Development of Rare Bacterial Monosaccharide Analogs for Metabolic Glycan Labeling in Pathogenic Bacteria

Article abstract of DOI:10.1021/acschembio.6b00790

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

Full text of DOI:10.1021/acschembio.6b00790

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Article
Development of rare bacterial monosaccharide analogs
for metabolic glycan labeling in pathogenic bacteria
Emily L. Clark, Madhu Emmadi, Katharine L. Krupp, Ananda R.
Podilapu, Jennifer D. Helble, Suvarn S. Kulkarni, and Danielle H. Dube
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00790 • Publication Date (Web): 21 Oct 2016
Downloaded from http://pubs.acs.org on October 23, 2016
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Development of rare bacterial monosaccharide analogs for metabolic
glycan labeling in pathogenic bacteria
Emily L. Clark,1 Madhu Emmadi,2 Katharine L. Krupp,1 Ananda R. Podilapu,2 Jennifer D. Helble,1
Suvarn S. Kulkarni,2* and Danielle H. Dube1*
1Department of Chemistry & Biochemistry, Bowdoin College, 6600 College Station, Brunswick, ME
04011 2Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400076,
India
*ddube@bowdoin.edu; *suvarn@chem.iitb.ac.in
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ABSTRACT
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Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to
pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing
molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the
presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents.
Thus, the development of chemical tools that label bacterial glycans is a crucial step toward
discovering and targeting these biomolecules. Here we explore the extent to which metabolic
glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and
symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant
monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into
bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility
of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare
deoxy amino D-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-
trideoxygalactose and established that these analogs are differentially incorporated into glycan-
containing structures in a range of pathogenic and symbiotic bacterial species. Further application
of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find
utility in treating a variety of infectious diseases with selectivity.
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INTRODUCTION
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One widespread challenge in the healthcare industry is the increasing ineffectiveness of
existing antibiotics due to the evolution of microbial resistance. Even when existing
antibiotics are effective at treating bacterial disease, these treatments are often broad-
spectrum and suffer from deleterious effects on our beneficial bacteria.1 Such disruptions to
the normal gut microbiome can precipitate long-term health problems2 including obesity,
autoimmune disorders, allergies, malnutrition, and expansion of enteric pathogens in the
gut.3,4 Thus, there is an urgent need to develop novel antibiotics that target specific bacterial
populations while leaving the rest of the microbiome intact.
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Bacterial glycoproteins serve as attractive targets of therapeutic intervention because
they are often linked to pathogenesis, their structures differ markedly from those found in
eukaryotes, and they contain distinctive monosaccharide building blocks that are
exclusively present on a small number of bacterial pathogens.5,6 For example, the gastric
pathogen Helicobacter pylori synthesizes glycoproteins that contain the rare amino- and
deoxy-monosaccharides pseudaminic acid, legionaminic acid, and bacillosamine (Figure
1).7,8 These sugars have limited expression on pathogenic bacteria, and there are no reports
of these three monosaccharides in nonpathogenic bacteria that dominate the human gut
microbiome3 (e.g. Bacteroides spp., Prevotella spp.). More broadly, Neisseria meningitidis
utilizes 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH),9 Pseudomonas aeruginosa
incorporates FucNAc residues,10 and the symbiotic Bacteroides fragilis appends 2-
acetamido-4-amino-2,4,6-trideoxygalactose (AAT)11 into its cell surface polysaccharides
(Figure 1). These distinctive building blocks appear to be exclusively present on a small
number of bacterial species.6,12
Despite the potential of distinct monosaccharides within bacterial glycans to serve as
molecular targets, the systematic discovery of bacterial glycoproteins is hampered by the
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presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding
reagents.13,14 In particular, lectins and glycosidases employed to detect and remove
eukaryotic glycans, respectively, often cannot be utilized to study bacterial glycans due to
their restricted binding specificity.15,16,17 The development of chemical tools that label
bacterial glycans is a crucial step toward discovering and targeting these biomolecules.
