John Albert4 1 Clinical Research Center, Analytical unit, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden;2Department of Chemistry, University of Peradeniya, Peradeniya, Sri La
Trang 1The structure of the O-chain of the lipopolysaccharide of a prototypal
Reine Eserstam1, Thushari P Rajaguru1,2, Per-Erik Jansson1, Andrej Weintraub3and M John Albert4 1
Clinical Research Center, Analytical unit, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden;2Department of Chemistry, University of Peradeniya, Peradeniya, Sri Lanka; 3 Karolinska Institute, Department of Microbiology, Pathology and Immunology, Division of Clinical Bacteriology, Huddinge University Hospital, Sweden; 4 Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait
The structure of the O-polysaccharide of the
lipopolysac-charide from a diarrheal strain isolated in Bangladesh
was studied with sugar, and methylation analysis, NMR
spectroscopy, mass spectrometry and partial acid hydrolysis
The strain was first designated as Hafnia alvei, but later
found to be a possible new species in the genus Escherichia
Two different polysaccharides were detected, a major and a
minor one.The structure of the major polysaccharide is
gi-ven below, while the structure of the minor one was not
investigated.The structure of the repeating unit was
estab-lished as
→6)-β-D-Galf-(1→3)-β-D-GalpNAc-(1→3)-β-D-Galp-(1→
α-NeuAc
↑6 2
The structure does not resemble any of the previously investigated lipopolysaccharide O-chains from Escherichia colior H alvei, but could fit in either group based on types of sugar residues and acidity
Phenotypic microbiological studies cannot definitely assign it to either species of the two genera.Genetic hybridization studies indicate that the Bangladeshi isolates may require a new species designation under the genus Escherichia
Keywords: lipopolysaccharide; Escherichia; Hafnia alvei; diarrhea; neuraminic acid
Hafnia alveiis a Gram negative bacterium and a member of
the family Enterobacteriaceae.There are reports of
associ-ation of H alvei with diarrhoea in Canada [1] and Finland
[2], but the mechanism of diarrhoea caused by this organism
in these locations remains unknown [3].However, some
isolates of a bacterium typed as H alvei from patients with
diarrhoea in Bangladesh produced diarrhoea in rabbits by
attaching and effacing (AE) lesions in the intestinal mucosa
that are characteristic of the lesions produced by
entero-pathogenic Escherichia coli [4].Like enteroentero-pathogenic
E coli, these H alvei isolates possess a homologous
patho-genicity island in the chromosome locus for enterocyte
effacement (LEE), which is responsible for producing
attaching and effacing lesions [5].LEE encodes a type III
secretory system [6].Secretion of the virulence factors leads
to effacement of the microvillus structure and reorganiza-tion of the actin cytoskeleton to form a pedestal-like structure, the attaching and effacing lesion [7].AE lesion formation is critical in mediating diarrhoea production in the host, but its exact role in disease is not known.Recent results from conventional biochemical analyses, testing of susceptibility to cephalothin, lysis by a Hafnia-specific phage, and amplification of the outer membrane protein gene phoE with species-specific primers support the identi-fication of these isolates as members of the genus Escheri-chiarather than Hafnia alvei [8].We studied the structure of the O-chain of the lipopolysaccharide of one them
M A T E R I A L S A N D M E T H O D S
Bacterium, cultivation and isolation
of lipopolysaccharide and O-specific polysaccharide The Hafnia alvei, strain number 10457, was from the culture collection of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka.This strain was isolated from a patient with diarrhoea and was positive for the AE property [15].The bacterium was grown
in TY-medium, and the lipopolysaccharide isolated by centrifugation and extraction of bacterial cells with hot aqueous phenol [16].The polysaccharide was analysed as
Correspondence to P.-E Jansson, Karolinska Institute,
Clinical Research Center, Novum, Huddinge University Hospital,
S-141 86 Huddinge, Sweden.
