Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O42

Article abstract of DOI:10.1016/j.carres.2014.05.003

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O42 has been investigated by NMR spectroscopy as the main method, which was complemented with sugar analysis, mass spectrometry, and analysis of biosynthetic information. The O-specific chain of the O-deacylated lipopolysaccharide (LPS-OH) consists of branched tetrasaccharide-glycerol repeating units joined by phosphodiester linkages. The lipid-free polysaccharide contains 0.8 equiv of O-acetyl groups per repeating unit and has the following teichoic acid-like structure: Based on biosynthetic aspects, this should also be the biological repeating unit. This O-antigen structure is remarkably similar to that of E. coli O28ac, differing only in the presence or absence, respectively, of a glucose residue at the branching point. The structural similarity explains the serological cross-reactivity observed between strains of these two serogroups, and also their almost identical O-antigen gene cluster sequences. → 2)-(R)-Gro-(1-P-4)-β-D-GlcpNAc-(1→3)-β-D-Galf2Ac-(1→3)-α-D-GlcpNAc-(1→α-D-Glcp-(1→3).

Full text of DOI:10.1016/j.carres.2014.05.003

Carbohydrate Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Structural studies and biosynthetic aspects of the O-antigen
polysaccharide from Escherichia coli O42
a
b
a,
⇑
Carolina Fontana , Andrej Weintraub , Göran Widmalm
a
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Karolinska Institute, Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University Hospital, S-141 86 Stockholm, Sweden
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure of the O-antigen polysaccharide (PS) from Escherichia coli O42 has been investigated by
NMR spectroscopy as the main method, which was complemented with sugar analysis, mass spectrom-
etry, and analysis of biosynthetic information. The O-specific chain of the O-deacylated lipopolysaccha-
ride (LPS-OH) consists of branched tetrasaccharide–glycerol repeating units joined by phosphodiester
linkages. The lipid-free polysaccharide contains 0.8 equiv of O-acetyl groups per repeating unit and has
the following teichoic acid-like structure: Based on biosynthetic aspects, this should also be the biological
repeating unit. This O-antigen structure is remarkably similar to that of E. coli O28ac, differing only in the
presence or absence, respectively, of a glucose residue at the branching point. The structural similarity
explains the serological cross-reactivity observed between strains of these two serogroups, and also their
almost identical O-antigen gene cluster sequences.
Received 7 April 2014
Received in revised form 6 May 2014
Accepted 10 May 2014
Available online xxxx
Keywords:
Escherichia coli
NMR
O-Acetylation
O-Antigen
→
2)-(R)-Gro-(1–P–4)-β-D-GlcpNAc-(1→3)-β-D-Galf2Ac-(1→3)-α-D-GlcpNAc-(1→
|
α-D-Glcp-(1→3)
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
Within gram-negative bacteria, the Enterobacteriaceae family
serogroups is of particular importance in epidemiological studies,
and three principal surface antigens have been employed to
differentiate them: (a) the lipopolysaccharide (LPS) O antigen,
(b) the capsular K antigen, and (c) the flagellar H antigen, and
the O:K:H combination is then referred as a serotype.3
comprises some of the most common pathogens usually found in
clinical specimens, such as those of the genera Escherichia, Klebsi-
ella, Salmonella, and Shigella. The Escherichia coli strains are rarely
harmful and they usually coexist with their host sharing mutual
benefits; however, some pathogenic strains have been implicated
in a wide range of intestinal diseases, urinary tract infections, sep-
sis, and meningitis. In particular, the diarrheagenic E. coli strains
that usually affect humans can be classified into six different path-
otypes: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli
The O-antigen polysaccharide (PS) is the outermost part of the
LPS and it usually consists of oligosaccharide repeating units. Since
diverse monosaccharides can be found in different arrangements
and linkages within and between repeating units, this is also con-
sidered the most variable portion of the LPS. The E. coli serogroups
are presently numbered from O1 to O181 according to their sero-
logical properties but, since seven of them have been removed,
1
4
,5
(
EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative
only 174 are currently in use. Moreover, slightly different O-anti-
gen structures can be identified within the same serogroup giving
E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent
E. coli (DAEC). The designation of E. coli strains in different
2
6,7
rise to different subgroups. The ECODAB (E. coli O-antigen data-
base) contains information about the structure, NMR chemical
shifts, and cross-reactivity of many E. coli O-antigens4 as well
as information about glycosyltransferases (GTs) involved in the
,8
⇑
Corresponding author. Tel.: +46 816 3742.
E-mail address: gw@organ.su.se (G. Widmalm).
9
biosynthesis of O-antigens.
http://dx.doi.org/10.1016/j.carres.2014.05.003
008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Fontana, C.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.003

2
C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
The genes responsible for the biosynthesis of E. coli O-antigens
medium. The LPS was delipidated under mild acid conditions to
yield the lipid-free PS, which was purified by size exclusion
are usually found in a single cluster in the bacterial chromosome
between the galF and gnd housekeeping genes10 with some known
exceptions being E. coli O8, O9, O55, O59, and O155 (in which some
1
13
chromatography. The H and C NMR spectra of this material
suggested the presence of an O-acetyl group (resonances indicated
with black filled triangles in Fig. 1a and c, respectively).