The chemical technique known as metabolic oligosaccharide engineering (MOE), which
was pioneered by Bertozzi,18,19 Reutter20 and others for the study of mammalian
glycoproteins, offers a powerful approach to label and detect bacterial glycoproteins.21 In
MOE, cells are supplemented with an unnatural sugar that is taken up, processed by
endogenous carbohydrate biosynthetic pathways, and installed into glycans in place of
natural monosaccharides. Using this approach, bio-orthogonal chemical reporters, such as
azides,22 can be incorporated into cellular glycans (Figure 2A). Once incorporated into
cellular glycoproteins, the azide acts as a chemical handle that undergoes covalent
elaboration with exquisitely selective reaction partners, including triarylphosphines23 and
cyclooctynes,24 for the attachment of biological probes25 (Figure 2A). Incorporation of azide-
containing sugars into bacterial glycans thus facilitates detection, enrichment, and
identification of bacterial glycoproteins, and enables targeting with covalent therapeutics to
incite cell damage.26
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MOE has been utilized to incorporate an azide-bearing analog of the common
monosaccharide N-acetylglucosamine (GlcNAc) into glycoproteins on the gastric pathogen
Helicobacter
pylori.21
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GlcNAc
analog
peracetylated-N-
azidoacetylglucosamine (Ac4GlcNAz (2), Figure 2B) was robustly incorporated into H.
pylori’s glycoproteins and facilitated the identification of 125 candidate H. pylori
glycoproteins.27 In addition to enabling bacterial glycoprotein discovery, Ac4GlcNAz could
form the basis of a bacterial glycan targeting strategy as it is minimally presented onto the
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surfaces of mammalian cells,28 despite its robust incorporation into mammalian O-GlcNAc
modified nuclear and cytosolic proteins.29 Indeed, our work led to the development of
immune-stimulant bearing phosphines that catalyzed the destruction of azide-covered H.
pylori in an immune-cell dependent manner.30 Thus, Ac4GlcNAz is a reasonable choice from
the perspective of discovering and targeting H. pylori glycoproteins and may have broad
utility for probing glycoproteins or other glycan-containing structures in diverse bacteria.
Here, we explored the extent to which MOE facilitates the study of glycoproteins in a
range of pathogenic and symbiotic bacterial strains. Our results demonstrate that relatively
few bacterial strains are azide-labeled upon treatment with Ac4GlcNAz, and thus suggest
that differential labeling of bacterial populations is possible. However, these results reveal a
need for additional azidosugar substrates to broaden the utility of MOE in bacteria. To meet
this need, we synthesized azide-containing analogs of rare bacterial monosaccharides
(Figure 2B; compounds 4, 5, 6) and established that pathogenic bacteria effectively process
these distinctive sugars. This work sets the stage for facilitating the study of glycoproteins in
diverse bacterial species and providing a means to inactivate select bacteria based on their
distinctive glycan coating.
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RESULTS AND DISCUSSION
To facilitate the studying and targeting of glycans in a range of pathogenic bacteria, we set
out to assess the metabolic incorporation of a range of azide-bearing monosaccharides in
diverse bacteria. Since incorporation of azide-containing sugars into a wide range of
bacterial glycoproteins is beneficial from the perspective of glycoprotein discovery but
detrimental for creating narrow-spectrum glycan-targeted therapeutics,6 we explored the
incorporation of azide-bearing analogs of both common and rare monosaccharide
substrates. We reasoned that rare monosaccharides with restricted metabolic fates (e.g.
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conversion to a single product) would be processed into glycans on fewer bacterial strains
than common monosaccharides with broad metabolic fates.
