Glycobiology vol. 16 no. 6 pp. 538–550, 2006
doi:10.1093/glycob/cwj094
Advance Access publication on February 20, 2006
Structure of the lipid A–inner core region and biological activity of Plesiomonas
shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide
Jolanta Lukasiewicz1,2, Tomasz Niedziela2,
Wojciech Jachymek2, Lennart Kenne3, and
Czeslaw Lugowski2
2
Department of Immunochemistry, Ludwik Hirszfeld Institute of
Immunology and Experimental Therapy, Polish Academy of Sciences,
R. Weigla 12, 53-114 Wroclaw, Poland; and 3Department of Chemistry,
Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
Received on January 19, 2006; revised on February 15, 2006; accepted on
February 16, 2006
Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of
diarrhea in humans. The extraintestinal infections caused
by this bacterium, for example, endopthalmitis, meningitidis,
bacteremia, and septicemia, usually have gastrointestinal
origin and serious course. The lipopolysaccharide (LPS,
endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and
especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated.
The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF
mass spectrometry, and the intact Kdo-containing core
region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS,
containing 4-substituted uronic acids, are usually very
complex and difficult to separate. Since Kdo residues, like
sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide
containing the intact Kdo region from the reaction mixture
by affinity chromatography. The major form of lipid A
was built of b-D-GlcpN4PPEtn-(1®6)-a-D-GlcpN1P
disaccharide substituted with 14:0(3-OH), 12:0(3-OH),
14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2,
O-3, N-2¢, and O-3¢, respectively. This is a novel structure
among known lipid A molecules. Analysis of intact Kdo-lipid
A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides
O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo
activity of P. shigelloides and Escherichia coli LPS are
discussed.
Key words: lipid A/lipopolysaccharide/MALDI-TOF
mass spectrometry/NMR spectroscopy/Plesiomonas
shigelloides
1To
whom correspondence should be addressed; e-mail:
[email protected]
Introduction
Plesiomonas shigelloides is a Gram-negative, facultatively
anaerobic and flagellated rod, belonging to the Enterobacteriaceae family and is the only species in the genus Plesiomonas (Garrity et al., 2004). P. shigelloides is not a part of
the normal human fecal flora. These bacteria are found in
the aquatic environment in the tropical and subtropical
regions, but also in cold climate. Bacteria occur as free-living
cells in water or in water creatures, such as fish, crabs,
prawns, mussels, and oysters. Thus water- and food-borne
outbreaks of intestinal infections due to P. shigelloides have
been reported. It was ranked third among etiological agents
in outbreaks of travellers’ diarrhea in Japan and China
(Stock, 2004). Various studies have reported on the invasive
nature of the infection, which may cause a cholera-like illness (Wong et al., 2000).
P. shigelloides is also responsible for various extraintestinal infections of gastrointestinal origin, particularly in neonates and immunosuppressed adults or people with an
underlying disease. The cases of cellulitis, cholecystitis, peritonitis, proctitis, pyosalpinx, septic arthritis, endopthalmitis, meningitidis in neonates, bacteremia, and septicemia
were reported for P. shigelloides as an etiological agent (Wong
et al., 2000; Stock, 2004). The outbreaks of P. shigelloidesrelated sepsis and meningitidis are associated with the serious course and high fatality rate (Stock, 2004). P. shigelloides
adheres to and subsequently enters the human intestinal
Caco-2 cells in vitro through phagocytic-like process. Moreover, it was found that live bacteria escape from cytoplasmic
vacuoles (Theodoropoulos et al., 2001) and induce apoptotic cell death (Tsugawa et al., 2005). To date several possible virulence factors of P. shigelloides have been discovered:
cholera-like toxin (Gardner et al., 1987), thermostable and
thermolabile toxins (Matthews et al., 1988; Sears and Kaper,
1996), β-hemolysin (Janda and Abbott, 1993), and cytotoxin
complex (Okawa et al., 2004). The cytotoxin having the
potential role in enteropathogenicity of P. shigelloides is a
heat-stable complex of lipopolysaccharide (LPS) and anticholera toxin-reactive proteins—LPS–ACRP complex
(Okawa et al., 2004).
