Probing the mycobacterial trehalome with bioorthogonal chemistry

Article abstract of DOI:10.1021/ja3062419

Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways

Full text of DOI:10.1021/ja3062419

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Communication
Probing the Mycobacterial Trehalome with Bioorthogonal Chemistry
Benjamin M. Swarts, Cynthia M. Holsclaw, John C Jewett, Marina Alber, Douglas
M. Fox, M. Sloan Siegrist, Julie A Leary, Rainer Kalscheuer, and Carolyn R Bertozzi
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 14 Sep 2012
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Probing the Mycobacterial Trehalome with Bioorthogonal Chemistry
†
†,#
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¶
∥,
⊥
Benjamin M. Swarts, Cynthia M. Holsclaw, John C. Jewett, Marina Alber, Douglas M. Fox, M. Sloan
†
,†,‡,§
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Siegrist, Julie A. Leary,∥ Rainer Kalscheuer, and Carolyn R. Bertozzi*
Departments of †Chemistry, ‡Molecular and Cell Biology, and §Howard Hughes Medical Institute, University of California, Berkeley,
California 94720, United States
Department of ∥Molecular and Cellular Biology, ⊥Campus Mass Spectrometry Facilities, University of California, Davis, California
95616, United States
¶Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Duesseldorf, 40225 Duesseldorf, Germany
Supporting Information Placeholder
ABSTRACT: Mycobacteria, including the pathogen Mycobacte-
rium tuberculosis, use the non-mammalian disaccharide trehalose as
a precursor for essential cell wall glycolipids and other metabolites.
Here we describe a strategy for exploiting trehalose metabolic
pathways to label glycolipids in mycobacteria with azide-modified
trehalose (TreAz) analogues. Subsequent bioorthogonal ligation
with alkyne-functionalized probes enabled detection and visualiza-
tion of cell-surface glycolipids. Characterization of the metabolic
fates of four TreAz analogues revealed unique labeling routes that
can be harnessed for pathway-targeted investigation of the myco-
bacterial trehalome.
Mycobacterium tuberculosis (Mtb), the causative agent of tubercu-
losis, currently infects 2 billion people worldwide and causes ap-
Figure 1. (A) Common trehalose glycolipids in mycobacteria. (B)
proximately 2 million deaths annually.1 The success of Mtb as a
Synthetic TreAz analogues used in this study. (C) TreAz-based
pathogen is in large part due to its complex cell wall, which is a
bioorthogonal chemical reporter strategy.
formidable barrier to antibiotics and residence to many biomole-
cules that are directly involved in pathogenesis. The mycobacterial
alternative for investigating glycoconjugates in living organisms.10
cell wall features a unique outer membrane – the “mycomembrane”
Here we report that trehalose glycolipids can be metabolically la-
(MM) – that is composed of long chain (C60-C90) mycolic acids
beled with azide-modified trehalose (TreAz) analogues (Figure
covalently bound to the underlying peptidoglycan-arabinogalactan
1B) in live mycobacteria, enabling bioorthogonal ligation with
polymer, as well as an array of additional free intercalating lipids
alkyne-functionalized fluorescent probes (Figure 1C). We capital-
and glycolipids.2,3
ized on the conserved pathways for mycobacterial trehalose me-
Trehalose-containing glycolipids are abundant, virulence-
tabolism shown in Figure 2. While trehalose glycolipids reside in
associated constituents of the MM that have essential roles in cell
the MM, they originate in the cytoplasm, where free trehalose is
synthesized through metabolic cycles involving either glucose (via
wall biosynthesis and disease progression. Trehalose mono- and
dimycolate (TMM and TDM), bearing 6-O- and 6,6’-di-O-mycolyl
substituents, respectively, are produced by all mycobacterial species
the OtsAB/trehalase enzymes) or α-glucans (via the TreYZ/TreS
enzymes).11-14 Trehalose and mycolic acid combine in the cyto-
(Figure 1A). TMM is an essential mediator of MM biosynthesis4-6
plasm to form TMM, which is then translocated across the plasma
(see Figure 2) and is also the precursor to TDM, an immunomodu-
membrane by MmpL3.5,6 Subsequently, the antigen 85 (Ag85)
latory molecule that promotes Mtb infectivity and survival within
complex mediates the transfer of mycolate from TMM to either
host macrophages.7-9 Along with TMM and TDM, numerous other
arabinogalactan, which forms covalently bound mycolates that
complex metabolites constitute the mycobacterial “trehalome.”
make up the foundation of the MM, or to another molecule of
Unfortunately, the various roles played by these metabolites in Mtb
physiology and pathogenesis are challenging to study using tradi-
TMM, which generates TDM.15,16 Both processes release free treha-
lose, which is recycled by the trehalose-specific transporter Sug-
tional genetic and biochemical methods, which generally require
ABC- LpqY.4 Species-dependent trehalose metabolism pathways
laborious radiolabeling, extraction, and purification procedures that
(not shown in detail) can generate a range of additional metabo-
are incompatible with in vivo experimentation.