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Differential labeling of bacterial glycoproteins with Ac4GlcNAz. To build upon the
established utility of Ac4GlcNAz (2),29 an azide-containing analog of the common
monosaccharide GlcNAc, for discovering27 and targeting30 Helicobacter pylori’s
glycoproteins, we first sought to investigate the utility of this substrate to study
glycoproteins in bacteria beyond H. pylori. We began by assessing the levels of incorporation
of Ac4GlcNAz into bacterial glycans in a range of species including pathogenic and symbiotic
bacteria. In particular, we focused on incorporation into the gram-negative pathogens
Campylobacter jejuni and Pseudomonas aeruginosa, close relatives of pathogens
Burkholderia thailandensis and Mycobacterium smegmatis, the plant pathogen Ralstonia
solanacerum, and the symbiotic bacterium Bacteroides fragilis. These bacteria were selected
as their ability to produce glycosylated proteins has been previously established.31-37 To
gauge metabolic incorporation, bacteria were treated with 1 mM Ac4GlcNAz (2) or the azide-
free control sugar peracetylated N-acetylglucosamine (Ac4GlcNAc (1), Figure 2B), then their
proteins were harvested by cell lysis and reacted with a phosphine probe comprising a
FLAG peptide38 (Phos-FLAG). In all Western blot analyses, lysates from H. pylori treated with
Ac4GlcNAz followed by Phos-FLAG were included as a positive control.21 Analysis of proteins
from treated H. pylori and R. solanacerum yielded an array of azide-labeled glycan-
containing structures that were detected by Western blot analysis with anti-FLAG antibody
(Figure 3). In contrast, similar treatment of the other bacterial strains with Ac4GlcNAz led to
no detectable azide-labeled glycoproteins (Figure 3). Control samples from cells treated
with the azide-free compound peracetylated GlcNAc (Ac4GlcNAc, 1) yielded no azide-
dependent signal, as expected. Zinc or Coomassie staining of electrophoresed samples
confirmed that all samples contain proteins (Supplementary Figure 1), indicating that the
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lack of detectable azides in some samples was not due to low protein levels. Counter to our
expectation, these data reveal that metabolic incorporation of azides into bacterial glycans
upon treatment with Ac4GlcNAz is not a widespread phenomenon but rather appears
limited to select bacterial strains.
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Synthesis of azide-containing variants of rare bacterial monosaccharides. Due to the
observed narrow incorporation of Ac4GlcNAz into bacterial glycans, we wondered whether
analogs of rare bacterial monosaccharides might facilitate glycoprotein discovery while
setting the stage for novel antibiotic strategies that interfere with select pathogenic strains
but leave the microbiome largely intact. Therefore, we designed a panel of azide-containing
variants of the rare bacterial sugars FucNAc, bacillosamine, and DATDG (Figure 2B, 4-6).
Given that a variety of carbohydrate biosynthetic pathways are permissive of N-azidoacetyl
substituents in place of the natural N-acetyl moieties,18,26 we reasoned that the requisite
bacterial enzymes would likely process analogs bearing N-azidoacetyl groups. Further, we
chose to temporarily mask the hydrophilic hydroxyl groups of these analogs with
hydrophobic acetyl groups39 to maximize efficiency of uptake by bacterial strains.21
To access these compounds, we built upon a strategy developed by Kulkarni and
coworkers to synthesize rare bacterial amino and deoxy monosaccharide building blocks
including FucNAc, bacillosamine, and DATDG.40,41 In this strategy, changing the sequence of
addition of nucleophiles in a series of one-pot double serial and double parallel
displacement reactions using azide, phthalimide and nitrite ions as nucleophiles and
mannose-derived 2,4 bis-trifluoromethanesulfonates (triflates) as electrophiles enabled
rapid access to various orthogonally protected rare deoxy amino sugar building blocks.40,41
Accordingly, the 2,4-diol substrates (Scheme 1, compound 7 and Scheme 2, compound 8) for
the one-pot double displacement reaction were first prepared starting from cheaply
available D
-mannose in six steps.41
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Transformation of D-rhamnosyl 2,4-diol 7 into D-fucosamine derivative 4 is shown in
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Scheme 1. Following our earlier established procedure, compound 7 was converted to the
corresponding 2,4-bistriflate derivative, which upon a brief work-up was subjected to a one-
pot, regioselective, nucleophilic displacement reaction. Accordingly, a sequential C2-OTf
displacement of the crude bistriflate with a stoichiometric amount of tetrabutylammonium
azide (TBAN3) at –30 °C followed by water mediated intramolecular displacement of the C4-
OTf by C3-OAc, via the ring opening of the corresponding orthoester intermediate, resulted
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in the formation of the C4-OAc derivative of D
-fucosamine 9 in 62% yields over three steps.41
The remaining 3-OH group was capped as an acetate to form 10 (95%), which upon azide
reduction under hydrogenation conditions, followed by EDC mediated coupling of the so-
formed amine with azidoacetic acid,19 afforded the azido derivative 11 in 88% yield over
two steps. Finally, hydrolysis of the thioglycoside by using NBS and water generated the
corresponding hemiacetal which upon subsequent treatment with acetic anhydride in the
presence of trielthylamine afforded triacetate compound 4 (α/β =2:1)(Ac3FucNAz, Fuc) in
76% yield over 2 steps.