Fax: + 46 8 58583820, Tel.: + 46 8 58583821,
E-mail: pererik.jansson@kfc.hs.sll.se
Abbreviations: AE, attaching and effacing; Hex, hexose; DEPT,
dis-torsionless enhanced polarization transfer; HMBC, heteronuclear
multiple-bond correlation; HSQC, heteronuclear single-quantum
coherence; LEE, locus for enterocyte effacement; TMS, trimethylsilyl.
(Received 19 March 2002, revised 6 May 2002,
accepted 21 May 2002)
Trang 2with (+)-2-butanol, as described previously, but with the
modification that acetates were used [9–11].Neuraminic
acid methyl glycoside methyl ester was analyzed as the
trimethylsilyl (TMS)-derivative and authentic reference
A colorimetric test for Kdo using thiobarbituric acid was
also made [17]
Methylation analysis
Methylation was carried out with methyl iodide in dimethyl
sulfoxide in the presence of sodium methylsulfinylmethanide
[18].The methylated polysaccharide was purified using Sep–
Pak C18 cartridges.Hydrolysis was performed as described
for sugar analysis; partially methylated monosaccharides
were converted into alditol acetates and analyzed by GLC
and GLC-MS on a Hewlett Packard 5890 chromatograph
equipped with a NERMAG R10–10 L mass spectrometer,
using the above conditions.Identification was made using
reference data
NMR spectroscopy
1H- and 13C-NMR spectra were recorded with a JEOL
GSX-270 or JEOL JNM ECP500 instruments or solutions
in2H2O at 70 or 85C.Chemical shifts are reported with
internal acetone (dH2.25, dC 31.00) as reference Mixing
times of 30–160 ms were used in TOCSY experiments, and
for NOESY 100 and 300 ms
MALDI mass spectrometry
MALDI mass spectrometry in the positive mode was run on
a Finnigan Lasermat instrument using dihydroxybenzoic
acid acid as matrix.Between 10 and 20 scans were
accumulated and added.The neuraminic-acid-free
polysac-charide was treated with 0.01Macetic acid for 1 h at 65C
and then neutralized with dilute sodium hydroxide solution
R E S U L T S A N D D I S C U S S I O N
Hafnia alveistrain number 10457 was grown in
tryptone-yeast (TY) medium and harvested by centrifugation
Extraction with hot phenol/water (1 : 1, v/v) gave a
lipo-polysaccharide in the aqueous phase, which was recovered
and freeze dried.Ultracentrifugation of the
lipopolysaccha-ride gave a pellet and an upper phase, the latter containing
most of the material.SDS/PAGE of the two materials
(Fig.1) in the upper phase and the pellet showed identical patterns and it was therefore concluded that the same polysaccharide was present.A hydrolysate of the upper phase, analyzed as alditol acetates, revealed asD-glucose,
D-galactose, D-galactosamine, L-glycero-D-manno-heptose, and D-glucosamine in the proportions 6 : 65 : 19 : 6 : 3 The relatively high proportion of heptose may be the result
of short chains.It is not a component in the polysaccharide
as demonstrated in the MS analysis (see below).The absolute configurations of the sugars were established by GLC analysis of the acetylated (+)-2-butyl glycosides [9–11].Methanolysis of the sample and analysis by
GLC-MS gave, in addition to the sugars mentioned, neuraminic acid.The pellet showed essentially the same compounds
To verify that the material was an O-polysaccharide or exclude the possibility, the content of Kdo was checked with the thiobarbiturate method.It showed that the content of Kdo in the crude material, the pellet, and the supernatant was 11, 15, and 12 lgÆmg)1, respectively, corresponding to one Kdo per 70 sugars or two per 35, i.e a significant amount of Kdo
Treatment of the upper phase with acetic acid buffer of
pH 4.2 followed by gel chromatography on a Sephadex G-50 column gave a major O-polysaccharide peak at the void volume (O-polysaccharide) and a minor peak just after The material in the major peak was devoid of neuraminic acid.The second minor peak, which was included in the column and had four signals in the anomeric region of the
1H-NMR spectrum at d 5.07, 4.92, 4.80, and 4.49, clearly different from the major compound.The presence of two different polysaccharides in Hafnia has not been observed before.It was not clear whether it was an lipopolysaccharide
or a capsular polysaccharide and the fraction was not further investigated.The proportion of the minor polysac-charide was indicated by the size of the minor peaks in the
1H-NMR spectrum (Fig.2), especially as the peak near
d 5.0 was separate enough to be able to make a quantitative estimation, approximately 5%
In the methylation analysis of the O-polysaccharide, derivatives corresponding to 6-substituted galactofuranose, 3-substituted galactopyranose and 3-substituted
galactosa-ultracentrifugation of the Hafnia alvei lipopolysaccharide For com-parison lipopolysaccharide from a smooth (Shigella flexneri, 3) and a rough bacterium (Salmonella typhimurium Ra, 4) was run simulta-neously.
