LETTERS

Analyzing the dynamic bacterial glycome with

© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

a lectin microarray approach

Ku-Lung Hsu1, Kanoelani T Pilobello1 & Lara K Mahal2

Glycosylation of bacterial cell surfaces is emerging as a critical
factor in symbiosis, pathogenesis, cell-cell interactions and
immune evasion1–3. The lack of high-throughput analytical
tools to examine bacterial glycans has been a major obstacle to
the field and has hindered closer examination of the dynamics
of carbohydrate variation. We have recently developed a lectin
microarray for the analysis of glycoproteins4. Herein we
present a rapid analytical system based on this technology for
the examination of bacterial glycans. The glycosylation pattern
observed distinguishes closely related Escherichia coli strains
from one another, providing a facile means of fingerprinting
bacteria. In addition, dynamic alterations in the carbohydrate
coat of a pathogenic E. coli strain are readily observed. The
fast evaluation of real-time alterations in surface-carbohydrate
epitopes allows examination of the dynamic role of
bacterial sugars in response to external stimuli such as
the immune system.
this assay is not suitable for high-throughput analysis of bacterial
glycans, as it requires painstaking attachment of multiple lectin-
modified membrane disks to electrodes for each bacterial strain and
large amounts of both lectin and bacteria18. Herein, we present
a strategy for quickly assessing bacterial cell-surface carbohydrate
composition based on lectin binding in an array format (Fig. 1).
Our updated lectin microarray consisted of a panel of 21 lectins,
chosen because of their use in agglutination assays (Supplementary
Table 1 online)16,17,19. For our arrays, each modified glass slide was
printed with 14 isolated subarrays, containing a minimum of five
spots per lectin, with a SpotBot Arrayer (95 mm spot size, Telechem).
Lectin activity for each print was verified by hybridization of Cy5-
labeled glycoprotein standards to subarrays on each slide. This allowed
us to compensate for any variations in printing or lectin activity
between slide batches. To test the feasibility of studying bacterial
glycosylation through a lectin microarray approach, we examined the

In Gram-negative bacteria, the majority of carbohydrates are found in a Labeled

the lipopolysaccharide (LPS), although an increasing number of bacteria
glycoproteins are being discovered1,5. LPS consists of a core lipid– Carbohydrates
on bacterial
anchored saccharide attached to a repeating sugar unit referred to as surface
the O-antigen5. There are at least 167 distinct O-antigens found in
E. coli alone. Many of the genes involved in LPS modifications are Lectin
phase variable, that is, their expression turns on and off in response to
Lectin microarray Array slide
various stimuli6. In addition, some Gram-negative bacteria can
become enveloped by capsular polysaccharides, further altering their b
carbohydrate composition7,8. The heterogeneity of these bacterial
surface carbohydrates presents a major challenge for analysis. Bacteria 1
Current methods for bacterial glycan analysis include mass spectro-
Incubate with Observed
metry, NMR, HPLC-based separations, thin-layer chromatography, Fluorescently dye array binding pattern
and chemical analysis9–12. Although these techniques reveal molecular (SYTO 85)
detail, they require a significant investment of time. Recently there has
been increased interest in using lectin-based strategies for structural
analysis of glycans4,13–15. Lectins are carbohydrate-binding proteins Bacteria 2 Fluorescently
labeled bacteria
that have been used as a profiling tool to identify and differentiate
bacterial strains, typically through agglutination assays16,17. These Figure 1 Lectin array. (a) Bacteria bind to the lectin microarray through the
assays are disadvantaged by a subjective visual readout and lack interaction of bacterial surface carbohydrates with the lectin. (b) Fluorescent
of sensitivity. Recent work updated this method, substituting an labeling of bacterial strains and hybridization to the array give distinct
electrochemical readout for the visual. Although more quantitative, binding patterns.