For the synthesis of Ac2Bac-diNAz (Bac) 5, we started from 3-OBz derivative 8 (Scheme
2). Diol 8 upon similar triflation followed by sequential, one pot SN2 reactions with TBAN3
and with tetrabutyl ammonium nitrite (TBANO2), at C2 and C4 respectively, afforded D-
fucosamine derivative 12 in 60% overall yield.41 Concomitant triflation of the axial 4-OH
group and its displacement with NaN3 furnished bacillosamine 13.41 Reduction of 2,4-azido
groups in 13 followed by EDC coupling with azidoacetic acid afforded 14 in 72% yield over
two steps. Debenzoylation, conversion of anomeric thioglycoside to the corresponding
hemiacetal with NBS/water and acetylation afforded the target molecule Ac2Bac-diNAz
(Bac) 5 (α/β = 1:0.1) in 68% yield over three steps.
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The synthesis of an analog of the rare sugar DATDG is outlined in Scheme 3. A double
displacement of the 2,4-bistriflate, obtained from 8, with excess NaN3 cleanly afforded di-
azido derivative 15 in excellent overall yield.41 The 3-O-benzoate group was cleaved using
NaOMe to obtain 16 (96%). This step was deemed necessary in order to avoid the possible
migration of benzoate (C3→C4) during the ensuing reduction of azides to corresponding
amines. The azides were then reduced under hydrogen atmosphere and so formed amines
were coupled with the azidoacetic acid using EDC as the coupling reagent. In this reaction,
along with the desired di-N-acylated product, we also obtained fully acylated product.
Treatment of the mixture of acylated products with NaOMe in MeOH gave the desired
product 17 in 74% yield over 3 steps. Thioglycoside was treated with NBS/water and the so
formed hemi acetal was acetylated to give the desired derivative Ac2DATDG-diNAz (DAT) 6
(α/β = 2:1) in 72% yield over 2 steps.
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Rare bacterial monosaccharide analogs are incorporated into diverse bacterial
pathogens. With access to these rare bacterial monosaccharide analogs, we performed
metabolic labeling experiments to assess incorporation into diverse bacterial species. We
first evaluated metabolic incorporation into bacterial pathogens or close relatives of
pathogens. For these experiments, bacteria were grown in media supplemented with one of
each of the panel of rare bacterial monosaccharide analogs (Ac3FucNAz (4), Ac2Bac-diNAz
(5), or Ac2DATDG-diNAz (6)), or with one of three GlcNAc analogs (Ac4GlcNAz (2),28 the
peracetylated 6-azido GlcNAc analog Ac36AzGlcNAc (3),42 or the azide-free control
Ac4GlcNAc (1); Figure 2B) that are not appreciably incorporated onto mammalian cell
surfaces.28,42 After metabolic labeling, proteins were harvested by cell lysis and reacted with
Phos-FLAG for Western blot analysis with anti-FLAG antibody to yield a snapshot of all
(“total”) azide-labeled glycans from the bacteria. Alternatively, intact cells were rinsed and
reacted with the cell impermeable compound Phos-FLAG to probe for the presence of
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surface accessible (“surface”) azides on the bacteria;30 these surface accessible azide-labeled
species were then detected by flow cytometry or Western blot analysis with an anti-FLAG
antibody.