Trang 3mine were detected, thus indicating a linear polysaccharide
consisting of repeats with three sugar residues.This was
corroborated by the 1H-NMR spectrum, which showed
signals for anomeric protons at d 5.11 (J small), 4.76
(J 7.7 Hz) and 4.46 (J 7.7 Hz), for ring protons, and for an
N-acetyl methyl group at d 2.05 The first chemical shift
should belong to a furanoside as these normally have small
J-values, the second and third signal have typical J-values
for b-linked sugars with galacto-pyranose configuration
The absence of NeuAc was evident as no
methylene-deoxyresonances could be detected.The13C-NMR
spec-trum showed signals inter alia for anomeric carbons at d
109.9, 104.1 and 103.6 The first value is very high and
characteristic of a b-furanosidic sugar.A distorsionless
enhanced polarization transfer (DEPT) spectrum revealed
that the substituted hydroxymethyl group, C-6 of the
galactofuranose, is located at d 71.8, thus among those of
secondary carbons.The 1H- and13C-NMR spectra were
assigned with 2D NMR spectra including COSY, TOCSY,
NOESY, HSQC, and HMBC.Overlap in the spin systems
were in some cases a problem, but could be overcome with a combination of the spectra.Residues and spin systems are denoted A–C in Table 1.Indeed, the A residue was the furanoside as evident from the correlation in the HSQC-spectrum between d 5.11/109.9 The possibility to trace signals beyond that H-2 was limited, but two of the signals
in the13C-NMR spectrum at 82–84 p.p.m could be shown
to derive from C-2 and C-4 in A, and to correlate to proton signals at approximately d 4.07 B could be assigned to the GalNAc residue as evident from the chemical shift of C-2 signal, which appeared at d 52.2, typical for C-N signals A downfield shift of the signal for C-3 signal to d 78.7 corroborated the linkage position.Residue C gave in the TOCSY spectrum three correlations, up to H-4, which had
a resonance at d 4.16, from the high value it was confirmed that it was galactose.In addition, the couplings of H-4 (1D/ 2D) were small, indicative of the galacto configuration.In the13C-NMR spectrum, it was evident that two signals were present at d 75.6, the second being assigned to C-5 in residue
C, close to the value in the monomer.The sequence of
Fig 2 The 1 H-NMR spectrum of the Hafnia
alvei lipopolysaccharide in D 2 O.
Table 1.1H- and13C-NMR chemical shifts (d, p.p.m.) for different H alvei polysaccharides The N-acetyl group in the GalNAC residue appears at d 2.05/22.7/176.1 in the O-polysaccharide and in the GalNAc and NeuAc residues at d 2.05/22.8–22.9/175.8 in the lipopolysaccharide.
