A Fluorogenic Trehalose Probe for Tracking Phagocytosed Mycobacterium tuberculosis

Article abstract of DOI:10.1021/jacs.0c07700

Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 μM. CDG-Tre fluoresces upon activation by BlaC, the β-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and postphagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.

Full text of DOI:10.1021/jacs.0c07700

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Communication
A Fluorogenic Trehalose Probe for Tracking
Phagocytosed Mycobacterium tuberculosis
Tingting Dai, Jinghang Xie, Qihua Zhu, Mireille Kamariza, Ke Jiang, Carolyn R Bertozzi, and Jianghong Rao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c07700 • Publication Date (Web): 19 Aug 2020
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A Fluorogenic Trehalose Probe for Tracking Phagocytosed
Mycobacterium tuberculosis
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Tingting Dai1†, Jinghang Xie2†, Qihua Zhu2,3,4, Mireille Kamariza5, Ke Jiang2, Carolyn R. Bertozzi1,6,
Jianghong Rao1,2*
1Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
2Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA
94305, USA.
3State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 211198, China.
4Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical
University, Nanjing 211198, China.
5Department of Biology, Stanford University, Stanford, CA 94305, USA.
6Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
†These authors contributed equally to this work.
ABSTRACT: Tuberculosis (TB) disease is a global epidemic
caused by the pathogenic Mycobacterium tuberculosis (Mtb).
Tools that can track the replication status of viable Mtb cells
within macrophages are vital for the elucidation of host-
pathogen interactions. Here, we present a cephalosphorinase-
dependent green trehalose (CDG-Tre) fluorogenic probe that
enables fluorescence labeling of single live Bacille Calmette-
Guérin (BCG) cells within macrophages at concentrations as
low as 2 µM. CDG-Tre fluoresces upon activation by BlaC, the
enzymatic labeling with small molecule fluorescent probes.11
Since its introduction, the genetic inheritance of fluorescent
proteins has been favored in monitoring gene expression and
tracking live organisms, including Mtb.12,13 Nonetheless, the
long half-life of fluorescent proteins prevents real-time
monitoring of cellular dynamic changes. As well, such genetic
manipulation in mycobacteria may compromise cell virulence
significantly.14–16 Genetic labeling has not yet been
demonstrated to report on cell wall biosynthesis, a critical
component for understanding Mtb pathogenesis and the
development of effective antibiotics.17,18 Metabolic engineering
for labeling the bacterial cell surface in macrophages19–22
includes the first use of fluorescent trehalose analogs, FITC-
Tre,23 and recently, DMN-Tre.24,25 These trehalose derivatives
are processed by antigen 85 (Ag85) enzymes, and incorporated
into the mycolyl arabinogalactan (mAG) layer metabolically.26
Their generally poor membrane permeability and strict
requirement of high working concentration (e.g. FITC-Tre, 200
μM; DMN-Tre, 100 μM), however, raised concerns in their
potential toxicity in macrophages. Enzyme-activated
fluorescent small molecule probes have shown great promise
for sensitive Mtb detection.11 CDG-DNB3, for example,
enabled rapid and specific labeling of single live Mtb by taking
the advantage of two enzymes expressed by Mtb: β-lactamase
C (BlaC) that activates the probe to restore fluorescence, and
decaprenylphosphoryl-β-D-ribose 2’-epimerase (DprE1) that
traps the fluorescent product via a covalent linkage.27 Unlike
metabolic labeling, this probe is not incorporated into cell wall;
however, its efficacy in labeling of post-phagocytosed Mtb
remains to be demonstrated.
β
-lactamase uniquely expressed by Mtb, and the fluorescent
product is subsequently incorporated within the bacterial cell
wall via trehalose metabolic pathway. CDG-Tre showed high
selectivity for mycobacteria over other clinically prevalent
species in the Corynebacterineae suborder. The unique labeling
strategy of BCG by CDG-Tre provides a versatile tool for
tracking Mtb in both pre- and post-phagocytosis and elucidating
fundamental physiological and pathological processes related to
the mycomembrane.