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As depicted in Figure 4, the gastric pathogen H. pylori robustly incorporates Ac4GlcNAz (2),
Ac2Bac-diNAz (5), and Ac2DATDG-diNAz (6) into surface glycans, as revealed by Western blot
(Figure 4A) and flow cytometry (Figure 4B; Supplemental Figure S2B) analysis. In contrast,
Ac3FucNAz (4) is minimally incorporated into H. pylori’s glycans and H. pylori treated with
Ac4GlcNAc (1) remain azide-free (Figure 4A, B). The gut pathogen C. jejuni displays a different
metabolic incorporation phenotype; Western blot analysis reveals robust processing of only Ac2Bac-
diNAz (5) and Ac2DATDG-diNAz (b) into select primarily surface-accessible glycans in C. jejuni
(Figure 4C). The opportunistic pathogen B. thailandensis incorporates Ac2Bac-diNAz (5) but no
other azidosugars into its glycan-containing structures (Figure 4D); follow up analysis of the
Ac2Bac-diNAz (5) labeled species from B. thailandensis indicates that they are not surface accessible
(Figure 4E). Finally, the plant pathogen R. solanacearum exhibits the broadest incorporation profile,
with treatment with Ac4GlcNAz (2), Ac2Bac-diNAz (5), Ac2DATDG-diNAz (6), Ac36AzGlcNAc (3) and,
to a lesser extent, Ac3FucNAz (4) yielding surface accessible azides detectable by Western blot
(Figure 4F) and flow cytometry (Supplementary Figure 3). In all cases Coomassie staining of
electrophoresed samples confirmed equivalent protein levels (Supplementary Figure 2), indicating
that the lack of detectable azides in some samples was not due to low protein content. Further, the
negative control samples from cells treated with Ac4GlcNAc (1) consistently lacked azide-dependent
signal. Taken together, these data suggest that the extent of incorporation into bacterial glycans is
dependent upon the structure of the metabolic input and upon the bacteria under study. Relative to
Ac4GlcNAz, analogs of rare bacterial monosaccharides appear better suited to study bacterial
glycans.
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Rare bacterial monosaccharide analogs are not incorporated onto symbiotic or
mammalian cells. With compelling evidence that this panel of azidosugars could be used to
study or target glycans in a variety of bacterial pathogens, we next explored whether these
azidosugars are incorporated into the beneficial bacteria Bacteroides fragilis. Such
information is critical to assess potential undesired impacts on innocent bystanders of a
rare bacterial monosaccharide-based antibiotic strategy. B. fragilis supplemented with the
panel of azide-containing sugars did not incorporate azides into glycoproteins at a
detectable level by Western blot analysis (Figure 5A) despite high protein content in these
samples (Supplementary Figure 4A). In contrast, Western blot and flow cytometry analyses
revealed that the azidosugar peracetylated N-azidoacetylgalactosamine (Ac4GalNAz),43 an
analog of N-acetylgalactosamine, was robustly displayed in B. fragilis surface glycans (Figure
5B,C; Supplementary Figure 4B); this result is consistent with a recent demonstration by
Kasper and co-workers that Ac4GalNAz is processed into polysaccharide A in B. fragilis.44
These data indicate that, though it is possible to incorporate azides into B. fragilis surface
glycans, such incorporation is negligible with the rare bacterial monosaccharide analogs
reported here. Thus, narrow range antibiotics based on differential incorporation of azides
onto the surface of pathogenic but not symbiotic bacteria may be possible.
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To explore whether these rare bacterial monosaccharide analogs may have the potential
to underpin a covalent antibiotic strategy aimed at gastrointestinal pathogens, we set out to
evaluate the incorporation of these analogs in mammalian cells. As a model system, we
turned to the well-studied Madin Darby Canine Kidney (MDCK) epithelial cell line, as this
cell line is an established model for epithelial cells that line the gastrointestinal tract. In
experiments analogous to those described above, MDCK cells were treated with each of a
panel of azidosugars, and metabolic incorporation of azides into cellular glycans and onto
cell surfaces was assessed using Phos-FLAG and anti-FLAG antibody.19 Western blot and
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flow cytometry analyses revealed that only the positive control sugar Ac4GalNAz, which is
robustly incorporated into mucin-type O-linked glycoproteins,43 led to significant delivery of
azides onto the surface of MDCK cells (Figure 5D, E). In contrast, none of the other
azidosugars resulted in appreciable display of surface-accessible azides (Figure 5E, “surface”
lanes); Coomassie staining confirmed that the differential azide labeling was not due to
appreciable differences in protein loading across samples (Supplementary Figure 4C). As
expected based on previous literature reports, Ac4GlcNAz (2) and Ac36AzGlcNAc (3) were
incorporated into internal MDCK glycoproteins, consistent with the metabolic incorporation
of these sugars into nuclear and cytosolic O-GlcNAc residues (Figure 5E, “total” lanes).42
Unexpectedly, bacillosamine analog 5 was processed by MDCK cells and led to installation of
azides into some internal MDCK structures (Figure 5E, “total lane”). This result is surprising
due to the assumed lack of metabolic enzymes to process this exclusively bacterial sugar
analog. Regardless, the finding that none of the rare bacterial monosaccharide analogs led to
appreciable presentation of azides on the surface of MDCK cells bodes well for the potential
of these sugars to form the basis for selective covalent antimicrobial applications.