Subsequently, the O-deacylated LPS (LPS-OH) was obtained upon
treatment of the LPS under alkaline conditions, and purified by size
exclusion chromatography. The aforementioned resonances from
of the genes involved in the biosynthesis of the O-antigen can also
be found between the gnd gene and his operon).1
1–14
The genes
found in the O-antigen gene cluster can be classified as follows:
i) genes involved in the biosynthesis of sugar precursors (and
(
1
13
additional non-sugar substituents when they are present), (ii)
genes involved in the O-unit formation (e.g., GTs, glycero-
phosphotransferases and O-acetyltransferases) and (iii) O-antigen
processing genes (e.g., genes encoding for the Wzx-flippase and
Wzy-polymerase). The first group of genes is highly conserved
among species, and the major structural differences observed in
the O-antigens are attributed to variations in genes encoding
for glycosyltransferases and the polymerase. Furthermore, genes
encoding for proteins that modify the O-specific chain after
polymerization (such as O-acetyl- or glucosyltransferases) may be
the O-acetyl group were absent in the H and C NMR spectra of
this material (Fig. 1b and d, respectively). The sugar analysis of
the LPS-OH revealed glucose, galactose, and 2-amino-2-deoxyglu-
cose in a relative ratio 1.2:1.0:2.5, respectively. The determination
of the absolute configuration of the monosaccharide components
utilized authentic standards and showed that the aforementioned
residues all have the D-configuration.
1
The H NMR spectrum of the LPS-OH (Fig. 1b) revealed four res-
onances in the anomeric region that were denoted by A–D in order
1
of decreasing H chemical shifts (5.329, 5.076, 5.043, and 4.701,
15–18
found outside the O-antigen gene cluster.
This is the case for
H
respectively). Resonances were present, inter alia, at d 2.103
E. coli O17, O44, O73, O77, and O106, which share the same
O-antigen backbone (encoded by genes found in their almost
identical O-antigen gene clusters), but they differ in the lateral
glucosylation pattern (presumably caused by GTs genes located
outside the respective O-antigen gene clusters).19 In the case of
E. coli O44, the gene that encodes for the GT responsible for the lat-
and 2.077 (corresponding to 3H each), indicating that the two
2-amino-2-deoxyglucoses detected in sugar analysis are N-acety-
lated. This is also consistent with the resonances in the 13C NMR
C 3 3
spectrum found at d 22.98 (CH ), 23.46 (CH ), 175.05 (CO), and
13
175.15 (CO). Furthermore, the splitting of the C resonances at
79.67, 78.12, 75.80, 74.92, and 65.53 ppm indicated the presence
1
9
31
eral glucosylation of the PS was found within a putative prophage.
of phosphorous. The P NMR spectrum of the LPS-OH contained
Likewise, E. coli O13, O129, and O135 exhibit a common O-antigen
backbone (formed by the action of enzymes encoded in their almost
identical O-antigen gene cluster) that in the different serogroups is
decorated with distinctive O-acetylation and glucosylation patterns
a
b
(
created by enzymes that are not encoded in their O-antigen gene
2
0
clusters).
E. coli O42 strains have been reported to cause diarrhea in both
humans and animals, and they have been classified either as EIEC or
2
1–24
ETEC, respectively.
Strains belonging to this serogroup were
reported to give positive results in PCR assays and oligonucleo-
tide-based DNA microarrays targeting the wzx and wzy genes of
E. coli O28ac (and vice versa), and serological cross-reactivity was
also observed between strains of these two serogroups.2
5,26
The
partial O-antigen gene cluster of E. coli O42 sequenced by Fratamico
2
5
et al. consisted of: (i) the wzx and wzy O-antigen processing genes
encoding for the flippase and polymerase, respectively), (ii) genes
(
encoding for three GTs (wbeS, wbeX and wbeY) and an acetyltrans-
ferase (wbeZ) and (iii) the partial sequence of a gene encoding for a
UDP-galactopyranose mutase (glf), which is involved in the biosyn-
thesis of a monosaccharide precursor (Galf). This partial O-antigen
gene cluster is almost identical to a region of the E. coli O28ac
O-antigen gene cluster, differing only in the substitution of a single
nucleotide in the case of wbeX gene and two nucleotides in the case
of the wbeY gene. These mutations are responsible for the respec-
tive differences in the amino acid sequences of the proteins (that
is, one and two amino acids are substituted in the WbeX and WbeY
GTs, respectively). Since just a few amino acid substitutions may be
enough to change the specificity of a glycosyltransferase, thereby
altering the structure of the O-antigen,27,28 a comparison of the
structures of the O-antigen PSs of E. coli O42 and O28ac would help
to reveal whether these mutations are responsible for the slightly
different serological responses observed between these serogroups
or not. Herein, we investigate the structure of the repeating unit of
the O-antigen PS of E. coli O42, and identify the structural difference
with respect to that of the E. coli O28ac O-antigen PS.
