The structure of the O-antigen of Cronobacter sakazakii HPB 2855 isolate involved in a neonatal infection

Article abstract of DOI:10.1016/j.carres.2010.06.020

Strains of Cronobacter sakazakii (previously known as Enterobacter sakazakii) are medically recognized important Gram-negative bacterial pathogens that cause enterocolitis, septicemia, and meningitis, with a high mortality rate in neonates. The structure of their O-antigens, that form part of their somatic lipopolysaccharide (LPS) components, is of interest for their chemical and serological identification and their relationship to virulence. The O-polysaccharide (O-PS) of C. sakazakii HPB 2855 (SK 81), a strain isolated from an infant at the Hospital for Sick Children in Toronto in 1981, was shown to be a polymer of a partially O-acetylated-repeating hexasaccharide unit composed of d-glucose, d-galacturonic acid, 2-acetamido-2-deoxy-d-galactose, and l-rhamnose (1:1:1:3). From composition and methylation analysis, and the application of 1D and 2D ¹H and ¹³C NMR spectroscopy, the O-PS was determined to be a polymer of a repeating oligosaccharide unit having the structure: [figure persented]

Full text of DOI:10.1016/j.carres.2010.06.020

Carbohydrate Research 345 (2010) 1932–1937
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The structure of the O-antigen of Cronobacter sakazakii HPB 2855 isolate
involved in a neonatal infection
a
a
b
b
a,*
Leann L. MacLean , Evgeny Vinogradov , Franco Pagotto , Jeffrey M. Farber , Malcolm B. Perry
a
Institute for Biological Sciences, National Research Council, Ottawa, Canada K1A 0R6
Bureau of Microbial Hazards, Health Products and Food Branch, Food Directorate, Health Canada Ottawa, Canada K1A 0K9
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2010
Received in revised form 7 June 2010
Accepted 29 June 2010
Available online 6 July 2010
Strains of Cronobacter sakazakii (previously known as Enterobacter sakazakii) are medically recognized
important Gram-negative bacterial pathogens that cause enterocolitis, septicemia, and meningitis, with
a high mortality rate in neonates. The structure of their O-antigens, that form part of their somatic lipo-
polysaccharide (LPS) components, is of interest for their chemical and serological identification and their
relationship to virulence. The O-polysaccharide (O-PS) of C. sakazakii HPB 2855 (SK 81), a strain isolated
from an infant at the Hospital for Sick Children in Toronto in 1981, was shown to be a polymer of a par-
Keywords:
tially O-acetylated-repeating hexasaccharide unit composed of D-glucose, D-galacturonic acid, 2-acetam-
Cronobacter sakazakii
Lipopolysaccharide
Polysaccharide
O-Antigen
ido-2-deoxy-D-galactose, and L-rhamnose (1:1:1:3). From composition and methylation analysis, and the
application of 1D and 2D 1H and C NMR spectroscopy, the O-PS was determined to be a polymer of a
13
repeating oligosaccharide unit having the structure:
NMR spectroscopy
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1
. Introduction
The taxonomy of Enterobacter sakazakii initially described as a
merase chain reaction, and differential selective agars have proven
useful for its identification. In view of the demonstrated usefulness
of LPS O-antigen typing systems for other Gram-negative bacterial
species we have undertaken the structural analysis of the O-PS of
selected Cronobacter spp. with a view to identifying particular O-ser-
otypes with pathogenicity. With the limited number of Cronobacter
O-PS antigen structures reported to date, the variety in composition
and structure suggests that many O-serotypes will be discovered.
The current investigation of the O-PS structure of the C. sakazakii
strain 2855, the causal agent of invasive infection in an infant in
Toronto, determined it to be a polymer of a repeating hexasaccha-
ride unit. Interestingly, this O-PS is structurally similar to the
repeating heptasaccharide unit found in the O-PS C. sakazakii strain
motile peritrichous Gram-negative bacteria has recently, on the
basis of phenotypic and genotypic characterization and other
observations, been assigned a novel genus Cronobacter with a divi-
1
2
sion of species based on previously described biogroups. As such,
there are now six distinct genomospecies comprising Cronobacter
sakazakii, (formerly E. sakazakii), Cronobacter malonaticus, Cronob-
acter turicensis, Cronobacter genomospecies 1, Cronobacter muytjen-
sii, and Cronobacter dublinensis. While a rare infection, C. sakazakii
can cause life-threatening neonatal meningitis, sepsis, and necro-
tizing enterocolitis and is thus an emerging pathogen of medical
interest.