LPS (endotoxin), built of O-specific chain, core oligosaccharide, and lipid A, is the main constituent of the outer
membrane of Gram-negative bacteria. LPS plays an important role for the effective barrier properties of the outer
membrane (Nikaido and Nakae, 1979). It constitutes a
‘pathogen-associated molecular pattern’ for host infection
by Gram-negative bacteria and is one of the most powerful
natural activators of the innate immune system. LPS plays
a key role during severe Gram-negative infections, sepsis,
and septic shock. Lipid A is an immunomodulatory center
© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
538
Plesiomonas shigelloides O54 lipid A–inner core region
of endotoxin and is recognized by different classes of
receptors, including Toll-like receptors (Alexander and
Zähringer, 2002).
Except for the detailed serotyping schemes of P. shigelloides
(Aldova, 1992), the LPSs of these bacteria have not been
extensively investigated (Linnerborg et al., 1995). Recently,
we have presented the structure of the core oligosaccharide
and the biological repeating unit of the O-antigen of P.
shigelloides O54 LPS (strain CNCTC 113/92) (Czaja et al.,
2000; Niedziela et al., 2002). The characteristic feature of
the core decasaccharide isolated by mild acid hydrolysis of
P. shigelloides O54 LPS is the lack of phosphate groups and
the presence of GalA (Niedziela et al., 2002). The intact
Kdo (3-deoxy-D-manno-oct-2-ulosonic acid)-containing
core region, the lipid A structure, and the linkage between
them have not been investigated to date. Due to the role of
LPS in P. shigelloides pathogenesis during intestinal and
extraintestinal infections, it was important to determine the
structure of its lipid A, completing the structural investigation of P. shigelloides O54 LPS.
The structural analysis of the intact Kdo-containing core
region and its linkage with the lipid A requires de-N,Oacylation of LPS. The products formed by deacylation
of smooth-type LPS, containing 4-substituted uronic
acids, are usually complex mixtures of polysaccharides and
oligosaccharides resulting from the β-elimination of the
substituent at O-4 (Holst, 2000). It has been shown that
sialic acids, Kdo residues, and Kdo-containing oligosaccharides derived from LPS form complexes with serotonin
(Sturgeon and Sturgeon, 1982; Rybka and Gamian, 2002).
Previously, the immobilized serotonin was used to isolate
free N-acetylneuraminic acid and oligosaccharides,
polysaccharides, and glycoproteins, containing this sugar
(Sturgeon and Sturgeon, 1982). We applied this technique
for the one-step isolation of Kdo-containing oligosaccharides from the de-N,O-acylation products of LPS by affinity chromatography with immobilized serotonin.
The structure of lipid A–inner core region of P. shigelloides
O54 LPS was investigated by chemical analysis combined with
NMR spectroscopy and MALDI-TOF mass spectrometry,
using positive and negative ion modes. Thus this study completes the structural analysis of the P. shigelloides O54
lipopolysaccharide and reports on its biological in vivo and
in vitro activity.
Results
Isolation and NMR analysis of intact Kdo-containing core
region of LPS
LPS of P. shigelloides O54 (strain CNCTC 113/92) was isolated by phenol/water extraction and purified as previously
reported (Petersson et al., 1997) (yield, 2% of dry bacterial
mass).
LPS was de-N,O-acylated by hydrazinolysis and KOH
treatment (Holst, 2000). As shown by NMR analysis, the
deacylation of P. shigelloides O54 LPS yielded a mixture of
incomplete core oligosaccharide linked to the lipid A carbohydrate backbone and fragments of core oligosaccharide
substituted with different number of O-specific repeats
(Figure 1A). The heterogeneity of the de-N,O-acylation
products of LPS was a result of the β-elimination of the
substituent at O-4 of α-D-GalpA, a constituent of the core
oligosaccharide of P. shigelloides O54 LPS (Niedziela et al.,
2002). The mixture (8 mg) was fractionated by affinity
chromatography on a serotonin–Sepharose 4B column.