Tuberculosis (TB) disease, caused by Mycobacterium
tuberculosis (Mtb), remains the leading cause of mortality by a
single pathogen.1 Upon inhalation of Mtb-containing droplets,
Mtb cells invade host lung macrophages where they have
evolved a variety of strategies to persist and replicate.2 In some
cases, these Mtb cells will eventually induce host cell death and
escape innate immune defenses.3–5 The ability to track Mtb
metabolic status, cell growth and division, at the single-cell
level, with high temporal and spatial resolution could greatly
facilitate our understanding of the host-pathogen interactions
and fuel innovations in both clinical detection and discovery of
novel anti-TB therapeutic strategies.6,7
In this work, we developed a fluorogenic probe--a conjugate
of cephalosporin and trehalose, named CDG-Tre--to improve
the labeling of phagocytosed Mtb (Fig. 1). By employing a
cephalosporin analog to cage 2-Me TokyoGreen (TG)
fluorophore conjugated to trehalose, we envision that the lactam
Several technologies have been reported for labeling and
tracking phagocytosed Mtb, from genetic labeling with
fluorescent reporter proteins,8,9 metabolic labeling with
fluorophores conjugated to cell wall building blocks,10 to
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ring will be hydrolyzed by BlaC.28,29 In the presence of a
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leaving group at the 3’ position, the hydrolyzed product will
proceed with spontaneous fragmentation, resulting in the
release and fluorescence turn-on. Subsequently, the fluorescent
trehalose fragment will be processed via the Ag85-mediated
trehalose pathway, resulting in the metabolic labeling of the cell
wall (Fig. 1). We characterized CDG-Tre in a series of
experiments and demonstrated the BlaC/Ag85 dependent
labeling mechanism. Importantly, we show efficient labeling of
phagocytosed Mtb using very low concentrations of CDG-Tre
(2 μM).
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Figure 2. Fluorescence analysis of different species of bacteria
and fungi labeled with 4-TG-Tre or 6-TG-Tre. (a) Confocal
images of freshly cultured E. coli (TOP10), S. aureus, C.
diphtheriae and M. smegmatis stained with 50 µM of 4-TG-Tre
and 6-TG-Tre in PBS for 2 h at 37 °C. For each set of images, the
top row = bright field; bottom row = green fluorescence, 63x/oil,
Ex-490 nm/Em-520 nm. Scale bars indicate 5 µm. (b-c) Green
fluorescence activated flow cytometry analysis of (b) freshly
cultured C. albicans, E. coli (TOP10), S. aureus, C. diphtheriae
and M. smegmatis with 50 µM of 6-TG-Tre in PBS at 37 °C for
2h; or (c) M. smegmatis stained with 50 µM of 4-TG-Tre and 6-
TG-Tre in PBS at 37 °C for 1, 2, 4, 6 h. The autofluorescence of
unlabeled bacteria (F0) was utilized to normalize the intensity.
N=3, error bars represent standard deviation.
Figure 1. Structures of fluorescent trehalose derivatives and the
proposed labeling of Mtb phagocytosed within macrophages.
CDG-Tre probes are taken up by macrophages into phagosomes,
then reach the periplasmic layer of Mtb cells. [Inset] The probes
are activated by BlaC and processed by Ag85 enzymes into 6-
TG-Tre monomycolate (6-TG-Tre-MM) with a mycolate chain
transferred from another molecule of trehalose monomycolate
(TMM). 6-TG-Tre-MM was further incorporated into the outer
leaflet of the capsule.
Cu-catalyzed azide-alkyne cycloaddition (CuAAC) with
trehalose, to yield 4-TG-Tre and 6-TG-Tre analogs (Fig. S1).
The uptake of 4-TG-Tre and 6-TG-Tre was evaluated in
several representative species of bacteria and fungi including
Candida albicans (C. albicans, the most prevalent cause of
fungal infection in human32), Escherichia coli (E. coli, a
Different positions on the trehalose molecule have been
decorated, resulting in varying efficacy for mycobacterial cell
wall labeling.23 It has been shown that the Ag85 complex
processes modifications at the trehalose 4- or 6-hydroxyl group
position more efficiently.30 In this study, TG, a fluorescein
derivative where the carboxylic group was replaced with a
methyl group, was chosen as the fluorophore. This probe has
great photostability and is highly fluorescent in its anionic form
while the quantum yield is near zero when the hydroxy group is
alkylated because the fluorescence is quenched via
intramolecular photoinduced electron transfer (PeT).27,31
Therefore, we coupled TG with 6-heptynoic acid, followed by
commonly
used
gram-negative
bacteria
model),
Staphylococcus aureus (S. aureus, frequently found in the
respiratory tract of patients with pulmonary diseases),
Corynebacterium diphtheriae (C. diphtheriae) and
Mycobacterium smegmatis (M. smegmatis). Corynebacterium
commonly colonizes the skin and mucous membranes of human
with C. diphtheriae as one of the most pathogenic species. M.
smegmatis has been widely used as a laboratory model
organism for Mtb studies due to the genetic similarity and its
rapid growth compared to Mtb. Both C. diphtheriae and M.
smegmatis belong to Actinobacteria phylum and carry the mAG
layer. Following incubation with 50 μM of 4-TG-Tre or 6-TG-
Tre at 37 °C for 2 h, C. diphtheriae and M. smegmatis exhibited
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strong green fluorescence labeling while little uptake was
observed in C. albicans, E. coli, and S. aureus due to the lack
of Ag85 enzymes and mAG structure (Figs. 2a & S3).