5.0
4.5
4.0
3.5
1H /ppm
3.0
2.5
2.0
c
d
160
140
120
100
80
60
40
20
13C /ppm
2
. Results
Figure 1. Diffusion filtered 1H NMR and 13C NMR spectra of the lipid-free PS (a and
c, respectively) and O-deacylated LPS (b and d, respectively) from Escherichia coli
O42. Resonances from the O-acetyl groups are indicated with black filled triangles.
The LPS of E. coli O42 was obtained by hot phenol/water extrac-
tion of the bacteria, which were grown in Luria–Bertani (LB)
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C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
3
a single signal at d
group.
P
À0.2 ppm, consistent with a phosphodiester
The 13C NMR spectra of the LPS-OH (Fig. 1d) revealed 31 reso-
nances, in agreement with the number of carbons of the oligosac-
charide material determined by MS. The multiplicity-edited
2
9–31
The LPS-OH was subjected to dephosphorylation with aqueous
8% hydrogen fluoride and, after purification by gel permeation
1
13
4
H, C-HSQC spectrum of the LPS-OH (Fig. 3a) showed in the region
chromatography, the resulting oligosaccharide material was ana-
lyzed by mass spectrometry (MS). The HR-MS spectrum in the
positive mode of the underivatized oligosaccharide showed, inter
alia, a peak at m/z 845.3004 corresponding to a compound of
for the anomeric resonances three cross-peaks corresponding to
hexopyranosyl residues (residues A, C and D) and one cross-peak
corresponding to a hexofuranosyl residue (residue B). In addition,
six hydroxymethyl groups were identified (shown in red color in
the spectrum of Fig. 3a) consistent with the presence of four hex-
oses and one glycerol moiety, as anticipated from the MS analysis.
54 2
molecular formula C31H N NaO23 (calculated value 845.3010)
+
that was attributed to the pseudomolecular ion [M+Na] . This
information, combined with the above sugar analysis of the
LPS-OH, revealed that the oligosaccharide is composed of one
Glc, one Gal, and two GlcNAc residues, as well as one residue of
1
13
The H and C resonances of the different residues were assigned
1
1
using a combination of 2D NMR experiments such as H, H-TOCSY,
1
1
1
1
13
1
13
H, H-NOESY, multiplicity-edited H, C-HSQC, H, C-H2BC, and
13
1
13
3
formula C H
O
8 3
that may be attributed to a glycerol moiety. Infor-
H, C-HMBC; the H and C the chemical shifts for each residue
-linked since
H1,H2 are 3.5–3.8 Hz and JC1,H1 are 171–172 Hz, and residue D is
mation about the monosaccharide sequence in the oligosaccharide
was obtained by MS/MS in both the positive and negative modes
from the precursor pseudomolecular ions m/z 845.3 (Fig. 2a) and
are compiled in Table 1. Residues A and C are a
3
1
J
3
1
35
b-linked since JH1,H2 is 8.5 Hz and JC1,H1 is 161 Hz. The correla-
1
1
8
21.3 (Fig. 2b), respectively, which produced the corresponding
daughter ions via A
-, B-, and C-type cleavages.32 In the case of
the MS/MS spectrum recorded in positive mode, the A -type
tion patterns observed in the H, H-TOCSY spectra indicate that res-
idues A, C, and D have the gluco-configuration, since all the protons
in the spin system can be traced from H1. Moreover, residues C and
D have C2 chemical shifts that correspond to N-acetylhexosamines
1
1
cleavages are only observed at the glycosidic linkages involving
GlcNAc residues, which reflects the higher stability of the HexNAc
3
3
oxonium ions with respect to those originating from hexoses.
Additionally, the fragment m/z 591.2 in the MS/MS spectrum
of Figure 2a could be attributed to a double cleavage process (i.e.,
a B-type cleavage of the fragment m/z 753.3) sometimes observed
for underivatized carbohydrates,32 and the fragments m/z 641.3
and 438.2 in the MS/MS spectrum of Figure 2b could be formed
by loss of one molecule of water from the m/z 659.3 and 456.2 ions,
a
60
E1b E1a
70
3
4
respectively.
D4
E2
C3
D3
80
bcdeB
m/z 683.2
cdeB
deB
[M+Na]+
m/z 845.3
B4
B3
B2
a
m/z 480.2 m/z 318.1
9
0
Hex- -GlcNAc- -Hex- -GlcNAc- -Gro
C1
A1
100
abC
abcC
abcdA1
m/z 753.3
D1
m/z 406.1 m/z 568.2
abA1
m/z 388.1
B1
110
6
83.2
4
2
0
8
45.3
4
80.2
b
60
70
3
88.1
568.2
591.2
4
06.1
318.1
753.3
400
500
600
700
800
m/z
−
bcdeB
m/z 659.2
cdeB
deB
[M−H]
m/z 821.3
80
b
m/z 456.2 m/z 294.1
B2
B3
B4
Hex- -GlcNAc- -Hex- -GlcNAc–Gro
90
abC
m/z 382.1
100
110
6
59.3
B1
1
0
5
0
821.3
800
4
38.2
641.3
600
2
94.1
3
82.1 456.2
5
.0
4.5
4.0
3.5
1H /ppm
300
400
500
m/z
700
Figure 3. Selected region of the multiplicity-edited 1H,13C-HSQC spectrum of O-
deacylated LPS (a) and lipid free PS (b) from Escherichia coli O42, showing the region
for resonances from the anomeric and ring atoms, as well as the hydroxymethyl
groups (in which the cross-peaks from the latter appear in red). Anomeric and
substitution positions are annotated in panel a, whereas cross-peaks with signif-
icant chemical shift changes upon O-deacetylation (B1, B2, B3, and B4) are
annotated in both panels (a and b).