Five fractions were eluted by a discontinuous ammonium
acetate gradient and checked by NMR spectroscopy. The
fraction eluted with 0.05 M ammonium acetate (3 mg) contained a decasaccharide built of incomplete core oligosaccharide with intact Kdo-containing region and lipid A
carbohydrate backbone (Figure 1B). The sugar residues of
the decasaccharide are notified by capital letters as shown
in the structure (Figure 4). These letters refer to the corresponding residues through the entire text, tables, and figures, and the labeling is consistent with that used previously
(Niedziela et al., 2002).
The affinity-purified decasaccharide was further investigated, and monosaccharides were identified on the basis of
characteristic signals of deoxy protons of Kdo (residues A
and Z), two signals of nitrogen-bearing carbons from aminosugars (residues X and Y), the characteristic resonance of
H-4, C-4 (δ 5.79 and 109.1 ppm) belonging to the β-elimination
product, 4-deoxy-β-L-threo-hex-4-enopyranosyl (residue
G), and the absence of signals of the O-specific chain constituents (Czaja et al., 2000) in the NMR spectra (Figures 1
and 2B and C). Chemical shifts of the identified residues
(Table I) were compared with previously published NMR
data for respective monosaccharides (Jansson et al., 1989).
Chemical shift values of the →2,3,7)-L-α-D-Hepp (D),
→3,4)-L-α-D-Hepp (B), terminal L-α-D-Hepp (F), terminal
β-D-Glcp (E), and terminal β-D-Galp (C) were in agreement
with those previously described as components of core
oligosaccharide obtained by mild acid hydrolysis of
P. shigelloides O54 LPS (Niedziela et al., 2002).
Residue G with the H-1/C-1 signals at δ 5.50/101.3 ppm,
JH-1,H-2 < 2 Hz was assigned as the terminal 4-deoxy-β-Lthreo-hex-4-enopyranosyl (α-ΔGalpA), on the basis of the
characteristic four-proton-spin system with the high chemical shift of the H-4 signal (δ 5.79 ppm) and the C-5 signal
(δ 146.2 ppm).
Residue A was identified as the →4,5)-α-Kdo-(2→ on the
basis of characteristic deoxy proton signals at δ 1.88 ppm
(H-3ax) and δ 2.12 ppm (H-3eq) (Figure 3B), and a relatively high chemical shifts of the C-4 (δ 71.9 ppm) and C-5
(δ 70.4 ppm) signals.
Residue Z was identified as the terminal α-Kdo-(2→
on the basis of similar chemical shifts as those reported
(Birnbaum et al., 1987; Müller-Loennies et al., 2002)
(Figure 3B).
Residue X with the H-1/C-1 signals at δ 5.67/92.7 ppm,
JH-1,H-2 < 3 Hz was assigned as the →6)-α-D-GlcpN1P
based on the typical chemical shifts of the H-1, C-1, and
C-2 (δ 55.8 ppm) signals, the high chemical shift of the C-6
signal (δ 71.2 ppm), and the large vicinal couplings between
H-2, H-3, H-4, and H-5 (JH-2,H-3, JH-3,H-4, JH-4,H-5 ∼10 Hz).
1
H, 31P-correlation experiment showed the connectivity
between the phosphate monoester peak at δ 3.45 ppm and
H-1 (δ 5.67 ppm) and H-2 (δ 3.42 ppm) of residue X (Figure
2E and F).
Residue Y with the H-1/C-1 signals at δ 4.81/100.7 ppm,
1
JH-1,H-2 ∼8 Hz was assigned as the →6)-β-D-GlcpN4P-(1→
539
J. Lukasiewicz et al.
Fig. 1. 1H-NMR spectra of the de-N,O-acylated Plesiomonas shigelloides O54 LPS (strain CNCTC 113/92) (A) and affinity-purified decasaccharide
(B). * denotes CH3 signals of the α-L-Rhap characteristic for the O-specific polysaccharide of P. shigelloides O54 LPS. The capital letters refer to
carbohydrate residues as shown on the structure in Figure 4, and the numbers refer to protons in the respective residue.
based on the low chemical shift of the C-2 signal (δ 57.0
ppm) and the large vicinal couplings between all ring protons. The chemical shift of the C-6 signal (δ 63.8 ppm) indicates the substitution by a Kdo residue (Bock et al., 1992;
Oertelt et al., 2001). 1H, 31P-correlation experiment revealed
the connectivity between the phosphate monoester peak at
δ 1.77 ppm and H-4 (δ 3.80 ppm) of residue Y (Figure 2F),
indicating the substitution of →6)-β-D-GlcpN-(1→ at O-4
by the phosphate group. The chemical shift values of residues X and Y were similar to these reported (Vinogradov and
Bock, 1999).