1Department of Chemistry and Biochemistry, and 2Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Institute for Cellular and

Molecular Biology, University of Texas at Austin, 1 University Station, A5300, Austin, Texas 78712-0265. Correspondence should be addressed to L.K.M.
(lmahal@cm.utexas.edu).
Received 7 December 2005; accepted 13 January 2006; published online 5 February 2006; doi:10.1038/nchembio767

NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 3 MARCH 2006 153

 LETTERS

reproducible differences in their lectin finger-

a 1,100
JM101
HB101 b AAA
AIA
prints. Although both strains showed strong
LT2 BPA
1,000 Con A
Uptake (fluorescence (arbitrary

900
RS218
binding to lectins GS II, HPA and WGA, only
DBA
DSA
800 JM101 gave moderate binding to MAA and
ECA
GNA
weak but reproducible interactions with BPA,
GS I
units)/OD600))

700 GS II
HPA
600 DBA and ECA (see Methods for complete
LcH
500 Lotus A
names of lectin sources). The two could not
MAA
400 PAA
300
be distinguished by the traditional hemagglu-
PNA
SBA
200 tination assay, wherein only the strong signals
SNA
STA
100 of GS II, HPA and WGA were obtained for
UEA I
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology

WGA
0 both strains (Supplementary Table 3 online).
BSA
0 10 20 30 40 50 60 JM101 HB101 RS218 LT2
SYTO 85 (µM) The MAA binding is interesting, as a-2,3-
sialic acid residues have been shown to exist
Figure 2 Glycopatterns of pathogenic and nonpathogenic bacterial strains. (a) Uptake of SYTO 85 dye as a terminal epitope on the O-antigens of
by bacterial cell lines. Dye uptake is defined as the absolute fluorescence of the cells at excitation of some pathogenic E. coli variants, yet the K12
545 nm and emission of 590 nm as a function of OD600. (b) Bacterial glycopatterns of Escherichia coli
strains are believed to have lost all O-antigen
K12 strains HB101 and JM101, neonatal meningitis E. coli RS218 and Salmonella typhimurium LT2. 21,22. Binding
Data shown are from two different slides, one for nonpathogenic HB101 and JM101 strains and one for synthesis because of mutations
the pathogens RS218 and LT2, and are representative of a minimum of three replicates for each strain. indicative of both a-2,3-(MAA) and a-2,6-
Approximately 108 cells in the exponential growth phase were hybridized per subarray, and BSA was (SNA)-sialic acid residues was observed in the
included as a negative control. two pathogenic bacteria examined, E. coli
RS218, a neonatal meningitis pathogen, and
the enteropathogen Salmonella typhimurium
binding of whole, fluorescently labeled bacteria to our microarray LT2. In addition, the two bacteria showed a-1,2-fucose binding as
(Fig. 1b). We used the nucleic acid–staining dye SYTO 85 (Molecular indicated by UEA I. We did not observe any binding by the fucose-
Probes) to fluorescently label the bacteria20. One concern with this specific AAA lectin, although the hybridization of glycoprotein stan-
type of staining is that differences in bacterial uptake of the dye could dards to the array confirmed activity (data not shown). The binding
skew our results. Therefore, we determined the fluorescence (545 nm specificities of AAA and UEA I are reported to be very similar, but they
excitation, 590 nm emission) standardized to the optical density at do show some differences in their recognition of a-1,2-fucose
600 nm (OD600), defined here as uptake, as a function of dye motifs23,24. The selectivity of UEA I for a-1,2-fucose has recently
concentration for the bacterial cell lines used in this study (Fig. 2a). been reconfirmed by carbohydrate microarray studies from the Con-
No significant differences in uptake were observed (one-way ANOVA, sortium for Functional Glycomics25. Both sialic acid and fucose have
P ¼ 0.94). In addition, the uptake at the concentration of dye used in been implicated in immune evasion by pathogens26,27. RS218 also
our assays (20 mM) was not significantly different between strains showed strong binding to BPA, DBA, ECA and GS I, whereas LT2
across multiple experiments (one-way ANOVA, P ¼ 0.24). showed mild interactions with these same lectins. To validate the
This contrasts sharply with the defined differences in both binding specificity of these interactions, we inhibited bacterial binding to select
patterns and levels observed for the individual bacterial strains lectins on the arrays using the disaccharide lactose, a known inhibitor.
(Fig. 2b, Supplementary Table 2 online). Notably, the closely related Representative data for JM101, HB101, RS218 and LT2 are given
K12-derived E. coli strains JM101 and HB101 showed consistent and (Fig. 3). Lactose (galactose-b-1,4-glucose) notably inhibited the lectins