+
Figure 2. The MS/MS spectra of the: (a) pseudomolecular [M+Na] ion m/z 845.3
À
and (b) pseudomolecular [MÀH] ion m/z 821.3 recorded in positive and negative
modes, respectively. The detected ions and the resulting sugar sequence of the
tetrasaccharide–glycerol compound, obtained after cleavage of the phosphodiester
group in the LPS-OH, are shown on the top of each spectrum.
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4
C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
Table 1
1
1
13
1
1
H and C NMR chemical shifts (ppm) at 42 °C of the O-antigen repeating unit from Escherichia coli O42 O-deacylated LPS and inter-residue correlations from H, H-NOESY and
13
H, C-HMBC spectra
Sugar residue
1H/13
C
Correlation to atom (from anomeric atom)
1
2
3
4
5
6
Me
CO
NOE
HMBC
a
-
D
-Glcp (1?
A
B
C
D
E
5.329 [3.8]
3.495
3.693
3.457
3.638
ꢀ3.813
H3, D
Me, D
C3, D
(
1
0.10)
00.00 {171} 72.52
(À0.04) (À0.03) (0.04)
(À0.20)
73.36 69.89
(À0.42) (À0.82) (0.76)
4.205
(0.11)
85.36
73.13
60.99
(À0.85)
3.656, 3.688
H3, D
(
7.01)
(0.05)
?
?
?
?
3)-b-
D
-Galf-(1?
5.076 <3.8>a
4.031
(0.01)
80.76
(À1.60) (8.42)
4.081
(0.20)
53.92
(À1.08) (6.85)
4.181
(0.13)
82.53
3.915
(0.11)
71.29
H3, C
C3, C
H3, C
(
1
(
À0.14)
09.08
7.06)
63.85
(À0.65) (À0.40) (0.20)
3.493
(0.00)
69.19
3)-
a-
D-GlcpNAc-(1?
5.043 [3.5]
À0.17)
7.91{172}
6.14)
3.852
(0.10)
78.59
3.878
(0.02)
73.12
3.811, 3.870 2.103
(0.04)
61.48
(À0.30)
3.830, 3.919 2.077
(0.02)
H2, E
H1b, E
C2, E
H2, E
(
9
22.98 175.05
(0.11) (À0.08)
(
(À2.07) (0.61)
3,4)-b-
D-GlcpNAc-(1?
4.701 [8.5]
3.818
(0.17)
3.969
(0.41)
79.67c
4.222
(0.76)
74.92d
(3.86)
3.588
(0.13)
H3, B
H2, B
C3, B
H3, B
(
1
À0.02)
e
01.28 {161} 55.53
75.80
61.35
23.46 175.15
(0.36) (À0.34)
(
5.43)
(À2.33) (4.86)
3.931
78.12
(À1.02) (À0.50)
2)-(R)-Gro-(1-P–b
4.036, 4.084
ꢀ3.771
f
g
6
5.53
62.13
(
1.73)
(4.82)
(À1.67)
3JH1,H2 and 1
JC1,H1 values are given in Hertz in square brackets and braces, respectively.
37,38
39
Chemical shift differences as compared to corresponding monosaccharides
and glycerol are given in parentheses.
a
Full width at half maximum.
b
d
P
= À0.2 ppm.
c
d
e
f
3
J
P,C3 = 3.7 Hz.
P,C4 = 5.8 Hz.
P,C5 = 2.6 Hz.
P,C1 = 5.1 Hz.
P,C2 = 6.9 Hz.
2
3
2
3
J
J
J
J
g
(
53.92 and 55.53 ppm, respectively) and thus are
b- -GlcpNAc; residue A is then -Glcp and residue B is a
tofuranose. The H and C chemical shifts of the latter are almost
identical to those reported for the b- -Galf residue of the O-deacet-
ylated O-antigen PS of E. coli O28ac, thus indicating that residue B is
a
-
D
-GlcpNAc and
3JP,C3(D) = 3.7 Hz, 2JP,C4(D) = 5.8 Hz, and 3
J
P,C5(D) = 2.6 Hz). Addition-
1
31
D
a
-
D
D
-galac-
ally, the H, P-hetero-TOCSY spectrum with a mixing time of
54 ms (Fig. 4b) allowed tracing of the full proton spin system of
the glycerol residue (H1–H3 in residue E) and the protons H1–H5
in the spin system of residue D.