Metabolic labeling with unnatural sugar substrates is a powerful
lites in Mtb and other mycobacteria.
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Figure 2. Trehalose metabolism in mycobacteria. Exogenous
TreAz can label glycolipids via the Ag85 or recycling pathways.
AG, arabinogalactan; CL, capsular layer; MM, mycomembrane;
PG, peptidoglycan; PM, plasma membrane. Exact extracellular
location of Ag85 is unknown.
In recent work, Backus et al. demonstrated that a fluorescein-
conjugated keto-trehalose analogue (FITC-Tre) is incorporated
into TDM via the Ag85 complex.17 This observation underscores
one possible route by which unnatural substrates might access tre-
halose glycolipids: after crossing through the MM, likely by a porin-
mediated process,18 the unnatural analogue could be processed by
Ag85 and incorporated into TMM or TDM outside of the cell
(Figure 2, orange dotted arrow). Alternatively, labeling could occur
via the trehalose recycling pathway, in which analogues would be
internalized by the SugABC-LpqY transporter and incorporated
into glycolipids from the inside-out (Figure 2, blue dotted arrow).
Access to the recycling pathway has not been reported for any
chemical probes to date but is essential for investigating processes
that originate in the cytoplasm, such as de novo biosynthesis of gly-
colipids and MM, as well as species-dependent trehalose metabo-
lism.
We hypothesized that TreAz analogues, in which a hydroxyl
group is replaced with a relatively small, minimally perturbing azido
group, would be well-tolerated by the mycobacterial biosynthetic
machinery, allowing unprecedented access to the trehalose recy-
cling pathway. In addition, the modular nature of bioorthogonal
ligation affords numerous advantages over other labeling strategies,
including choice of bioorthogonal reaction and probe type, tem-
poral control of probe delivery (permitting pulse-chase biological
experiments19), and the ability to minimize background signal by
use of low probe concentrations.
Figure 3. (A) Flow cytometry analysis of TreAz-labeled Msmeg
reacted with BARAC-Fluor. Error bars denote the standard devia-
tion of three replicate experiments. (B) Fluorescence microscopy
of TreAz-labeled Msmeg reacted with alk-AF488 via CuAAC. Scale
bars, 5 µm.
flow cytometry revealed that all four TreAz analogues labeled
Msmeg, albeit with different efficiencies (Figure 3A). 2- and 6-
TreAz labeled cells robustly at low concentrations (5–25 µM),
while labeling with 3- and 4-TreAz required higher concentrations
(250–500 µM). At these doses, no changes in bacterial growth were
observed. The described flow cytometry assay was also used to
evaluate the dependence of Msmeg labeling on analogue concentra-
tion, culture time, and exogenously added free trehalose (i.e., com-
petition studies; Figures S1–S3).
Next, fluorescence microscopy was used to visualize azide-
bearing cell-surface glycolipids (Figure 3B). Msmeg was cultured
with TreAz, fixed, and then reacted with alkyne-functionalized
Alexa Fluor 48821 (alk-AF488) via Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC).22,23 For all TreAz analogues, azide-specific
fluorescence was observed uniformly throughout the bacterial pop-
ulation, and signal was predominantly localized to the cell surface,
which is in agreement with glycolipids inhabiting the MM. Interest-
ingly, fluorescence was frequently concentrated at the bacterial
poles, consistent with a polar growth model for mycobacteria.24
We synthesized a series of trehalose analogues containing azido
groups at all possible positions with native stereochemistry [Figure
1B; refer to the Supporting Information (SI) for schemes and pro-
cedures]. The compounds were initially evaluated for metabolic
incorporation into glycolipids in the model organism M. smegmatis
mc2155 (Msmeg). Briefly, bacteria were cultured with TreAz until
logarithmic phase (typically 12-16 h), then reacted with BARAC-
Fluor (1 µM, 30 min), a fluorescein-conjugated biarylazacy-
clooctynone used for rapid Cu-free click chemistry.20 Analysis by
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In addition to fluorescence detection, we directly characterized
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TreAz-labeled glycolipids by chemical methods. Analysis of partial-
ly purified chloroform–methanol lipid extracts by TLC and high-
resolution mass spectrometry confirmed that all four TreAz ana-
logues produced azide-labeled glycolipids in Msmeg (Figure S4).
To provide further direct evidence that TreAz was incorporated
into glycolipids, petroleum ether lipid extracts from Msmeg cul-
tured with 2- or 6-TreAz (or vehicle) were deacylated by treatment
with NaOMe at 60 °C. The released free sugars were analyzed by
high-pH anion exchange chromatography with pulsed amperomet-
ric detection (HPAEC-PAD), which showed peaks for 2- and 6-
TreAz in the corresponding samples but not in vehicle-treated or
non-deacylated control samples (Figure S5). Comparison of peak
integrations indicated that 2- and 6-TreAz replaced approximately
20% and 70% of natural trehalose, respectively, in surface-exposed
glycolipids extracted from TreAz-treated cells.