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Discussion. Here we explore and expand the scope of metabolic glycan labeling across
diverse bacterial species. Presumably due to the distinctive monosaccharide building blocks
employed by bacteria, the analog Ac4GlcNAz (2) appears to have some but limited utility for
metabolic labeling of bacterial glycoproteins. To address this limitation, we produced a
series of novel bacteria-specific azidosugar substrates that are robustly incorporated into
glycans in a variety of pathogenic bacteria. Our data indicate that the choice of azidosugar
substrate dictates which bacterial species process azides into their glycans.
The ability of bacterial strains to process monosaccharide analogs into their glycans
could reflect underlying differences in glycosylation machinery present within each species.
For example, H. pylori and C. jejuni have biosynthetic pathways that convert bacillosamine to
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pseudaminic acid. A likely metabolic fate of Ac2Bac-diNAz (5) in these bacteria, therefore, is
conversion to azido-pseudaminic acid and installation onto flagellin glycoproteins. In
contrast, B. fragilis does not have the pseudaminic acid biosynthetic pathway enzymes, nor
does it process Ac2Bac-diNAz into its glycans. Thus, metabolic incorporation of azidosugars
into bacterial glycans is linked to which carbohydrate active enzymes are present within
each species. The differences in glycan labeling across bacterial species observed in this
work could also be a reflection of the labeling conditions used. Due to the dependence of
glycan expression on a variety of factors (e.g. cell cycle, the environment), bacterial strains
might exhibit a different metabolic incorporation phenotype at different growth stages or in
different environments. For example, under different environmental conditions bacterial
strains might incorporate monosaccharide analogs that were not displayed in their glycans
in the conditions used in this study. Moreover, exploring a range of sugar analog
concentrations (e.g. higher and lower than 1 mM) has the potential to broaden or narrow
metabolic incorporation into glycans. Even within the scope of this work, where fixed
concentrations of sugars were utilized for fixed periods of time, subtle differences in
glycoprotein fingerprint were observed across samples; Ac4GlcNAz-treated H. pylori
displayed altered labeling profiles (Figure 3), likely due to phase variation causing changes
in protein and glycan expression levels.
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Metabolic incorporation sets the stage to use the azide as a chemical handle to enrich
these labeled species, ultimately enabling glycoproteomic analyses.19 Indeed, Woo et al.
recently reported an azido-glycoproteomic method that could be applied to identify which
bacterial proteins are glycosylated and to elucidate the structures of the modifying
glycans.45 Moreover, metabolic labeling of bacterial glycoproteins provides an approach to
monitor bacterial glycan dynamics; time- and environment-dependent changes in glycan
expression have the potential to yield a dynamic picture of bacterial glycosylation,
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ultimately providing insight into the functions of these critical biomolecules. Expanding the
scope of metabolic glycan labeling to a range of bacterial species is therefore an important
step to facilitate the study of bacterial glycoproteins.
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In addition to easing bacterial glycoprotein discovery, this work sets the stage for
covalent targeting of distinctive bacterial glycans with molecules that incite damage or
permit visualization.6 Our data indicate that the choice of azidosugar substrate dictates
which bacterial species become azide-covered. Differential incorporation of azides across
bacterial cells could form the basis for narrow-spectrum antibiotics that induce killing of
pathogenic bacteria yet leave symbiotic bacteria unharmed. For example, azide-coated
bacteria could be targeted with immune stimulant-bearing phosphines via Staudinger
ligation30 or with copper-free click-chemistry46 delivered payloads. Analogously, azide-
reactive molecules bearing imaging agents47,48 could be employed to track bacterial infection
in an animal model. Taken together, the work presented here provides expanded tools for
metabolic labeling of bacterial glycans, which might be harnessed for glycoprotein
discovery, targeted cell killing, or molecular imaging.