Each disaccharide element in the decasaccharide was
identified by HMBC (Figure 2A and D) and NOESY
experiments (Table II), providing the sequence of sugar
monomers in the decasaccharide. The HMBC spectrum
showed cross-peaks between the anomeric proton and the
carbon at the linkage position (Figure 2A) and between the
anomeric carbon and the proton at the linkage position
(Figure 2D), which confirmed the structure (Figure 4). The
inter-residue NOEs were found between H-1 of E and H-2
of D, H-1 of G and H-3 of D, H-1 of F and H-7a,b of D, H1 of D and H-3 of B, H-1 of B and H-5 of A, H-1 of C and
H-4 of B, and H-1 of Y and H-6a,b of X (Table II). The
inter-residue NOE signals between H-6 of residue Z and H3ax (weak signal) and H-3eq (strong signal) of residue A
further supported the disaccharide element: α-Kdo-(2→4)α-Kdo (Birnbaum et al., 1987) (Figure 3A). These results
suggest the following structure of the decasaccharide
540
isolated from the LPS of P. shigelloides O54 containing the
intact Kdo and lipid A carbohydrate backbone regions
(Figure 4).
Isolation and compositional analysis of lipid A
The lipid A fraction was isolated by mild acid hydrolysis of
P. shigelloides O54 LPS. Qualitative analysis of fatty acids
was done separately for amide- and ester-bound fatty acids,
using chemical analysis followed by GC-MS. The (R)-3hydroxytetradecanoic acid (14:0(3-OH)) was identified as
amide-linked fatty acid. Dodecanoic acid (12:0), (R)-3hydroxydodecanoic acid (12:0(3-OH)), tetradecanoic acid
(14:0), and trace amount of hexadecenoic acid (16:1) were
detected as ester-bound fatty acids. Identification of the
methoxy derivative—methyl ester of 3-methoxy-dodecanoic
acid, among fatty acid methyl esters obtained by transesterification with sodium methanolate—showed also the presence
of 12:0(3-OH), which was substituted by ‘secondary’ fatty
acid in the native lipid A. The absolute configuration of
GlcN residues in lipid A was determined as D on the deacylated, defosforylated lipid A fraction, using (R)-2-butanol as
previously described (Gerwig et al., 1978, 1979).
MS analysis of lipid A
P. shigelloides O54 lipid A isolated by mild acid hydrolysis
and native LPS were analyzed using MALDI-TOF MS
(Figure 5; Table III).
Plesiomonas shigelloides O54 lipid A–inner core region
Fig. 2. Selected 1JH,C, 3JH,C, and 3JH,P connectivities in 600 MHz HSQC-DEPT (B and C), HMBC (A and D), and 1H,31P HSQC (E and F) spectra of the
decasaccharide isolated from LPS of Plesiomonas shigelloides O54 (strain CNCTC 113/92). The spectra were recorded at 30°C. The capital letters refer to
carbohydrate residues as shown on the structure in Figure 4, and the numbers refer to protons and carbons in the respective residue.