3
4
767, an isolate from a neonatalmeningitis death in Paris. This is the
first reported finding of a Cronobacter spp. O-PS having strikingly
similar structures. Unlike the close structural relationship between
the O-PS antigens of C. sakazakii strains 767 and HPB 2855, previ-
Currently no agreed-upon typing protocol has been developed to
trace C. sakazakii, however, biochemical profiling, real-time poly-
5
ously determinedO-PS structures of C. sakazakii HPB3290, C. muytj-
ensii HPB 3270, and C. malonaticus HPB 3267 are composed of
6
7
*
Corresponding author. Tel.: +1 613 990 0837; fax: +1 613 941 1327.
E-mail address: malcolm.perry@nrc-cnrc.gc.ca (M.B. Perry).
structurally unrelated repeating oligosaccharide units.
0
008-6215/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.06.020

L. L. MacLean et al. / Carbohydrate Research 345 (2010) 1932–1937
1933
2
2
. Experimental
trifluoroacetic acid (120 °C, 2 h), reduced (NaBD
4
), acetylated, and
analyzed by GLC–MS (as described above) using a temperature gra-
dient 170 °C (4 min delay) to 260 °C at 4 °C/min. Retention times of
derivatives are quoted relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-
methylglucitol (TGM = 1.0).
.1. Bacterial growth and LPS and O-PS production
C. sakazakii HPB 2855 (NRCC 6464) was grown in 3.7% brain–
heart infusion broth (Difco) at 3% oxygen concentration and
3
7 °C in a New Brunswick 25 L fermenter. The bacteria were killed
2.5. NMR Spectroscopy and mass spectrometry
with phenol (1% final concentration at 4 °C, 2 h) and collected by
centrifugation as a wet paste (295 g). Saline-washed cells (250 g)
were extracted by vigorous stirring in 50% aqueous phenol (1.2 L)
at 65 °C for 15 min. The water phase of the cooled (4 °C) extract
was dialyzed against running tap water until free from phenol,
and then lyophilized. The lyophilizate dissolved in 0.02 M sodium
phosphate buffer (pH 7.0, 160 mL) was treated sequentially with
deoxyribonuclease, ribonuclease, and proteinase K (2 h each at
1H and 13C spectra were recorded at 42 °C using a Varian Inova
2
500 MHz with samples dissolved in 99% D O and internal acetone
1
13
standard (2.225 ppm for H and 31.45 ppm for C) employing
standard parameters for COSY, TOCSY (mixing time 100 ms),
1
13
ROESY (mixing time 400 ms), H, C-HSQC, and heteronuclear cor-
relation gHMBC, for 6 Hz long-range coupling constants.
3
7 °C) and the solution was then subjected to ultracentrifugation
2.6. Reduction of O-PS by the carbodiimide-borodeuteride
method
(
150,000 g, 10 h, 4 °C) and the precipitated LPS was taken up in
water (80 mL) and lyophilized (yield 1.70 g).
Following the method of Taylor and Conrad13 a solution of O-PS
(70 mg) in water (10 mL) was treated with 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (250 mg) and the stirred mixture was
maintained over 4 h at pH 4.5 by adjustment with 0.05 M HCl.
In a parallel study, an identical growth and extraction from a cell
3
paste (533 g) of C. sakazakii 767 (HPB 5723) afforded LPS (5.6 g), a
portion of which was used to produce an O-PS under the same con-
ditions as those described below for C. sakazakii HPB 2855.
LPS from C. sakazakii HPB 2855 (1.0 g) was hydrolyzed with 2%
acetic acid for 2 h at 100 °C and insoluble lipid A (145 mg) was re-
moved from the cooled hydrolyzate by low speed centrifugation.