Tre incubation. Together, these results have established the
mechanism of the uptake, activation, and metabolic
incorporation of CDG-Tre by mycobacteria.
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We quantitated the mean fluorescence intensity of each
labeled species by flow cytometry. Within 2 h, 6-TG-Tre treated
M. smegmatis showed approximately 80-fold increase in
fluorescent intensity over unlabeled bacteria in phosphate
buffered saline (PBS). Similarly, C. diphtheriae showed a 50-
fold increase. For C. albicans, E. coli, and S. aureus, the
fluorescence intensity was only 2-, 7- and 1-fold higher than the
background, respectively (Fig. 2b). As illustrated in Fig. 2c, the
labeling efficiency of 6-TG-Tre was higher than that of 4-TG-
Tre. A longer incubation of M. smegmatis with 6-TG-Tre for 4
and 6 h barely improved the total uptake. To confirm Ag85
processing of 6-TG-Tre, we purified Mtb Ag85c (Fig. S4) and
resorufin butyrate was used as an acyl donor in the assay as
previously reported (Fig. S5 and S6).33 Mass spectrometry
analysis confirmed the addition of butyrate on 6-TG-Tre by
Ag85c. Collectively, these data suggested that 6-TG-Tre is a
suitable candidate for the specific labeling of mycobacteria and
corynebacteria.
Next, the cephalosporin scaffold was coupled with TG to
generate CDG-Tre (Fig. S2). The fluorogenic property of CDG-
Tre was evaluated with recombinant BlaC (Fig. S7). In the
presence of BlaC, the fluorescence of CDG-Tre was rapidly
turned on (within 20 minutes) and the hydrolyzed product 6-
TG-Tre was confirmed by liquid chromatography-mass
spectrometry (LCMS) (Fig. S8). We further tested if CDG-Tre
could be processed by Ag85c as an acyl acceptor before the
cleavage by BlaC. Surprisingly, unlike 6-TG-Tre, the product
with butyrate addition was not found (Fig. S9). These data
suggested that in order to be processed and incorporated by
Ag85c, CDG-Tre must be first uncaged by BlaC.
The capability of CDG-Tre in differentiating cephalosporin
resistant mycobacteria was assessed in live bacteria and fungi.
Unlike C. diphtheriae which is sensitive to β-lactam antibiotics
and lacks the ability to activate CDG-Tre, M. smegmatis
naturally expresses a β-lactamase called BlaS which could
rapidly hydrolyze CDG-Tre and turn on fluorescence.28 As
shown in Figs. 3a, 3b and S10, up to a 55-fold increase in
fluorescence intensity was observed in M. smegmatis whereas
the uptake by C. albicans, E. coli, S. aureus and C. diphtheriae
was minimal. Inhibitors of Ag85 enzymes and BlaC were used
to confirm the labeling mechanism of CDG-Tre. Both trehalose
and ebselen (Ag85 inhibitors) inhibited the labeling of M.