1
13
D
2
9
1
13
also b-linked. A regular aliased H, C-HMBC spectrum was used
to assign the resonances of the carbonyl groups via two- and three-
bond protonAcarbon couplings. The resonance at 175.05 ppm has a
correlation to the protons at 2.103 (N-acetyl methyl protons) and
The sequence of the sugar residues in the O-antigen repeating
1
13
1
1
unit was determined from H, C-HMBC and H, H-NOESY experi-
ments. Three-bond heteronuclear correlations were observed from
the anomeric carbon of each monosaccharide residue to the
respective proton atoms at the substitution positions (Fig. 4c) as
well as correlations from anomeric protons to the glycosyloxylated
carbons (Table 1). Consequently, the structure of the repeating unit
of the O-deacylated O-antigen PS of E. coli O42 is:
4
.081 ppm (H2 in residue C) whereas the resonance at 175.15 has
a correlation to the protons at 2.077 (N-acetyl methyl protons)
and 3.818 ppm (H2 in residue D), confirming the N-acetyl groups
at the C2 positions in residues C and D, respectively.
The substitution positions for the sugar residues were identified
?2)-Gro-(1-P-4)[
a-D-Glcp-(1?3)]-b-D-GlcpNAc-(1?3)-b-D-
1
3
36
13
from C NMR glycosylation shifts; the C chemical shifts of
-GlcpNAc, b- -GlcpNAc, -Glcp, b- -Galf, and glycerol were used
Residues B and C are both 3-substituted, with
8.42 and 6.85, respectively; thus these res-
-Galf-(1? and ?3)- -GlcpNAc-(1?, respec-
Galf-(1?3)- -GlcpNAc-(1?
a-D
1
1
a-
D
D
7–39
a-D
D
The inter-residue correlations observed in the H, H-NOESY
spectrum (Table 1), and the sequence of monosaccharide residues
determined by MS/MS (Fig. 2), are both consistent with the
proposed structure, which is remarkably similar to that of the
3
as references.
glycosylation shifts
idues are ?3)-b-
Dd
C
D
a-D
2
9
tively. Residue D has glycosylation shifts of 4.86 and 3.86 ppm at
O-deacetylated PS of E. coli O28ac:
C3 and C4, respectively, showing that this residue is disubstituted
?2)-(R)-Gro-(1-P-4)-b-
GlcpNAc-(1?
D-GlcpNAc-(1?3)-b-D-Galf-(1?3)-a-D-
and corresponds to ?3,4)-b-
D
-GlcpNAc-(1?. Residue A does not
show any significant C glycosylation shifts for the C2–C6 atoms
and thus it is a terminal -Glcp (1? group. Residue E (glycerol)
1
3
Comparison of the 13C chemical shifts of the ?3)-
a
-D
-GlcpNAc-
(1?2)-Gro-(1– moiety in both polymers showed a maximum
C(O42–O28ac) deviation of 0.2 ppm for C1 in -GlcpNAc and
a
-D
1
3
is substituted at C2, with a C glycosylation shift of 4.82 ppm. This
D
d
a-D
residue is also 1-O-phosphorylated, since correlations were
À0.2 ppm for C1 in glycerol that may be attributed to long range
perturbations due to the presence/absence of the glucose residue
in the E. coli O42/O28ac O-antigens, respectively. Having consid-
ered this, and the biosynthetic aspects that will be discussed
below, the stereogenic center in the glycerol moiety can be antici-
pated to have the R configuration.
1
31
observed in the H, P-HMBC spectrum (Fig. 4a) from the phospho-
rous atom to the H1 protons in the glycerol moiety (residue E) and
to H4 in residue D. Consequently, residue E is ?2)-Gro-(1-P– and it
is connected to C4 of residue D via a phosphodiester linkage. This
was corroborated by the splitting of the 13C resonances of C1 and
C2 in residue E and C3, C4, and C5 of residue D observed in the
The H and 13C NMR spectra of the lipid-free PS showed reso-
1
1
13
H-decoupled C NMR spectrum due to two- and three-bond
nances corresponding to an O-acetyl group: d
C
21.06 (CH
3
) and
2
3
1
13
phosphorousAcarbon couplings ( JP,C1(E) = 5.1 Hz and JP,C2(E) = 6.9 Hz,
H 3
173.41 (CO), and d 2.147 (CH ). The H and C chemical shift
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C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
5
assignments of each monosaccharide spin system were carried
c
C2
E2
D3
1
1
1
1
out using 2D H, H-TOCSY, H, H-NOESY, multiplicity-edited
C1
9
8
1
13
1
13
H, C-HSQC, and H, C-HMBC experiments, and are compiled in
Table 2. The O-acetylated residue could be readily identified by
A1
D1
100
1
13
comparison of the multiplicity-edited H, C-HSQC of the lipid-free
PS with that of the LPS-OH (Fig. 3b and a, respectively), since only
1
1
1
02
04
06
B3
D2
C3
the chemical shifts of the
D-Galf residue (B) represented significant
chemical shift differences that could be attributed to perturbations
4
0,41
due to O-acetylation.
The exact location of the O-acetyl group
1
13
was corroborated by an H, C-HMBC correlation from the reso-
nance at 173.41 ppm (CO) to the protons at 2.147 (O-acetyl methyl
protons) and 4.861 ppm (H2 in residue B), confirming that the
B2
108
10
B1
galactofuranosyl residue is 2-O-acetylated. Integration of the ano-
1
1
meric resonances of both
D
-Galf and
D
-Galf2Ac in the H NMR spec-
trum of the lipid-free PS showed that the degree of O-acetylation
was 80%. Thus, the structure of the teichoic acid like tetrasaccha-
ride–glycerol repeating unit of the O-antigen PS of E. coli O42 is
as shown in Figure 5.