Figure 3 Carbohydrate inhibition. (a) Lactose a Lactose

competes for lectin-binding sites. The array is Carbohydrates
preincubated with lactose before bacterial on bacterial
surface
hybridization. (b) Lactose inhibition data for
nonpathogenic bacterial strains JM101 and Altered binding
HB101. The effect of lactose inhibition is shown Hybridization pattern
Inhibited
as a percentage of control signal (the signal from lectin
the lactose-incubated array divided by the signal Lectin binding
from a PBS-treated control array
100) for each Array slide
Lectin array
positive lectin. Data shown are from the same
slide with five replicate spots per sample. The b 100 c 100
RS218 LT2
data are consistent with data obtained from JM101 HB101
Percentage of control signal

multiple repeat experiments. Grubbs’ test (http://

Percentage of control signal

graphpad.com) was used to determine outliers in 75 75

data sets (n ¼ 5, alpha ¼ 0.05). The error was
calculated by propagation of error based on the
50 50
s.d. (see Methods for details). (c) Lactose
inhibition data for pathogenic bacteria RS218
and LT2. Data shown are from two different 25 25
slides with five replicate spots per sample.
The data are consistent with data obtained
from multiple repeat experiments. The error 0 0
A
BA

M I
AA

A
BA

M I
AA

A
A

A
PA

AA

PA
II
II

BP

BP
S

S
EC

SN

EC

SN
G

was calculated by propagation of error based

S
S

G
H

D
M
W

W
G
G

on the s.d. (see Methods for details).

154 VOLUME 2 NUMBER 3 MARCH 2006 NATURE CHEMICAL BIOLOGY

 LETTERS

shown. We observed lower inhibition of the RS218 lectin signals using
the same level of lactose (200 mM), although at least 25% inhibition
was observed. This may be due to the higher affinity of RS218 to
the lectins, caused by greater expression of the appropriate carbo-
hydrate epitopes, as reflected in the stronger binding signals. One note
about these data: the error for lactose inhibition of SNA for LT2 as
calculated by propagation of error (see Methods) is very high because
of one anomalous point in the positive control data, which could not
be discarded using the Grubbs’ outlier test (data not shown). This
error is not representative of the data observed in other replicates of
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
lectin binding levels was observed for all positive lectins (BPA, DBA,
ECA, GNA, GS I, MAA, PAA, SBA, SNA and UEA I, Fig. 4). A
graphical depiction of the data for three representative lectins is given
(Fig. 4b–d). This decrease was reproducible and not caused by a
change in dye uptake (Supplementary Fig. 2 online). Preliminary
experiments using a two-color system in which growth-dependent
bacterial samples were competitively hybridized against bacteria from
a single time point also support this trend (Supplementary Figs. 3
and 4 online). In addition, the general decrease in glycosylation
observed does not appear to be medium dependent, as cells grown

this experiment.
The ability of this technology to rapidly evaluate glycosyla-
tion enables us to easily monitor dynamic changes in bacterial sur-
face glycans. Alterations in bacterial carbohydrates are known to
occur in response to environmental stimuli, according to genetic
evidence. These changes have a role in the pathogenesis of many
bacteria, including E. coli and Salmonella strains2. Growth-dependent
variation in the invasiveness of E. coli RS218 led us to examine the
possibility that the glycosylation of RS218 also changes with growth28.
Therefore, we monitored the glycosylation of RS218 as a function
of growth (Fig. 4). Cells at the early time points (OD600 o 0.4)
appeared to be in lag phase, cells with OD600 between B0.4 and
2.0 were considered in exponential growth phase and those with an
OD600 of 2.0 or higher approached stationary phase (Supplementary
Fig. 1 online). We were unable to grow RS218 beyond an OD600 of
B2.5 in the Rich Defined medium used29. A progressive decrease in
in Luria broth (LB) showed similar results (Supplementary Fig. 5
online). Given that microbial surface carbohydrates are known to
influence bacterial adhesion and invasion, our results suggest a
possible role for glycosylation in growth-dependent invasion by
RS218 (ref. 2).
Several limitations to our lectin microarray technology deserve
mention. First, only accessible carbohydrate motifs, rather than the
entire glycome, are visualized using whole bacteria on the arrays.
However, because the glycans relevant to immune recognition, inter-
microbial interactions and other biological phenomena are typically
accessible surface epitopes, the most biologically relevant portion of
the glycome is surveyed. A second limitation is the lack of availability
of lectins or other binding proteins that recognize sugars unique to
bacteria. One advantage of our technology is that as the repertoire of
carbohydrate-binding proteins that recognize bacterial epitopes
increases, the detection capabilities of our array can be expanded by