smegmatis by CDG-Tre (Fig. 3c & 3d). Two β-lactamase
inhibitors, clavulanic acid and avibactam,34,35 reduced the
fluorescence of labeled M. smegmatis by 33% and 40%,
respectively (Fig. 3d). Furthermore, studies have reported that
cephalosporin antibiotics could be taken up through porins.36,37
We pretreated M. smegmatis with spermidine, a polyamine
compound known to interfere with the bacterial porin
function.38 Dose-dependent inhibition in fluorescent labeling
was observed, confirming the involvement of porins in the
uptake of CDG-Tre (Fig. 3d). We also applied CDG-Tre for
drug susceptibility test. In particular, we sought to visualize
changes in cells when treated with front-line TB drugs, such as
rifampicin (RIF), a transcriptional inhibitor that targets
specifically the RNA polymerases of bacteria, and isoniazid
(INH), a mycolic acid biosynthesis inhibitor.39As presented in
Fig. 3e, M. smegmatis cells treated with RIF (0.2 µg/mL) or
INH (10 µg/mL and 100 µg/mL) for 3 h exhibited a
significantly reduced mean fluorescence intensity after CDG-
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Figure 3. Characterization of CDG-Tre labeling. (a) Confocal
images (top row = bright field and bottom row = green
fluorescence, Ex-490 nm/Em-520 nm) of freshly cultured E. coli
(TOP10), S. aureus, C. diphtheriae and M. smegmatis incubated
with 50 µM of CDG-Tre in PBS at 37 °C for 2 h. Scale bars
indicate 5 µm. (b) Flow cytometry analysis of bacteria and fungi
shown in (a). The unlabeled bacterial autofluorescence in PBS
(F0) was utilized to normalize and calculate the relative
fluorescence intensity. (c) Inhibitory study with increasing
concentrations of free trehalose at 1, 10, 100, 1000-fold over
CDG-Tre (100 µM) with M. smegmatis. (d) Inhibitory study with
clavulanate (2 mg/ml), avibactam (2 mg/ml), ebselen (200 μg/ml)
and spermidine (2.5, 5, 7.5 mg/ml) in the presence of 50 µM
CDG-Tre with M. smegmatis. M. smegmatis treated with CDG-
Tre exhibited a 60-fold increase of mean fluorescence intensity
over PBS, which was arbitrarily set as 1 to normalize the test
samples and show percentage inhibition. (e) M. smegmatis was
treated with rifampicin (RIF, 0.2 µg/mL) or isoniazid (INH, 10
µg/mL or 100 µg/mL) for 3 h before incubation with CDG-Tre
(100 µM) for 1 h at 37 °C. Mean fluorescent intensity of CDG-
Tre was arbitrarily set as 1 to normalize other samples with drugs
treatment and show percentage inhibition. All error bars represent
standard deviation of three independent measurements.
Lastly, we investigated whether CDG-Tre could label BCG
cells phagocytosed within macrophages. Freshly cultured BCG
were pre-labeled with TAMRA-Tre40 overnight then washed
and incubated with mouse RAW macrophage at a ratio of
multiplicities of infection (MOI) 10 to 1 in complete medium
for 6 h. The macrophages were washed, then incubated with
CDG-DNB3 (20 μM), 6-TG-Tre (20 μM) or CDG-Tre (20 μM)
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for 4 h in serum free medium (Fig. S11). CDG-DNB3 has been
background. Staining of macrophages that had phagocytosed
pre-killed BCG did not show any fluorescent labeling (Fig.
S12).
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utilized for pre-labeling live BCG and tracking the process of
phagocytosis in macrophages.27 Nonetheless, a post-labeling
within 4 h generated significant non-specific green fluorescent
background in macrophages. 6-TG-Tre presented clean
background in macrophages, yet no probe uptake was observed
in BCG. Considering the rapid uptake of 6-TG-Tre by
mycobacteria alone (Fig. 2c), we hypothesized that the
availability of 6-TG-Tre within live macrophages, especially in
phagosomes, must be highly compromised. In CDG-Tre treated
macrophages, green fluorescent BCG cells were observed
(overlaying with the red fluorescence from TAMRA-Tre), in
For 6-TG-Tre, under the same conditions, some weak
fluorescent labeling of phagocytosed live BCG could be
observed when the probe concentration was over 100 μM (Fig.
4a). Both 6-TG-Tre and CDG-Tre displayed high stability in
serum free medium at 37 °C (Fig. S13), and comparable toxicity
to macrophages (Fig. S14). However, the required high labeling
concentration of 6-TG-Tre (100 μM) made it toxic to
macrophages. We performed super resolution structured
illuminated microscopy (SR-SIM) analysis in an attempt to
examine if CDG-Tre was incorporated into the bacterial cell
walls. As shown in Fig. 4b, single BCG bacilli localized within
macrophages presented bright green fluorescent cell wall
structure. Interestingly, the green fluorescence from CDG-Tre
staining did not completely overlay with the red fluorescence in
BCG (from pre-labeling by TARMA-Tre). This fluorescence
was preferentially localized to the polar regions of the bacilli in
an asymmetric manner. In a previously proposed model,
mycobacteria are believed to grow and elongate asymmetrically
with new biosynthetic materials enriched in the poles.21
Therefore, CDG-Tre fluorescence is observed at poles where
the newly biosynthesized cell wall membrane of live BCG is
located. Taken together, these results demonstrate the
advantages of CDG-Tre for labeling live BCG cells
phagocytosed in macrophages.