4.2
4.1
4.0
1H /ppm
3.9
3.8
b
-
1
E2 E3
D3 D2
3. Discussion and conclusions
0
D1
D5
The O-antigen PSs of E. coli O28ac and O42 are remarkably sim-
ilar, sharing the same trisaccharide–glycerol backbone (Fig. 6 top
and bottom, respectively). The only difference between these two
1
a
-
1
polysaccharides is that the b-D-GlcpNAc residue in the latter is
3
-substituted with a glucose residue, which is absent in the
0
1
O-antigen of E. coli O28ac. The functions of the glycosyltransferases
involved in the biosynthesis of these polymers were previously
D4 E1b E1a
25
predicted using the respective O-antigen gene clusters and BLAST
9
similarity searches. The names of the GTs anticipated to catalyze
4.6
4.4
4.2
1H /ppm
4.0
3.8
3.6
the formation of the different linkages are annotated in Figure 6,
in the proximity of the respective glycosidic linkages. Since the
polymerase (Wzy) was proposed to catalyze the formation of the
Figure 4. The 1H,31P-HMBC (a) and 1H,31P-hetero-TOCSY (
of the O-deacylated LPS from Escherichia coli O42 showing correlations from the
phosphorous atom. Selected region of the H, C-HMBC spectrum (c) showing intra-
and inter-residue correlations from anomeric carbons.
s
mix = 54 ms) (b) spectra
a-
D-GlcpNAc-(1?2)-Gro moiety, the biological repeating unit is
1
13
defined by having a 3-substituted -GlcpNAc residue at its
a
-D
Table 2
1
1
13
1
1
H and C NMR chemical shifts (ppm) at 42 °C of the O-antigen repeating unit from Escherichia coli O42 lipid free PS and inter-residue correlations from H, H-NOESY and
13
H, C-HMBC spectra
Sugar residue
1H/13
C
Correlation to atom (from anomeric atom)
1
2
3
4
5
6
Me
CO
NOE
HMBC
a
-
D
-Glcp (1?
A
B
C
D
E
5.320 [4.0]
3.491
3.691
3.456
3.636
ꢀ3.810
H3, D
C3, D
(
1
(
0.09)
00.01
7.02)
(À0.05) (À0.03) (0.04)
(À0.20)
72.52
(0.05)
73.35
69.88
73.13
60.97
(À0.87)
3.660, 3.684 2.147
H3, D
(À0.43) (À0.83) (0.76)
?
?
?
?
3)-b-
D
-Galf2Ac-(1?
5.224 <3.2>a 4.861
4.349
(0.25)
83.40
(6.46)
4.269
(0.22)
83.30
(0.12)
3.933
(0.12)
71.11
H3, C
C3, C
H3, C
(
1
(
0.00)
06.81
4.79)
(0.84)
82.47
(0.11)
63.86
21.06 173.41
(À0.58) (0.21)
3.869 3.816, 3.859 2.060
3)-
a-
D-GlcpNAc-(1?
5.021 [3.5]
À0.19)
7.93
6.16)
4.067
(0.19)
53.98
(À1.02) (6.24)
3.808
(0.16)
55.45
(À2.41) (4.89)
3.870
(0.12)
77.98
3.482
H2, E
H1b, E
(
(À0.01) (0.01)
69.10 73.15
(À2.16) (0.64)
4.209
(0.75)
74.87b
(3.81)
(0.00)
22.97 174.82
(0.10) (À0.31)
9
61.46
(À0.32)
3.808, 3.897 2.081
(0.02)
61.33
H2, E
(
3,4)-b-
D-GlcpNAc-(1?
4.740 [8.6]
0.02)
00.99
5.14)
3.970
(0.41)
79.70
3.550
(0.09)
H3, B
H2, B
C3, B
H3, B
(
c
1
75.80
23.52 175.09
(0.42) (À0.40)
(
(À1.02) (À0.52)
2)-(R)-Gro-(1-P–
4.030, 4.074 3.920
ꢀ3.766
6
5.55d
1.75)
78.17
(4.87)
62.11
(
(À1.69)
3JH1,H2 and 1
JC1,H1 values are given in Hertz in square brackets and braces, respectively.
37,38
39
Chemical shift differences as compared to corresponding monosaccharides
and glycerol are given in parentheses.
a
Full width at half maximum.
b
c
2
3
2
J
J
J
P,C4 = 4.0 Hz.
P,C5 = 3.0 Hz.
P,C1 = 4.8 Hz.
d
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6
C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
been anticipated to perform the 2-O-acetylation of the Galf resi-
2
5
due. This acetyltransferase is identical to that of E. coli O42,
and a -Galf2Ac residue is indeed found in the repeating units of
D
both of the O-antigen structures.