The lyophilized water-soluble products dissolved in 0.05 M pyrid-
inium acetate (pH 4.5, 8 mL) were subjected to Sephadex G-50 col-
umn (2.5 Â 95 cm) chromatography using the same buffer. The
eluate was monitored by refractive index and colorimetrically for
The reaction mixture was reduced by the slow addition of NaBD
4
(100 mg) solution with a maintained pH ꢀ7.0 by M HCl addition.
After 16 h the mixture was acidified (AcOH), dialyzed against water,
and the reduced O-PS (55 mg) was recovered by lyophilization.
2.7. CE-MS analysis
neutral aldose, hexuronic acid,9 and 2-amino-2-deoxyhexose
8
10
CE-MS was accomplished using a Prince CE system coupled to a
4000 Q-trap mass spectrometer employing a sheath solution of 2:1
isopropanol–MeOH and a 5-kV electrospray-ionizing voltage for
the positive ion detection mode.
and collected fractions were lyophilized to yield O-PS (Kav 0.02,
2
89 mg) and core oligosaccharide (Kav 0.42, 32 mg).
2
.2. Gel electrophoresis
2
.8. Specific optical rotations
Deoxycholate–polyacrylamide gel-electrophoresis (DOC–PAGE)
Specific optical rotations were determined at 20 °C using a 10-
cm. microtube and a Perkin–Elmer 343 Polarimeter.
was performed on separating gels of 14% acrylamide and detection
was made by silver staining after periodate oxidation.11
2
.3. Monosaccharide analysis
3
. Results and discussion
O-PS samples (2–5 mg) were hydrolyzed with 4 M trifluoroace-
Fermenter-grown cells of C. sakazakii HPB 2855 were extracted
tic acid (115 °C, 2 h) and the concentrated residues were reduced
NaBH ) and acetylated as previously described. The acetylated
14
by a modified hot-aqueous phenol method and ultracentrifuga-
tion of the concentrated aqueous phase afforded LPS in 6% based
on dry cell mass. DOC–PAGE analysis of the LPS showed a typical
pattern for a smooth endotoxin product (Fig. 1) in which the bands
in the silver-stained gels were consistent for a LPS composed of a
high molecular mass O-PS chain composed of a repeating hexasac-
charide unit.
Mild acid hydrolysis of the LPS from C. sakazakii HPB 2855 affor-
ded precipitated lipid A (14% yield) and Sephadex G-50 column
chromatography of the water-soluble products afforded a high
(
4
alditol derivatives were analyzed by GLC using a HP5 capillary col-
umn (30 m  0.25 mm) with a flame ionization detector (Agilent
6
2
850 chromatograph) in a temperature gradient from 170 to
60 °C at 4 °C/min, and by GLC–MS using a Varian 200 ion-trap
instrument with the same column conditions. Mobilities are
quoted relative to hexa-O-acetylglucitol (TGA = 1.0).
Chromatographically pure samples of component aldose
hydrolyzates of O-PS (100 mg) were obtained by preparative
paper chromatography on water-washed Whatman 3MM filter
paper using 1-butanol–pyridine–water (10:3:3 v/v) mixture as the
eluant. Detection of glycoses on excised guide strips was made by
sprays of 2% p-anisidine HCl (in EtOH) and 2% ninhydrin (in ace-
molecular mass O-PS (56% yield) having [
and a core oligosaccharide (7% yield).
D
a] +36 (c 0.5, water)
Preparative paper chromatography of the acid (M H
hydrolyzed O-PS afforded chromatographically pure -rhamnose
-glucose (RGal 1.26), and 2-amino-2-deoxy- -glucose
Gal 0.56) that were identified from their proton NMR spectra, spe-
2 4
SO )-
L
tone). Chromatographic mobilities are quoted relative to D-galact-
ose (RGal = 1.0).
(
R
Gal 3.54),
D
D
(R
cific optical rotations, and by GLC of their acetylated alditol deriv-
atives. A hydrolyzate of the reduced (NaBD ) O-PS prepared by the
carbodiimide method afforded, in addition to the above-identi-
fied glycoses, the new derivative -galactose-6-d that was charac-
terized by proton NMR and by GLC–MS of its hexa-O-acetyl-
galactitol-6-d derivative (TGA 1.02), thus identifying the uronic
acid component of the O-PS as -galacturonic acid.