To establish whether TreAz incorporation into glycolipids oc-
curred via the Ag85 or recycling pathway, azide labeling was evalu-
ated in the Msmeg mutants ΔsugC and ΔlpqY, which lack the treha-
lose transporter,4 by reaction with BARAC-Fluor followed by flow
cytometry (Figure 4A). Fluorescence observed for 2-, 4-, and 6-
TreAz in the wild-type strain was completely abolished in the mu-
tants, demonstrating that metabolic labeling of glycolipids using
these analogues requires uptake into the cytoplasm and proceeds
through the recycling pathway. In contrast, labeling with 3-TreAz
was not reduced in the transporter mutants, indicating that this
compound likely incorporates into glycolipids extracellularly via
Ag85, similar to FITC-Tre.17
To confirm that 2-, 4-, and 6-TreAz enter the cytoplasm,
HPAEC-PAD was used to assess the presence of these analogues in
cytosolic extracts. As expected, 2-, 4-, and 6-TreAz were observed
in extracts from wild-type Msmeg, while 3-TreAz was not (Figure
4B); identically prepared samples from the ΔsugC mutant showed
no cytosolic TreAz (Figure S6). Taken together, these data reveal
that our TreAz analogues have the capacity to selectively label tre-
halose-containing metabolites via the Ag85 or recycling pathways,
and thus provide a platform for pathway-targeted probing of the
trehalome.
Our finding that 2-, 4-, and 6-TreAz enter the cell through the
recycling pathway in Msmeg raises the possibility that these com-
pounds might also be processed by trehalase or TreS (Figure 2).
We sought address whether these enzymes affect cell-surface label-
ing, either by diverting TreAz into cell-surface α-glucan (via TreS)
or by degrading TreAz (via trehalase). Msmeg ΔtreS14 and
ΔMSMEG_4535 (trehalase) mutants were generated (see SI) and
evaluated for TreAz labeling compared to wild-type Msmeg (Fig-
ures S7–S11). For the most part, no significant differences were
observed in the mutants, ruling out any effects on cell-surface label-
ing by these enzymes. However, we did observe a TreS-
dependency for 4-TreAz labeling in Msmeg (Figure S8). We are
currently investigating the intriguing possibility that TreAz ana-
logues are incorporated into cell-surface α-glucan, an experimental-
ly elusive25 cell wall structure that has been implicated in immune
evasion.14,26
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Figure 4. (A) Flow cytometry analysis of TreAz-labeled Msmeg
strains. Error bars denote the standard deviation from three repli-
cate experiments. * p < 0.05. (B) HPAEC-PAD analysis of cytosolic
extracts from TreAz-treated wild-type Msmeg. Dotted lines repre-
sent retention times for authentic standards (see SI for details).
Figure 5. Flow cytometry analysis of TreAz-labeled Mtb and BCG
strains. (A) Wild-type Mtb H37Rv and BCG; (B) wild-type BCG,
BCG ΔlpqY-sugC, and the corresponding complemented strain.
Error bars denote the standard deviation from three replicate ex-
periments.
dependent fluorescence in both species (Figure 5A). We also gen-
erated a BCG mutant ΔlpqY-sugC missing the trehalose transporter
and its corresponding complemented strain (see SI) to test the
route of TreAz labeling (Figure 5B). Consistent with the results
from Msmeg, 2- and 6-TreAz labeling was substantially reduced in
the mutant, suggesting that a recycling pathway mechanism for
these analogues may be conserved across mycobacterial species.
Finally, we tested the TreAz analogues in pathogenic mycobacte-
ria. Mtb H37Rv and the closely related avirulent species M. bovis
BCG-Pasteur (BCG) were assessed by flow cytometry as described
above for Msmeg, and all four compounds led to significant TreAz-
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ASSOCIATED CONTENT
Supporting Information. Experimental procedures, characterization
data, supporting figures, schemes, and tables. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
crb@berkeley.edu
Present Address
#Department of Chemistry and Biochemistry, University of Arizona,
Tucson, AZ 85721.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank M. Boyce, M. Breidenbach, and S. Canham for helpful dis-
cussions and for critical reading of the manuscript. S. Bauer is acknowl-
edged for providing assistance with HPAEC-PAD instrumentation.
This work was supported by a grant to C.R.B. from the NIH
(AI51622). B.M.S., J. C. J, and M. S. S. were supported by postdoctoral
fellowships from the American Cancer Society, and B.M.S. was also
supported by a fellowship from the Center for Emerging and Neglected
Diseases. C.M.H. was supported by the UC Davis Office of the Vice
Chancellor for Research. R.K. acknowledges support from the Juergen
Manchot Foundation.
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158x162mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
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158x147mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 11 of 11
Journal of the American Chemical Society
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89x35mm (300 x 300 DPI)
ACS Paragon Plus Environment