In the case of E. coli O42, the gene that encodes for the GT
involved in the formation of the b-(1?3)-linkage between
D-Glcp
and -GlcpNAc (branching point) was not present in the available
D
2
5
O-antigen gene cluster reported by Fratamico et al. One should
note that whereas the whole O-antigen gene cluster between the
galF and gnd genes has been sequenced for E. coli O28ac (GenBank:
DQ462205, comprising the wegS–wzx–wbeS–wzy–wbeX–wbeY–
wbeZ–glf genes), only a partial O-antigen gene cluster has been
reported for E. coli O42 (GenBank: FJ539194, comprising the region
from wzx and a truncated glf gene, in the same sequence order as in
E. coli O28ac). Consequently, the gene that encodes for the E. coli
O42 glucosyltransferase might be either in the remaining region
of the O-antigen gene cluster that is not available, or located out-
side the O-antigen gene cluster. With the knowledge of the struc-
Figure 5. Structure of the biological repeating unit of the O-antigen LPS from
Escherichia coli O42 in CFG-notation (top) and standard nomenclature (bottom). The
2
-O-acetylation of residue B in the lipid-free PS was ꢀ80%.
reducing end. Furthermore, since the polymerase and the predicted
glycerol phosphotransferase (WbeS)2 of E. coli O28ac are identical
to those of E. coli O42, the configuration of the stereogenic center in
the glycerol residue is expected to be the same in both polysaccha-
rides. The absolute configuration of glycerol moiety in the O-anti-
gen PS of E. coli O28ac has been determined previously29 and, thus,
the same R configuration is expected in the O-antigen PS of E. coli
O42. The very good agreement of the 13C chemical shifts observed
5
2
9
ture of both E. coli O28ac and O42 O-antigens (elucidated herein),
we have now identified another group of E. coli O-antigens sharing
a common backbone structure and almost identical O-antigen gene
2
5
clusters, in addition to the previously identified E. coli serogroups
1
9,20
O17/O44/O73/O77/O106 and O13/O129/O135.
for the carbon resonances of the ?3)-
a
-D
-GlcpNAc-(1?2)-
4. Experimental
Gro-(1– moiety in both polysaccharides supported this hypothesis.
Furthermore, the WbeX and WbeY glycosyltransferases were
predicted to catalyze the formation of the b-(1?3)-linkages
4.1. Bacterial strain, conditions of growth and preparation of
the lipopolysaccharide
between the
D-GlcpNAc/D-Galf and D-Galf/D-GlcpNAc residues,
respectively. The WbeX protein of E. coli O42 differs from that of
E. coli O28ac just by the substitution of a single amino acid,
whereas the WbeY protein differs by two amino acid substitutions.
E. coli O42:K-:H37, strain CCUG 11348, was obtained from Cul-
ture Collection, University of Göteborg, Sweden. The bacterium
was grown in LB broth and the LPS isolated as previously
2
7
43
Contrary to what happened in the case or E. coli O9 and O9a, and
described.
2
8
E. coli O107 and O117, these differences seem not to affect the
specificity of the enzymes for their substrates, catalyzing the for-
mation of the same structural elements in both E. coli O42 and
O28ac.
4.2. Preparation of the O-deacylated lipopolysaccharide (LPS-OH)
4
The LPS (30.7 mg) was treated with 2 mL of NH OH 12.5% for
The genes responsible for the biosynthesis of 2-acetamido-2-
deoxyglucopyranose, glucopyranose, galactopyranose and glycerol
derivatives are considered housekeeping genes shared by other
pathways and, thus, are located outside the O-antigen gene clus-
17 h at 37 °C. The solution was then neutralized with 4 M HCl, dia-
lyzed against distilled water for 3 days and lyophilized. The crude
material was purified by size exclusion chromatography on a
HiLoad™ 16/60 Superdex™ 30 column (GE Healthcare), eluted
1
5
À1
ter. The glf gene found in the O-antigen gene cluster of E. coli
O28ac has been predicted to encode for an UDP-galactopyranose
mutase using BLAST similarity searches; this is a protein composed
of 381 amino acid residues that catalyzes the conversion of UDP-
with 1% 1-butanol in water at a flow rate of 1 mL min using
an ÄKTA™ purifier system. RI and UV detection at 214 nm were
used to monitor elution. The purified fractions were combined
in two pools and lyophilized to yield a total of 18.5 mg of the
LPS-OH.
2
5,42
Galp to UDP-Galf.
A partial sequence of a glf gene, encoding
for the first 302-amino acid fragment of the aforementioned pro-
tein, has also been identified in the O-antigen gene cluster of
E. coli O42, which is consistent with the presence of Galf residues
in the O-antigen polysaccharide. Based on the structural features of
the O-antigen PS of E. coli O28ac, the WbeZ acetyltransferase has
4.3. Preparation of the lipid-free polysaccharide (PS)
2
5
The LPS (20 mg) was dissolved in 3 mL of 1% AcOH (pH 3)
and stirred for 90 min at 100 °C. The lipid A was removed by
Figure 6. Comparison of the structures of the biological repeating unit of the O-antigen PSs of E. coli O28ac and O42. The glycosyltransferases (green color) involved in the
biosynthesis of the different structural elements, and the O-acetyltransferase (magenta color), are annotated in the proximity of the respective linkages. With exception of
WbeY and WbeX, all the enzymes are identical for both E. coli O42 and O28ac serogroups (the WbeY proteins differ by two amino acids whereas the WbeX proteins differ by
just a single amino acid). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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C. Fontana et al. / Carbohydrate Research xxx (2014) xxx–xxx
7
centrifugation at 15000Âg for 20 min at 4 °C, and the supernatant
was adjusted to pH 5 by adding 1 M NaOH very slowly at 0 °C and
under continuous stirring. The solution was then dialyzed against
distilled water and the crude material purified by size exclusion
chromatography as described above to yield 5 mg of the purified
PS.