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a
The introduction of the cephalosporin molecule in CDG-Tre
endows the probe the fluorogenic feature and a dual activation
mechanism by both BlaC and Ag85 enzymes. Additionally,
better performance of CDG-Tre (compared to TG-Tre) in
labeling intracellular BCG cells in macrophages may be due to
CDG-Tre enhanced uptake by macrophages into the
phagosomes. We suspect the hydrophobic nature of the lactam
unit may be contributing to this effect. Indeed, a recent study
reported that lipid droplets in macrophages facilitated the
b
transferring of bedaquiline,
a lipophilic antibiotic, to
intracellular Mtb.41 The hydrophobic lactam unit in CDG-Tre
may promote its association with lipid droplets in macrophages,
resulting in more efficient uptake and transferring to
phagosomes for labeling of BCG (compared to more
hydrophilic TG-Tre). Further investigation is required to
confirm the underlying mechanism.
In summary, we report a new dual targeting fluorogenic
probe CDG-Tre that can be sequentially activated by two target
enzymes expressed in Mycobacterium tuberculosis: BlaC and
Ag85s. We investigated the mechanism of the uptake,
activation, and metabolic incorporation of CDG-Tre by
mycobacteria and demonstrated the advantages of CDG-Tre
over previous methods. CDG-Tre was shown to report changes
on bacteria viability 3 h following drug treatment, while genetic
labeled bacteria cells usually require longer incubation time to
reflect the changes (24 h for M. smegmatis and 5 d for Mtb).42
In compared to other trehalose fluorescent probes that are
generally poor cell permeable and work at high concentrations
(typically at 100-200 µM), CDG-Tre enabled high contrast
imaging at a low working concentration that displayed low
toxicity to the macrophages in labeling phagocytosed BCG cells
within macrophages. The post-phagocytosis labeling by CDG-
Tre may provide a new approach to tracking the kinetics of a
single Mtb cell within macrophages and probe their interactions
with high temporal and spatial resolution.
Figure 4. Imaging of live BCG cells within macrophage
phagosomes with CDG-Tre. a) Freshly cultured BCG was pre-
labeled with TAMRA-Tre (50 µM in PBS, red fluorescent)
overnight then washed and incubated with macrophages at a 10-
to-1 ratio in complete medium for 6 h. Macrophages were washed
with HBSS three times, incubated with 6-TG-Tre or CDG-Tre in
serum free medium at different concentrations for 22 h, then
further incubated in complete medium overnight. b)
Phagocytosed BCG cells were post-labeled by CDG-Tre (10 μM)
and imaged with a superresolution structured illuminated
microscope. Blue: Hoechst; Green: CDG-Tre or TG-Tre; Red:
TAMRA; Deep Red: Membrane stain. White arrows point to the
asymmetrical labeling of a single BCG. Scale bars indicate 10
addition to green punctuates. When the incubation time of
CDG-Tre increased to 22 h, followed by overnight culturing in
complete medium, the fluorescence labeling intensity of
phagocytosed live BCG was significantly enhanced, and the
green punctuates disappeared to produce a very clean
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ASSOCIATED CONTENT
Supporting Information
(8)
Experimental methods, supporting figures, and chemical
characterizations (HRMS, 1H and 13C NMR spectra). This material
is available free of charge via the Internet at http://pubs.acs.org.
(9)
AUTHOR INFORMATION
Corresponding Author
(10)
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Jianghong Rao - Departments of Radiology and Chemistry,
Molecular Imaging Program at Stanford, Stanford University
School of Medicine, Stanford, CA 94305, USA.
Email: jrao@stanford.edu
(11)
(12)
Kong, Y.; Yang, D.; Cirillo, S. L. G.; Li, S.; Akin, A.;
Francis, K. P.; Maloney, T.; Cirillo, J. D. Application
of fluorescent protein expressing strains to evaluation
of anti-tuberculosis therapeutic efficacy in vitro and in
vivo. PLoS One 2016, 11 (3), e0149972.
Author Contributions
† These authors contributed equally. The authors declare no
competing financial interest.
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We thank Stanford Cell Sciences Imaging Facility (CSIF) for help
on SR-SIM, Stanford Neuroscience Microscopy Service
(supported by NIH NS069375) for help on confocal imaging,
Stanford Shared FACS Facility for instrumentation and assistance
with flow cytometry, and Vincent Coates Foundation Mass
Spectrometry Laboratory, Stanford University Mass Spectrometry
for support on mass spectrometry analysis. This work was
supported by NIH grant R01AI125286. T.D. thanks the support
from the Center for Molecular Analysis and Design (CMAD) at
Stanford.
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