2
.4. Methylation analysis
4
1
3
Samples of O-PS (3 mg) and carboxylic acid-reduced (NaBD
4
) O-
D
2
PS (3 mg) dissolved in dimethylsulfoxide (1 mL) were methylated
by the addition of NaOH following the Ciucanu–Kerek procedure,
washed through Sep-Pak C18 cartridges, and hydrolyzed with 2 M
D-
1
2
2
D

1
934
L. L. MacLean et al. / Carbohydrate Research 345 (2010) 1932–1937
1
.27, 9H) attributed to the presence of three
single methyl signal (2.03 ppm, 3H) from an acetyl function of the
-GlcNAc component residue. Consistent with the proton NMR spec-
L-Rha residues, and a
D
1
3
trum the related C spectrum of the O-PS (Fig. 3) also showed six
anomeric carbon signals (104.0–97.2 ppm) inter alia with three
methyl resonances (17.9, 17,6, and 17.3 ppm) from the
dues, and a C-2 ring carbon resonance (57.2 ppm) of the acetam-
ido-substituted function in the -GlcNAc residue that was also
L-Rha resi-
D
associated with a methyl resonance (23.5 ppm) and carbonyl
resonance (175.2 ppm). A second carbonyl resonance at 173.8 ppm
(
pD 3.2) shifted upfield to 174.4 ppm at pD 8.5 was assigned to the
carboxyl group of the -GalA residue. The combined experimental
evidence leads to the O-PS composition as -Glc (1 part), -Rha
3 parts), -GlcNAc (1 part), and -GalA (1 part).
For a more detailed NMR analyses (Fig. 4) the component sugars
D
D
L
(
D
D
were assigned the letters A to F in order of their decreasing ano-
meric proton shifts. The small JH-1,H-2 (<1.5 Hz) coupling constants
of residues A, C, and D identified the three manno-configured res-
idues whose spin systems terminated with methyl groups revealed
in TOCSY and COSY spectra. Residue B had a galacto-configuration
3
determined from its JH,H coupling pattern and contained a carbox-
ylic group at the C-6 position (173.8 ppm), further confirming B as
a
D-GalpA residue. Residue F was confirmed as a GlcpNAc residue
with an amino function at C-2 (57.2 ppm). Residue E was identified
3
by its JH,H coupling constant pattern, as the a-D-Glcp residue.
1
1
Inter-residue correlations were made from H– H NOESY and
1H–13C HMBC spectra obtained for the de-O-acetylated O-PS. The
observed anomeric NOE proton connectivities were H-1A to H-4B
(4.5 ppm), H-1B to H-2C (4.30 ppm), H-1C to H-4E (3.64 ppm),
H-1D to H-3F (3.67 ppm), and H-1F to H-3C (3.85 ppm, weak
signal). HMBC spectra showed long-range connectivities of H-1A
to C-4B (77.1 ppm), H-1B to C-2 C (75.1 ppm), H-1C to C-4E
(78.5 ppm), C-1D to H-3F (3.67 ppm), and H-1F to C-3C. The spectral
data were consistent with the proposed sequence shown below [I].
Figure 1. DOC–PAGE analysis of the lipopolysaccharide of [1] C. sakazakii 767, [2] C.
sakazakii HPB 2855, and [3], S. milwaukee (standard reference).
2
2
1
Preliminary 1D H NMR of the O-PS indicated it to be significantly
O-acetylated and subsequent-described analyses were made on a
column chromatographically (Sephadex G-50) purified de-O-acety-
lated O-PS prepared by treatment with anhydrous hydrazine under
The anomeric configurations of the glycose residues were estab-
7
1
previously described conditions. The de-O-acetylated O-PS had [
lished by the coupling constants
J
H-1,C-1 determined in a HMQC
a]
D
+38 (c 0.8, water) and its 1H NMR spectrum (Fig. 2, Table 1)
experiment recorded without decoupling. Residues A to E having
1
showed six anomeric proton signals in the region 5.29–4.68 ppm
indicative of five residues having -configurations and one b-config-
uration. The spectrum showed inter alia three methyl signals (1.25–
J
H-1,C-1 values >170 Hz (Table 1) were assigned to
a
-configuration
-GlcpNAc residue F ( JH-1,C-1 165 Hz) was assigned the
b-configuration. A consideration of the above composition and
1
a
and the D
Figure 2. 1D Proton NMR spectra of the de-O-acetylated O-PS from [A] C. sakazakii 767 and [B] C. sakazakii HPB 2855.

