on cell wall dynamics during ethambutol (EMB) treatment. A)
Scheme depicting experimental workflow and labeling pattern expected for differential mycomembrane fluidity.
M. smegmatis was pre-labeled with 6-FlTre, then treated with EMB at different concentrations for 3 h. Cells
were washed to remove excess 6-FlTre and chased with TDL in the absence of drug for 20 min. Spatial
distribution of labeled glycolipids was evaluated by fluorescence microscopy after fixation (B). Representative
cells are shown in each panel. 6-FlTre and TDL signals are s in green and red channels, respectively, for
merged images (left panel). FRAP recovery traces (C) and fraction mobile (D) for cells treated with different
concentrations of EMB. Recovery trace lines represent the averaged signal of n ≥ 5 cells. Scale bar, 2 µm.
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