Conclusions
Metabolic labeling of bacterial glycoproteins is a relatively new strategy to study and target
these biomolecules. Prior to this work, common monosaccharide precursors were employed
to metabolically label glycoproteins in H. pylori27 and Bacteroidales species,49 and a
bacillosamine analog was employed as a committed pseudaminic acid precursor to label
flagellin glycoproteins in C. jejuni.50 The work reported here adds a new dimension to this
field by generating analogs of rare bacterial monosaccharides that are processed by a
variety of pathogenic but not symbiotic bacterial strains, thus offering the potential to
exploit the incorporated azides for glycoprotein discovery, targeted cell killing, and
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molecular imaging. Although we focus here on a relatively small panel of pathogenic and
symbiotic bacterial strains, this strategy has the potential to be applied to study, target, and
track distinctive glycans in complex microbial communities present in an animal infection
model. Thus, this strategy opens the door to discovering novel antibiotic targets and treating
a wide range of bacterial diseases in a glycosylation-dependent manner.
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METHODS
Materials and Chemical Synthesis. Organic chemicals and anti-FLAG antibodies were
purchased from Sigma-Aldrich. H. pylori strain G2751 was a gift of Manuel Amieva (Stanford
University), and M. smegmatis strain Mc2155::otsA treS treY52 was a gift from Peter Woodruff
(University of Southern Maine). Bacterial cells (C. jejuni ATCC 33560; B. thailandensis ATCC
700388; R. solanacearum ATCC 33291* (*incorrectly cataloged as C. jejuni; species
confirmed as R. solanacearum by 16S ribosomal RNA sequencing); B. fragilis ATCC 23745; P.
aeruginosa ATCC 39324) and MDCK cells (ATCC CCL-34) were purchased from ATCC and
grown according to the supplier’s instructions. Ac4GlcNAc (1), Ac4GlcNAz (2), Ac4GalNAz
and Phos-FLAG were synthesized as previously described.19,53 Ac36AzGlcNAc (3)42 was
kindly provided by Matthew Pratt (University of Southern California). Ac3FucNAz (4),
Ac2Bac-diNAz (5), and Ac2DATDG-diNAz (6) were synthesized using standard organic
1
chemistry procedures and characterized by standard techniques including H and 13C NMR
spectroscopy and mass spectrometry. Ac3FucNAz (4), Ac2Bac-diNAz (5), and Ac2DATDG-
diNAz (6) were purified using flash silica gel chromatography.
Metabolic Labeling. Bacterial cells were grown in rich liquid media supplemented with
1 mM21 Ac4GlcNAz (2), Ac36AzGlcNAc (3), Ac3FucNAz (4), Ac2Bac-diNAz (5), Ac2DATDG-
diNAz (6), Ac4GalNAz, or the azide-free control Ac4GlcNAc (1) for 2–5 days, depending on
the doubling rate of the organism. H. pylori, C. jejuni, and R. solanacearum were metabolically
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labeled for 5 days under microaerophilic conditions (14% CO2, 37 °C), B. fragilis were
metabolically labeled for 2 days under anaerobic conditions (created by a Becton Dickinson
EZ anaerobe Gaspack in an airtight container; 37 °C), and M. smegmatis, B. thailandensis, and
P. aeruginosa were grown in 1 mM sugar-containing media for 5 days under aerobic
conditions, as appropriate for each bacterial strain. MDCK were grown in Dulbecco’s
Modified Eagles Medium supplemented with 250 µM of Ac4GlcNAc (1), Ac4GlcNAz (2),
Ac36AzGlcNAc (3), Ac3FucNAz (4), Ac2Bac-diNAz (5), Ac2DATDG-diNAz (6), or Ac4GalNAz for
3 days. Cells were then harvested, rinsed with phosphate buffered saline (PBS), and reacted
with 250 µM Phos-FLAG for 5 hours at 37 °C for detection of cell surface azides; whole cells
were then lysed for Western blot, as described below. Alternatively, metabolically labeled
cells were harvested, rinsed with PBS, lysed, and then reacted with 250 µM Phos-FLAG
overnight at 37 °C. Protein lysates were standardized (BioRad’s DC protein concentration
assay) and analyzed by Western blot.