16,000
a AAA
b
AIA BPA
14,000
Fluorescence (532 nm, arbitrary units)

BPA
Con A
DBA 12,000
DSA
ECA
GNA 10,000
GS I
GS II
HPA 8,000
LcH
Lotus A
6,000
MAA
PAA
PNA 4,000
SBA
SNA
STA 2,000
UEA I
WGA 0
0.44 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50
OD600 0.64 1.0 1.7 2.2 2.4
Optical density (600 nm)

c 8,000
d 14,000

GS I MAA
Fluorescence (532 nm, arbitrary units)
Fluorescence (532 nm, arbitrary units)

12,000
7,000

6,000 10,000

5,000 8,000

4,000
6,000
3,000
4,000
2,000

1,000 2,000

0 0
0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50
Optical density (600 nm) Optical density (600 nm)

Figure 4 Growth-dependent variation of neonatal meningitis E. coli strain RS218 glycosylation. (a) RS218 was grown to different densities. Equal amounts
of cells (B108) were fluorescently stained and hybridized to the 21-lectin array. Representative data from the same array are shown. (b–d) Graphical
presentation of the mean fluorescence intensity as a function of OD600 for three representative lectins (BPA, GS I and MAA, highlighted in red) is shown.
Error bars represent the s.d. of the mean.

NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 3 MARCH 2006 155

 LETTERS
their inclusion. The present set of lectins binds to epitopes commonly
found on mammalian cells and on bacterial pathogens that mimic the
mammalian glycome26,30,31. The specificities of these lectins are
currently being more carefully defined through the use of carbohy-
drate arrays, improving the structural resolution of glycan analysis
obtained from our lectin microarrays25,32.
Glycosylation is involved in many of the seminal interactions
that define bacteria as pathogens and symbiotes2,6. By taking advan-
tage of lectin microarray technology developed in our laboratory,
we were able to fingerprint a series of bacterial strains based on their
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
carbohydrates. Pathogenic bacterial strains had distinct binding
signals and different patterns from those of the nonpathogenic strains
on our arrays, although they were similar to one another. This implies
that pathogenic strains may have a characteristic glycopattern that
could be used to identify ‘good’ from ‘bad’ bacteria. Most of the
knowledge we have about bacterial glycans comes from time-consum-
ing studies of isolated LPS, which present a static picture of bacterial
glycosylation and do not represent the dynamic alterations suggested
by the genetic data. The technology presented here provides a power-
ful method to quickly evaluate dynamic changes in surface glycosyla-
tion of bacteria in response to environmental stimuli. This is
demonstrated by our ability to observe growth-related alterations in
the surface glycosylation of the neonatal meningitis strain of E. coli.
Rapid evaluation of microbial carbohydrates enables us to study how
bacteria actively modulate their glycans to establish pathogenic, or
conversely, symbiotic relationships in living systems, a subject of
future work.
METHODS
Bacterial cultures. We grew Escherichia coli strains JM101, HB101, neonatal
meningitis RS218 and Salmonella typhimurium LT2 in EZ Rich Defined
medium (Teknova Inc.)29 at 37 1C with shaking (250 r.p.m.) to an OD600 of
B1.0 (B108 cells per ml). We initially recovered RS218 strain in LB medium
before dilution to an OD600 of 0.1 in the EZ Rich Defined medium for growth.
Fluorescent staining. We centrifuged 1 ml of culture for each bacterial strain
at 5,000g for 10 min, and resuspended it in an equal volume of phosphate
buffered saline (PBS, 100 mM sodium phosphate, 150 mM NaCl, pH 7.2).
This wash step was repeated twice. We then stained the cells with SYTO 85
nucleic acid dye (Invitrogen). The dye was added to a 500-ml suspension
of bacteria to a final concentration of 20 mM and shaken at 37 1C for 1 h
(250 r.p.m.). After incubation, we applied the cells directly to the array, as free
dye does not fluoresce20.
Dye uptake. We grew bacterial cultures in EZ Rich Defined medium at 37 1C
with shaking to OD600 of B1.0 (108 cells per ml, 250 r.p.m.). We stained cells
with SYTO 85 as previously described, with the exception that the amount of
dye was varied such that final concentration of dye ranged from 0 to 50 mM. We
measured the fluorescence (545 nm excitation per 590 nm emission) and
OD600 for 100-ml samples in a 96-well Fluotrac plate (USA Scientific) using the
Synergy HT microplate reader (BIO-TEK). Uptake was defined as the fluores-
cence signal (arbitrary units) per OD600.
Lectin arrays. Lectins from the species Anguilla anguilla (AAA), Artocarpus
integrifolia (AIA), Bauhinia purpurea (BPA), Canavalia ensiformis (Con A),
Dolichos biflorus (DBA), Datura stramonium (DSA), Erythrina crista-galli
(ECA), Galanthus nivalis (GNA), Griffonia simplicifolia I (GS I), Griffonia
simplicifolia II (GS II), Helix pomatia (HPA), Lens culinaris (LcH), Lotus
tetragonolobus (Lotus A), Maackia amurensis (MAA), Perseau americana
(PAA), Arachis hypogaea (PNA), Glycine max (SBA), Sambucus nigra (SNA),
Solanus tuberosum (STA), Ulex europaeus I (UEA I) and Triticum vulgare
(WGA) were purchased from EY Laboratories, Inc. We dissolved the lectins
in PBS to a concentration of 500 mg ml–1, except for AAA, ECA and LcH,
which were diluted to 1 mg ml–1. We added bovine serum albumin (BSA,