L. L. MacLean et al. / Carbohydrate Research 345 (2010) 1932–1937
1935
Table 1
NMR Chemical shifts and coupling constants for the de-O-acetylated O-polysaccharide of Cronobacter sakazakii HPB 2855
Glycose residue
Chemical shifts
H-3/C-3
H-1/C-1
H-2/C-2
H-4/C-4
H-5/C-5
H-6/C-6
[A]
[B]
[C]
[D]
[E]
[F]
a
-
L
-Rhap-(1?
?4)- -GalpA-(1?
?2,3)- -Rhap-(1?
-Rhap-(1?
-Glcp-(1?
-GlcpNAc-(1?
5.29 (<1.5)
4.05
71.7
3.93
69.2
4.30
75.1
3.85
78.2
3.55
72.8
3.72
57.2
3.83
71.3
4.10
71.8
3.85
80.3
3.90
70.6
3.84
72.7
3.67
82.4
3.36
73.5
4.50
77.1
3.57
72.2
3.48
73.1
3.64
78.5
3.50
69.7
3.82
69.8
4.68
73.1
4.02
70.4
4.03
70.7
4.08
71.8
3.37
77.0
1.26
17.1
1
01.5 (173)
5.04 (s)
7.2 (173)
5.04 (s)
8.7 (173)
4.95 (<1.5)
9.7 (170)
4.86 (ꢀ3)
8.9 (173)
4.68 (7.4)
04.0 (161)
a-D
9
175.7
1.26
17.1
1.26
17.1
3.80/3.8
61.2
3.74/3.85
62.4
a-L
9
?2)-
a
-
-
L
9
?4)-
a
D
9
?3)-b-
D
1
Chemical shifts are recorded in ppm and coupling constants in Hz are shown in parentheses.
Figure 3. 1D 13C NMR spectrum of the de-O-acetylated O-PS of C. sakazakii HPB 2855.
NMR data leads to the basic hexasaccharide-repeating unit of the C.
sakazakii HBP 2855 O-PS [I] having the structure:
1,2,3,5-tetra-O-acetyl-4-O-methylrhamnitol-1-d (TGM 1.17), 1,4,5-
tri-O-acetyl-2,3,6-tri-O-methylglucitol-1-d (TGM 1.44), 1,4,5,6-tetra-
Consistent with the O-PS structure [I] the positive mode ESI
mass spectrum of the C. sakazakii HBP 2855 de-O-acetylated O-PS
3
O-acetyl-2,3-di-O-methylgalactitol-1,6,6-d (TGM 1.91), and 1,3,5-tri-
O-acetyl-2-(N-methylacetamido)-2-deoxy-4,6-di-O-methyl-glucitol-
1-d (TGM 2.97). The identified methylated derivatives are consistent
with the proposed respective linked glycose units, Rhap-(1?, ?2)-
Rhap-(1?, ?2,3)-Rhap-(1?, ?4)-Glcp-(1?, ?4)-GalpA-(1?, and
?3)-GlcpNAc-(1?. The same methylated products were also identi-
fied by GLC–MS analysis in the hydrolysis products of the methylated
(
(
Fig. 5) contained fragment peaks corresponding to 204.0 amu
HexNAc), 350.0 amu (HexNAcdHex), 526 amu (HexNAcdHex
HexA), 834.5 amu (HexNAcdHex
M+1 = 980.0 Da confirming the proposed repeating unit structure
HexNAcdHex HexAHex; calculated mass 978.9 Da).