H alvei O-polysaccharide
Native H alvei lipopolysaccharide
Trang 4sugars was indicated by the following H/C correlations in
the HMBC spectrum, d 5.11/78.7 (A H-1/B C-3), d 4.73/82.6
(B H-1/C C-3), and d 4.44/71.8 (C H-1/A C-6).The
disaccharide elements A–B, B–C, and C-A were thus present
to make up the chain as
→6)-β-D-Galf-(1→3)-β-D-GalpNAc-(1→3)-β-D-Galp-(1→
The next step was to analyze the native
lipopolysaccha-ride.Methylation analysis of the native lipopolysaccharide
gave major GLC peaks corresponding to 6-substituted
galactofuranose, 3,6-disubstituted galactose, and
3-substi-tuted 2-acetamido-2-deoxy-D-galactose.In addition, smaller
peaks corresponding to the minor component
polysaccha-ride were observed.The repeating unit of the polysacchapolysaccha-ride
thus contains a terminal NeuAc, and the above mentioned
residues.A comparison to the methylation analysis data on
the O-polysaccharide, indicates that the terminal NeuAc
should be substituting the galactose residue in the
6-position
For the full characterization of the lipopolysaccharide,
with NeuAc still present, an NMR sample was prepared
from the native lipopolysaccharide.The spectrum had
broadened lines but were surprisingly good with resolved
couplings (Fig.2).The1H-NMR spectrum of the
lipopoly-saccharide showed signals for three anomeric protons at
d 5.11 (J small), 4.73 (J 7.7 Hz), 4.43 (J 7.7 Hz), thus close
to those observed for the O-polysaccharide.In the high field
region signals for a methylene group, assigned to CH2in
NeuAc were observed at d 2.75 and 1.68, the large difference
establishing the presence of an axial carboxyl group and an
a-linkage in the NeuAc residue.Signals for N-acetyl groups
deriving from NeuAc and GalNAc were present at d 2.05 In
the 13C-NMR spectrum, the corresponding signals were
present inter alia at d 109.9, 104.2, 103.7, and 101.2 for
anomeric carbons and at d 41.0 and 22.8–22.9 for methylene
and methyl groups, respectively.The spectrum resembled
that of the O-polysaccharide, but some changes were
evident.The signals at d 73.8, 69.0, 63.4, and 52.6 were higher than the others and were subsequently assigned to the NeuAc residue
Analysis of both of the1H- and the13C-NMR spectra using 1D and 2D techniques gave the data shown in Table 1, where the residues are referred to as A–D, D being the additional NeuAc residue.Most of the signals could be unambiguously assigned.The13C-NMR spectrum showed
28 signals of the possible 30.The assignment was made essentially as described for the O-polysaccharide and by comparison with the spectra of the O-polysaccharide Figures 3–6 show the COSY, TOCSY, HSQC and HMBC spectra, respectively
From the large glycosylation shifts of signals from C-6 in
A, C-3 and C-6 in B, and C-3 in C, the linkage positions were verified.The absence of any glycosylation shift in D further indicated that it was terminal.The chemical shift displacement of the C-6 signal in C to d 64.1, is small, typical for substitution with ketosides.An HMBC experi-ment showed the following inter-residue correlations from anomeric protons to linkage carbons: A H-1/B C-3 (5.11/ 78.8), B H-1/C C-3 (4.73/82.8) corroborating elements A–B and B–C.From the NOE spectrum the following inter-residue correlations between H-1 and protons on linkage carbons were observed: A H-1/B H-3 (5.11/3.82), demon-strating the element A–B Correlations 4.73/3.72 and 4.43/ 3.90 are in accord with elements B–C and C–A but ambiguous due to signal overlap.From the combined data, however, the following structure can be postulated for the repeating unit
→6)-β-D-Galf-(1→3)-β-D-GalpNAc-(1→3)-β-D-Galp-(1→
α-NeuAc
↑
6 2
D
Fig 3 The COSY spectrum the of the Hafnia alvei lipopolysaccharide showing the anomeric and the ring proton region.