500 mg ml–1) and the appropriate monosaccharide (final concentration 1 mM,
156
see Supplementary Table 1) to the print buffer (PBS) to improve spot
morphology. We arrayed the lectins on Nexterion H slides (SCHOTT North
America) using a SpotBot personal MicroArrayer with SMP3 pins (TeleChem
International, Inc.) at B50–60% humidity and 25–27 1C. A minimum of
five spots per lectin was printed in each of the 14 subarrays per slide. We used a
16-well FAST frame format (Schleicher & Schuell) with only 14 printed
subarrays due to the barcode. After printing, slides were allowed to incubate
at room temperature for 1 h to ensure maximum coupling efficiency to the
activated surface. We then submerged the slides in blocking solution (50 mM
ethanolamine, pH 8.0) for 1 h, washed three times in PBST (PBS + 0.05%
Tween 20 by volume), once with PBS and then dried them using a slide spinner
(Labnet International).
Lectin array hybridization and lactose inhibition. We used a 16-pad FAST
frame hybridization chamber (Schleicher & Schuell) to segregate the subarrays
for incubation. For inhibition assays, we preincubated each subarray with 50 ml
of 200 mM lactose dissolved in PBS for 1 h at room temperature with gentle
shaking before hybridization of the bacterial cells. We then added 50 ml of the
appropriate cell sample (B 108 cells) to each subarray and incubated for 1 h at
room temperature with gentle agitation. After incubation, we added 100 ml
PBST to individual subarrays and gently rocked the slides for 3 min. The PBST
wash was repeated three times. We then removed the hybridization chamber
and immersed the slides in PBS for 10 min with gentle shaking. We dried the
slides by centrifugation and scanned using a Genepix 4100A slide scanner
(Molecular Devices).
Phase variation. We first recovered E. coli RS218 in LB medium and then
diluted to an OD600 of 0.1 in EZ Rich Defined medium at 37 1C with shaking
(250 r.p.m.). Appropriate volumes of cells were taken at each time point so that
all aliquots had B108 cells per ml. We centrifuged culture aliquots for each
time point at 5,000g for 10 min and stained as previously described before
hybridization on the array.
Agglutination assay. We mixed bacterial cultures (40 ml, B108 cells per ml)
with 10 ml of lectin solution (500 mg ml–1 in PBS) or PBS alone (negative
control) in a Corning 96-well microtiter plate (round-bottom wells) and
allowed cells to settle overnight undisturbed at 20 1C. Results were read by
visual inspection: a positive reaction was determined by a carpet of aggregated
cells on the bottom of wells, and a negative reaction was indicated by a dot of
cellular material at the bottom of a well. Negative results were confirmed by
tilting wells at an angle 4451 and observing the movement of cellular material.
Data analysis. We used GenePix Pro 5.1 software for the fluorescence analysis
and extraction of data (Molecular Devices). Microsoft Excel and GraphPad
Prism 4 software were used for statistical analysis and the generation of graphs
and tables. We calculated the error for inhibition data using propagation of
error with the standard deviations by the following formula: Error ¼ (% of
control) [((s.d. inhibited value)/(mean inhibited value))2 + ((s.d. control)/
(average control))2]1/2.
Note: Supplementary information is available on the Nature Chemical Biology website.
ACKNOWLEDGMENTS
The authors thank D.E. Graham, Department of Chemistry and Biochemistry,
University of Texas at Austin, for JM101; S.M. Payne, Department of Molecular
Genetics and Microbiology, University of Texas at Austin, for LT2 and HB101;
R. Silver and L. Wright, University of Rochester Medical Center, for RS218;
I.J. Molineux, Department of Molecular Genetics and Microbiology,
University of Texas at Austin; and the Beckman Young Investigator
Award for financial support.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing interests.
Published online at http://www.nature.com/naturechemicalbiology/
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reprintsandpermissions/
1. Schmidt, M.A., Riley, L.W. & Benz, I. Sweet new world: glycoproteins in bacterial
pathogens. Trends Microbiol. 11, 554–561 (2003).
VOLUME 2 NUMBER 3 MARCH 2006 NATURE CHEMICAL BIOLOGY