The proposed structure was further confirmed from methylation
analysis. GLC–MS analysis of the reduced (NaBD ) and acetylated
hydrolysis products of the reduced (NaBD /THF) fully methylated
O-PS identified 1,5-di-O-acetyl-2,3,4-tri-O-methylrhamnitol-1d (TGM
.59), 1,2,5-tri-O-acetyl-3,4-di-O-methylrhamnitol-1-d (TGM 0.83),
2
HexAHex), and a molecular ion
(
3
4
O-PS preparation obtained by the reduction (NaBD ) of O-PS by the
4
carbodiimide method.
4
From integration of the methyl group resonances in the pro-
ton NMR spectra, the native O-PS was estimated to contain
approximately 1.6 O-acetyl functions per hexasaccharide-repeat-
0

1
936
L. L. MacLean et al. / Carbohydrate Research 345 (2010) 1932–1937
Figure 4. 1H,13C-HSQC NMR spectrum of the de-O-acetylated O-PS from C. sakazakii HPB 2855.
Figure 5. CE-MS spectrum of the de-O-acetylated O-PS from C. sakazakii HPB 2855.
ing unit. From the observed chemical resonances for the di-O-
glycosidic-substituted branch -Rhap [C] residue, its C-4 carbon
73.6 ppm), and H-4 (4.92 ppm) resonance chemical shifts to,
respectively, 72.2 ppm and 3.57 ppm in the de-O-acetylated O-
PS, it was concluded that residue C was fully acetylated at its
O-4 position. Similar non-stoichemic observed proton and carbon
resonance shifts indicated highly variable O-acetyl substitutions
at the O-2, O-3, and O-4 positions of the terminal
idue A.
It is interesting to note that the structure of the O-antigen of C.
sakazakii HPB 2855 has a close structural relationship to the re-
cently determined structure [II] of C. sakazakii 767, the essential
a-L-Rhap res-
L
(
difference being the addition, in the latter strain, of an
a-D-Glcp
residue at the O-4 position of the b- -GlcpNAc residue in the back-
D

L. L. MacLean et al. / Carbohydrate Research 345 (2010) 1932–1937
1937
bone chain of the repeating heptasaccharide unit in the C. sakazakii
In our comparison of the LPS preparations of C. sakazakii 767
and HPB 2855 strains it was found that the yield of cell mass of
C. sakazakii 767 was 70% greater than that of the C. sakazakii HPB
7
67 O-PS.
2
855 and that the relative respective yields of O-PS from the two
LPSs were approximately 2:1. Multiple preparations of O-PS from
the two LPSs were consistent in composition and position of
O-acetyl substitutions, as revealed by proton NMR spectra of the
native and de-O-acetylated polymers (Fig. 1) and that the
It is remarkable that the discussed O-antigens of C. sakazakii
were associated with neonatal infection in widely separated geo-
graphic locations.
It has been observed that in several O-antigens, the addition of
a-D-Glcp and b-D-Glcp residues to a specific basic oligosaccharide-
C. sakazakii HPB 2855 LPS did not have any
a-D-Glcp side chain
substitution. Future analysis of particularly virulent strains of
C. sakazakii may reveal whether they belong to an O-serogroup
related to the O-antigens of C. sakazakii 767 and HPB 2855.
repeating unit forming O-PS antigens is a common occurrence.
Acknowledgment
For example, the O-antigen of Salmonella O:30 contains two related
1
5,16
structures.
One structure is a polymer of a repeating tetrasac-
We thank Mr. Perry Fleming for the production of bacterial cell
mass and the use of the NRC Fermentation Unit and containment
facility.
charide (incidentally identical of the O-PS-repeating unit in Esche-
richia coli O157 LPS ) while the second subtype antigen is a
polymer of a pentasaccharide-repeating unit composed of the sub-
1
7
type tetrasaccharide by a b-D-Glcp attached in single non-reducing
residues to the polymeric chain. A similar O-antigenic mobility is
discussed in a paper on the sharing of a common repeating tetrasac-
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4
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0
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9
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2
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of the structure of the O-antigen of E. coli O70 serotype it was
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1
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NAc residues contained in the backbone chain of the basic E. coli
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a-D-Glcp residue in the C. sakazakii 767 LPS O-antigen may involve
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the C. sakazakii 2855 strain.
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