Avance III 700 MHz spectrometer equipped with a 5 mm TCI
1
13 15
À1
( H/ C/ N) Z-Gradient (53.0 G cm ) CryoProbe™ and the NMR
spectra of the lipid-free PS (5 mg, 75 mM buffer phosphate, pD 6)
were carried out on a Bruker Avance III 600 MHz spectrometer
À1
equipped with a 5-mm inverse Z-gradient (55.7 G cm ) TXI probe
1
13 31
( H/ C/ P). Chemical shifts are reported in ppm using internal
2
sodium 3-trimethylsilyl-(2,2,3,3- H
external 1,4-dioxane in D O (d 67.40) or 2% H
0.00) as references.
The 1D diffusion-filtered 1H NMR spectra were recorded on a
4
)-propanoate (TSP, d
H
0.00),
4
.4. Dephosphorylation of the LPS-OH
2
C
3
PO
4
in D
2
O
(d
P
The LPS-OH (9.9 mg) was treated with 2 mL of aqueous 48% HF
at 3 °C for 6 days. After evaporation of the solvent with a stream of
dry air, 2 mL of H O were added and the solution neutralized with
M NH OH; all these steps were carried out at 0 °C. The solution
Bruker Avance III 600 MHz spectrometer using the 1D stimulated
spin-echo pulse sequence with bipolar gradients and LED (led-
bpgp2s1d). Diffusion encoded sinusoidal gradients pulses (d/2)
2
4
5
1
4
was then concentrated to 0.5 mL and centrifuged; the supernatant
was purified by size exclusion chromatography on a Superdex™
Peptide 10/300 GL (Tricorn™) column (GE Healthcare), using the
same conditions as described above. Different fractions were col-
lected and analyzed by mass spectrometry, and the fully dephos-
phorylated oligosaccharide was identified in fractions eluting at
retention volumes between 14.6 and 17.6 mL.
of 1.8 ms and strength 50% of the maximum were used; the diffu-
1
1
46
sion time was set to 100 ms. The H, H-TOCSY experiments were
obtained employing echo/antiecho gradient selection, an MLEV-17
spin-lock of 10 kHz and mixing times of 20, 40, 60, and 100 ms. The
1
1
47
gradient selected H, H-NOESY experiments were recorded with
1
13
a mixing time of 100 ms. The multiplicity-edited H, C-HSQC
4
8
experiments
were recorded employing the echo/antiecho
1
13
49
method and the H, C-H2BC experiment was recorded with a
1
13
4
.5. Mass-spectrometry
constant-time delay of 22 ms. The gradient selected H, C-HMBC
5
0
experiments
were carried out with an evolution time of
The electrospray ionization high-resolution mass spectrum
ESI-HRMS) was recorded in positive mode using a MicrOTOF-
Q™ mass spectrometer (Bruker Daltonics) using as solvent water/
isopropanol (1:1) with 0.2% formic acid. An MS/MS spectrum in
positive mode was obtained from the m/z 845.3 ion precursor
ꢀ60 ms over a spectral region of 5.0–6.0 Â 120 ppm in F2 and F1,
13
(
respectively. The C offset was 60 ppm, and the correlations to
the carbonyl carbons were determined from the aliased spectrum.
3
1
The P-based NMR spectra of the LPS-OH were obtained on a Bru-
1
31
ker Avance III 600 MHz spectrometer. The H, P-HMBC spec-
5
0,51
(
sodium adduct) and a MS/MS spectrum in negative mode was
trum
was recorded with an evolution time of 50 ms. The
1
31
52
obtained from the m/z 821.3 ion precursor (MÀH). Nitrogen was
H, P-hetero-TOCSY experiments were carried out with mixing
times of 36 and 54 ms, using a DIPSI2 mixing sequence set at
.2 kHz on both channels.
used as the collision gas.
3
4
.6. Sugar analysis and absolute configuration
Acknowledgements
The LPS-OH (0.2 mg) was hydrolyzed with 2 M trifluoroacetic
acid at 120 °C for 2 h. The sample was subsequently reduced with
NaBH and acetylated. The mixture of alditol acetates was analyzed
by gas-liquid chromatography (GLC). The absolute configuration of
-Glc, -Gal, and -GlcNAc were determined by GLC analysis of
their acetylated (+)-2-butyl glycoside derivatives.
This work was supported by grants from the Swedish Research
Council and the Knut and Alice Wallenberg Foundation. The
research that has led to these results has received funding from
the European Commission’s Seventh Framework Programme FP7/
2007-2013 under grant agreement No. 215536.
4
D
D
D
44
4
.7. GLC analyses
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Please cite this article in press as: Fontana, C.; et al. Carbohydr. Res. (2014), http://dx.doi.org/10.1016/j.carres.2014.05.003