Trang 5MALDI-MS of the O-polysaccharide
The O-polysaccharide, i.e the desialylated polysaccharide
chain, was also characterized by MALDI-MS of the
fragmented chain, anticipated to be facile to cleave with
acid, as furanosides were present.Thus, the
O-polysaccha-ride was treated with aqueous 0.01M acetic acid and the
samples were withdrawn at different times.Good spectra
were obtained after approximately 1 h.The spectra showed
three series of ions, all sodiated, the first at m/z 1626, 2149,
2677, 3204, 3730, and 4254, the second at m/z 1825, 2352,
2881, and 3401, and the third at m/z 1946, 2475, 3001, 3528,
and 4054.All series are spaced with approximately
527 atomic mass units (amu), corresponding to the
molecu-lar mass of the repeating unit containing two hexoses and
one acetamidohexose.The first series corresponds to a
multiple of the repeating unit, thought to be derived from
the expected hydrolysis of the furanosidic linkage, thus (HexNAc-Hex-Hexf)n.The second series contains a mul-tiple of the repeat plus an additional acetamidohexose (203 amu) and the third contains a multiple of the repeat plus two additional hexose residues (324 amu).This implies that not only the furanosidic linkage is acid-labile, but also that of the b-D-GalNAc residue.Thus, assuming that the linkage second most easily hydrolyzed is that of the acetamidohexose, the ions in the second and the third series correspond to the formulas (HexNAc-Hex-Hex)n-HexNAc and Hex-Hex-(HexNAc-Hex-Hex)n and the sequence is further established
The initial phenotypic characterization of the strain 10457 with a commercial identification system, API-20 E identified the strain as Hafnia alvei [4].Additional phenotypic characterization and partial 16S rRNA sequencing of a set of isolates identified them not as typical Hafnia alvei, but
Fig 5 HSQC spectrum of the Hafnia alvei
lipopolysaccharide showing the anomeric and
the ring proton/carbon region and including
high resolution1H- and13C-NMR spectra.
Fig 4 TOCSY spectra of the Hafnia alvei
lipopolysaccharide showing the correlations
deriving from the anomericprotons Mixing
times were from 30 to 160 ms.
Trang 6unique isolates [12].Further phenotypic characterization
suggested that these isolates are neither Hafnia alvei nor
Escherichia coli, but closely related to the genus Escherichia
[8].DNA hybridization studies suggested that these isolates
deserve a new species name under the genus Escherichia
(J.Albert, Kuwait University, Safat, Kuwait, personal
communication).The unique structure of the O-chain of
one of these isolates further confirms this conclusion
The lipopolysaccharide structures of both H alvei and
E coli are known [13,14].More than 20 strains from
H alveihave been investigated and both amino sugars and
acidic sugars are frequent.Furanosidic sugars are also
observed.Neuraminic acid has been found once but only as
an internal residue.Of the more than 60 E coli strains that
have been investigated, many contain hexoses and
hexosa-mines, as well as acid functions.Also here, neuraminic acid
has been found only internally.Furanosidic galactose is
present, but not common.As a whole, the structural
features of the investigated strain cannot be related to any
particular structure among those studied of Hafnia and
Escherichia.The terminal neuraminic acid is, however, an
interesting feature, normally associated with glycoproteins
and glycolipids.The biological properties of the novel
lipopolysaccharide of this strain remain to be elucidated
A C K N O W L E D G E M E N T S
The authors thank Mrs G.Alvelius for help in mass spectrometry and
Mrs M.So¨rensson for microbiology work.This work was supported
by grants from the Swedish Natural Science Research Council, the
Swedish Medical Research Council (No.B95-16X-11227-01 A), and
the International Science Programs, Uppsala University, Sweden.Mrs
Farrah Vesali is thanked for some preliminary experiments.
R E F E R E N C E S
1.Ratnam, S.(1991) Etiologic role of Hafnia alvei in human
diarrheal illness J Clin Microbiol 59, 4744–4745.
2 Ridell, J., Siitonen, A., Paulin, L., Mattila, L., Korkeala, H &
Albert, M.J (1994) Hafnia alvei in stool specimens from patients
with diarrhoea and healthy controls J Clin Microbiol 32, 2335– 2337.