2. Lerouge, I. & Vanderleyden, J. O-antigen structural variation: mechanisms and possible
roles in animal/plant-microbe interactions. FEMS Microbiol. Rev. 26, 17–47 (2002).
3. Weintraub, A. Immunology of bacterial polysaccharide antigens. Carbohydr. Res. 338,
2539–2547 (2003).
4. Pilobello, K.T., Krishnamoorthy, L., Slawek, D. & Mahal, L.K. Development of a lectin
microarray for the rapid analysis of protein glycopatterns. ChemBioChem 6, 985–989
(2005).
5. Caroff, M. & Karibian, D. Structure of bacterial lipopolysaccharides. Carbohydr. Res.
338, 2431–2447 (2003).
6. van der Woude, M.W. & Baumler, A.J. Phase and antigenic variation in bacteria. Clin.
Microbiol. Rev. 17, 581–611 (2004).
7. Whitfield, C. & Roberts, I.S. Structure, assembly and regulation of expression of
capsules in Escherichia coli. Mol. Microbiol. 31, 1307–1319 (1999).
© 2006 Nature Publishing Group http://www.nature.com/naturechemicalbiology
8. Roberts, I.S. The biochemistry and genetics of capsular polysaccharide production in
bacteria. Annu. Rev. Microbiol. 50, 285–315 (1996).
9. Harvey, D.J. Matrix-assisted laser desorption/ionization mass spectrometry of carbohy-
drates. Mass Spectrom. Rev. 18, 349–450 (1999).
10. Szymanski, C.M. et al. Detection of conserved N-linked glycans and phase-variable
lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and
high resolution magic angle spinning NMR spectroscopy. J. Biol. Chem. 278, 24509–
24520 (2003).
11. Sletmoen, M., Maurstad, G., Sikorski, P., Paulsen, B.S. & Stokke, B.T. Characterisation
of bacterial polysaccharides: steps towards single-molecular studies. Carbohydr. Res.
338, 2459–2475 (2003).
12. Buttke, T.M. & Ingram, L.O. Comparison of lipopolysaccharides from Agmenellum
quadruplicatum to Escherichia coli and Salmonella typhimurium by using thin-layer
chromatography. J. Bacteriol. 124, 1566–1573 (1975).
13. Angeloni, S. et al. Glycoprofiling with micro-arrays of glycoconjugates and lectins.
Glycobiology 15, 31–41 (2005).
14. Zheng, T., Peelen, D. & Smith, L.M. Lectin arrays for profiling cell surface carbohydrate
expression. J. Am. Chem. Soc. 127, 9982–9983 (2005).
15. Kuno, A. et al. Evanescent-field fluorescence-assisted lectin microarray: a new strategy
for glycan profiling. Nat. Methods 2, 851–856 (2005).
16. Aabenhus, R., Hynes, S.O., Permin, H., Moran, A.P. & Andersen, L.P. Lectin typing of
Campylobacter concisus. J. Clin. Microbiol. 40, 715–717 (2002).
17. Hynes, S.O., Hirmo, S., Wadstrom, T. & Moran, A.P. Differentiation of Helicobacter
pylori isolates based on lectin binding of cell extracts in an agglutination assay. J. Clin.
Microbiol. 37, 1994–1998 (1999).
18. Ertl, P. & Mikkelsen, S.R. Electrochemical biosensor array for the identification of
microorganisms based on lectin-lipopolysaccharide recognition. Anal. Chem. 73,
4241–4248 (2001).
NATURE CHEMICAL BIOLOGY VOLUME 2 NUMBER 3 MARCH 2006
LETTERS
19. Annuk, H., Hynes, S.O., Hirmo, S., Mikelsaar, M. & Wadstrom, T. Characterisation and
differentiation of lactobacilli by lectin typing. J. Med. Microbiol. 50, 1069–1074
(2001).
20. Disney, M.D. & Seeberger, P.H. The use of carbohydrate microarrays to study
carbohydrate-cell interactions and to detect pathogens. Chem. Biol. 11, 1701–
1707 (2004).
21. Gamian, A., Kenne, L., Mieszala, M., Ulrich, J. & Defaye, J. Structure of the
Escherichia coli O24 and O56 O-specific sialic-acid-containing polysaccharides and
linkage of these structures to the core region in lipopolysaccharides. Eur. J. Biochem.
225, 1211–1220 (1994).
22. Yao, Z. & Valvano, M.A. Genetic analysis of the O-specific lipopolysaccharide biosynth-
esis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer
group 6 specificity to Shigella flexneri serotypes Y and 4a. J. Bacteriol. 176, 4133–
4143 (1994).
23. Wu, A.M., Wu, J.H., Singh, T., Liu, J.H. & Herp, A. Lectinochemical studies on the affinity
of Anguilla anguilla agglutinin for mammalian glycotopes. Life Sci. 75, 1085–1103
(2004).
24. Baldus, S.E. et al. Characterization of the binding specificity of Anguilla anguilla
agglutinin (AAA) in comparison to Ulex europaeus agglutinin I (UEA-I). Glycoconj. J.
13, 585–590 (1996).
25. Alvarez, R.A., Lee, A., Davis, C., Hoffmann, J. & Blixt, O. Defining the binding
specificity of commercially available plant lectins using a printed glycan array.
Glycobiology 15, 1207 (2005).
26. Coyne, M.J., Reinap, B., Lee, M.M. & Comstock, L.E. Human symbionts use a host-like
pathway for surface fucosylation. Science 307, 1778–1781 (2005).
27. Vimr, E.R., Kalivoda, K.A., Deszo, E.L. & Steenbergen, S.M. Diversity of microbial
sialic acid metabolism. Microbiol. Mol. Biol. Rev. 68, 132–153 (2004).
28. Badger, J.L. & Kim, K.S. Environmental growth conditions influence the ability of
Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum
resistance. Infect. Immun. 66, 5692–5697 (1998).
29. Neidhardt, F.C., Bloch, P.L. & Smith, D.F. Culture medium for enterobacteria.
J. Bacteriol. 119, 736–747 (1974).
30. Karlsson, K.A. The human gastric colonizer Helicobacter pylori: a challenge for host-
parasite glycobiology. Glycobiology 10, 761–771 (2000).
31. Friis, L.M., Pin, C., Pearson, B.M. & Wells, J.M. In vitro cell culture methods for
investigating Campylobacter invasion mechanisms. J. Microbiol. Methods 61, 145–
160 (2005).
32. Manimala, J.C., Li, Z., Jain, A., VedBrat, S. & Gildersleeve, J.C. Carbohydrate
array analysis of anti-Tn antibodies and lectins reveals unexpected specificities:
implications for diagnostic and vaccine development. ChemBioChem 6, 2229–2241
(2005).
157