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Western Blot. Reacted lysates from metabolically labeled cells were standardized to a
protein concentration of ~3 mg mL-1, loaded onto a 4–15% Tris–HCl SDS-PAGE gel,
separated by electrophoresis, and transferred to nitrocellulose paper. Anti-FLAG-HRP was
employed to visualize FLAG-tagged proteins via chemiluminescence.
Flow cytometry. Metabolically labeled intact cells were reacted with Phos-FLAG (250
µM) for 1 hour, then rinsed and treated with anti-FLAG-FITC to visualize FLAG-covered
cells. Alternatively, the presence of azides on cells was probed using Click-IT Alexa Fluor
488 DIBO Alkyne (20 µM) for 5 hours in the dark for copper free click chemistry detection of
azides (ThermoFisher Scientific; ex: 488/em: 519). Cells were analyzed by flow cytometry
on a FACSCalibur (BD Biosciences) instrument, with 20,000 live cells gated for each
replicate experiment.
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ASSOCIATED CONTENT
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Supporting Information Available:
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Detailed procedures for chemical syntheses and biological experiments, scanned NMR data for
synthesized compounds, and Supplementary Figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
ddube@bowdoin.edu; suvarn@chem.iitb.ac.in
ACKNOWLEDGEMENTS
We thank K. Moulton and A. McBride for helpful comments, M. Pratt and K. Chuh for
generously providing Ac36AzGlcNAc, and P. Woodruff and M. Amieva for sharing bacterial
strains. E.L.C. acknowledges support from a Surdna Foundation Undergraduate Research
Fellowship, K.L.K. thanks Maine-INBRE for a summer research fellowship, M.E. and A.R.P
thank CSIR-New Delhi for fellowships, and E.L.C., K.L.K., and J.D.H. received Grua/O’Connell
Research Awards. Research reported in this publication was supported by the National
Institutes of Health under grant number R15GM109397, by a Henry Dreyfus Teacher-
Scholar award to D.H.D., and by an Institutional Development Award (IDeA) from the
National Institute of General Medical Sciences of the National Institutes of Health under
grant number P20GM0103423, and by Science and Engineering Research Board,
Department of Science and Technology, Government of India (Grant No.
EMR/2014/000235).
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Graphical table of contents en
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Figure 1 Structures of rare, exclusively bacterial deoxy amino monosaccharides and examples of bacteria
that install them in their glycans.
Figure 1
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Figure 2 Schematic overview of metabolic oligosaccharide engineering (MOE) experiments performed, and
structures of monosaccharide analogs used in this study. A) In MOE experiments, cells are first metabolically
labeled with an unnatural azide-containing sugar. Azide-bearing glycans then undergo Staudinger ligation
with phosphine probes to enable their detection on cells or in cell lysates. B) Metabolic incorporation of
azide-containing analogs of the naturally abundant monosaccharide N-acetylglucosamine (GlcNAc) as well as
of the rare bacterial monosaccharides FucNAc (4), bacillosamine (5), and DATDG (6) were explored in this
work.
Figure 2
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Figure 3 Western blot analyses indicate that Ac4GlcNAz (2) supplementation leads to installation of azides
into glycoproteins in select bacterial pathogens. A) Helicobacter pylori (Hp), Campylobacter jejuni (Cj), B)
Pseudomonas aeruginosa (Pa), C) Burkholderia thailandensis (Bt), D) Mycobacterium smegmatis (Ms), and
E) Ralstonia solanacerum (Rs) were grown in media supplemented with 1 mM Ac4GlcNAz (2, Az) or the
azideꢀfree control sugar Ac4GlcNAc (1, Ac) for 5 days, then harvested by lysis. F) Due to its faster doubling
time, Bacteroides fragilis (Bf) was grown in media supplemented with 1 mM Ac4GlcNAz (2, Az) or the azideꢀ
free control sugar Ac4GlcNAc (1, Ac) for 2 days, then harvested by lysis. The presence of azides in cellular
glycoproteins was detected by reacting lysates with 250 ꢁM PhosꢀFLAG for 6ꢀ12 hours at 37 °C, and then
analyzing samples via Western blot with antiꢀFLAG antibody. The data shown are representative of replicates
(n≥3).
Figure 3
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Scheme 1. Synthesis of Ac3FucNAz (Fuc) 4
Scheme 1
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Scheme 2. Synthesis of Ac2Bac-diNAz (Bac) 5
Scheme 2
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