3 Ismaili, A., Bourke, B., de-Azavedo, J.C., Ratnam, S., Karmali, M.A & Sherman, P.M (1996) Heterogeneity in phenotypic and genotypic characteristics among strains of Hafnia alvei J Clin Microbiol 34, 2973–2979.
4 Albert, M J , Alam, K , Islam, M , Montanaro, J , Rahman, H , Haider, K , Hossain, M A , Kibriya, A K M G & Tzipori, S (1991) Hafnia alvei: a probable diarrheal pathogen of humans Infect Immun 59, 1507–1513.
5 McDaniel, T.K., Jarvis, K.G., Donnenberg, M.S & Kaper, J.B (1995) A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens Proc Natl Acad Sci 92, 1664– 1668.
6 Jarvis, K G , Giron, J A , Jerse, A E , McDaniel, T K , Donnenberg, M.S & Kaper, J.B (1995) Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation Proc Natl Acad Sci USA 92, 7996– 8000.
7 Rosenshine, I , Ruschkowski, S , Stein, M , Reinscheid, D J , Mills, S.D & Finlay, B.B (1996) A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation EMBO J 15, 2613–2624.
8 Janda, J.M., Abbott, S.L & Albert, M.J (1999) Prototypal diar-rheagenic strains of Hafnia alvei are actually members of the genus Escherichia J Clin Microbiol 37, 2399–2401.
9 Gerwig, G.J., Kamerling, J.P & Vliegenthart, J.F.G (1978) Determination of the D and L configuration of neutral mono-saccharides by high resolution capillary glc Carbohydr Res 62, 349–357.
10 Gerwig, G.J., Kamerling, J.P & Vliegenthart, J.F.G (1979) Determination of the absolute configuration of monosaccharides
in complex carbohydrates by capillary glc Carbohydr Res 77, 1–7.
11.Leontein, K , Lindberg, B.& Lo¨nngren, J.(1978) Assignment of the absolute configuration by glc of their acetylated glycosides formed from chiral alcohols Carbohydr Res 62, 159–162.
12 Ridell, J., Siitonen, A., Paulin, L., Lindroos, O., Korkeala, H & Albert, M.(1995) Characterization of Hafnia alvei with bio-chemical test, RAPD-PCR and partial sequencing of the 16S rRNA gene J Clin Microbiol 33, 2372–2239.
Fig 6 The full HMBC spectrum of the Hafnia alvei lipopolysaccharide.
Trang 713 Knirel, Y.A & Kochetkov, N.K (1994) The structure of
lipo-polysaccharides of gram-negative bacteria.III.The structure
of O-antigens Biochemistry 59, 1325–1383.
14.Jansson, P -E.(1999) The chemistry of O-polysaccharide chains in
bacterial lipopolysaccharides Endotoxin in Health and Disease
(Brade, H., Opal, S.M., Vogel, S.N & Morrison, D.C., eds), pp.
155–178.Marcel Dekker, New York.
15 Albert, M.J., Faruque, S.M., Ansaruzzaman, M., Islam, M.M.,
Haider, K., Alam, K., Kabir, I & Robins-Browne, R (1992)
Sharing of virulence-associated properties at the phenotypic and
genetic levels between enteropathogenic Escherichia coli and
Hafnia alvei J Med Microbiol 37, 310–314.
16.Westphal, O.& Jann, K.(1965) Bacterial lipopolysaccharides Extraction with phenol-water and further applications of the procedure Methods Carbohydr Chem 5, 83–91.
17 Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J & Carlo D.J (1978)
A new and improved microassay to determine 2-keto-3-deoxy-octonate in lipopolysaccharide of Gram-negative bacteria Anal Biochem 85, 595–601.
18 Jansson, P.-E., Kenne, L., Liedgren, H., Lindberg, B & Lo¨nng-ren, J.(1976) A practical guide to the methylation analysis of carbohydrates.In Chemical Communications.pp.1–75.University
of Stockholm, Stockholm.