The negative ion mode MALDI-TOF mass spectrum
obtained for LPS contained three regions of ions (lipid A
substituted with core oligosaccharide and one repeating
unit of O-specific chain, lipid A substituted with core oligosaccharide and lipid A). Main ions, present in the lower
mass range of the spectrum, corresponded to P. shigelloides
O54 lipid A (Figure 5A) and arose from fragmentation
between polysaccharide and lipid A due to the acid lability
of ketosidic bond in matrix solution. Observed ions
reflected heterogeneity of the lipid A in native LPS. On the
basis of the chemical composition, the most abundant ion
at m/z 1741.8 [M–H]− could be attributed to the hexaacylated lipid A, bisphosphorylated at O-1 and O-4′ and built
up of two amide-bound 14:0(3-OH) and four ester-bound
fatty acids: two 12:0(3-OH), one 12:0, and one 14:0, substituting the glucosamine backbone (Figure 5A, inset structure; Table III). Minor forms of lipid A were represented by
peaks at m/z 1714.1 and m/z 1767.0. Peak at m/z 1714.1
originated from the molecule possessing a shorter (–28 Da)
fatty acid. Peak at m/z 1767.0 was attributed to the presence
in lipid A of a longer unsaturated (+26 Da) fatty acid (16:1)
identified by chemical analysis. The ion at m/z 1864.4 corresponded to the lipid A molecule (major form) which was
hexaacylated, bisphosphorylated, and additionally substituted with a phosphoethanolamine (PEtn). The ion at m/z
1889.7 represented minor form of P. shigelloides O54 lipid
A, which was hexaacylated, bisphosphorylated, and substituted with PEtn.
The pattern of ions in negative ion MALDI-TOF mass
spectrum of the free lipid A (Figure 5B) was more complex
than that observed in the lower mass range of spectrum
obtained for the native LPS (Figure 5A). Ions at m/z 1741.8
and 1714.1 corresponding to bisphosphorylated, hexaacylated forms of P. shigelloides O54 lipid A were also identified. The presence of peaks with lower m/z values (Table III)
can be explained by partial degradation of lipid A during
541
J. Lukasiewicz et al.
Table I. 1H, 13C NMR chemical shifts (δ) of the decasaccharide isolated from Plesiomonas shigelloides O54 LPS (strain CNCTC 113/92)
Chemical shifts [ppm]
Residue
A
B
H-1/C-1
→4,5)-α-Kdo-(2→
JC-1,H-1 175 Hz
C
D
C-1,H-1 164 Hz
C-1,H-1
178 Hz
JC-1,H-1 163 Hz
C-1,H-1
174 Hz
JC-1,H-1 176 Hz
Y
C-1,H-1
174 Hz
C-1,H-1 166 Hz
α-Kdo-(2→
3.49
72.5
5.30
4.19
101.4
80.2
4.51
3.17
104.4
74.8
4.89
3.99
102.9
71.9
5.50
101.3
3.79
72.0
5.67
92.7
→6)-β-D-GlcpN4P-(1→
1J
Z
4.43
→6-α-D-GlcpN1P
1J
4.05
72.2
104.3
α-ΔGalpAb-(1→
1
X
5.25
100.2
L-α-D-Hepp-(1→
1J
G
35.9
β-D-Glcp-(1→
1
F
(1.88, 2.12)
101.0
→2,3,7)-L-α-D-Hepp-(1→
1J
E
–
ND
β-D-Galp-(1→
1J
H-3/C-3 (H-3ax/eq)
–
→3,4)-L-α-D-Hepp-(1→
1
H-2/C-2
3.42
55.8
4.81
3.08
100.7
–
57.0
–
4.11
75.7
3.63
73.5
4.23
77.9
3.46
76.4
3.83
71.0
4.41
67.3
3.89
70.8
3.86
73.3
(1.75, 2.13)
36.1
H-4/C-4
H-5/C-5
4.11
4.23
71.9
70.4
4.23
4.19
74.9
73.6
3.87
3.62
70.0
76.5
4.03
3.64
67.9
73.9
3.41
3.54
70.7
76.7
3.84
3.63
67.6
5.79
109.1
3.60
71.0
3.80
75.7
4.01
67.9
73.1
H-6a,b/C-6
H-7a,b/C-7
3.67
73.4
3.83
72.0
4.15
70.2
3.66, 3.71
H-8a,b/C-8
3.77, 3.95
3.59, 3.87
65.2
–
65.5
–
–
62.5
4.20
69.2
3.67, 3.74
72.5
3.80, 3.75
62.1
3.97
71.6
3.65, 3.71a
64.4
–
146.2
4.10
74.0
3.72
75.2
4.04
67.7
3.76, 4.27
71.2
3.42, 3.65
63.8
3.60
73.6
4.00
70.5
3.70a, 3.94
64.5
ax, axial position; eq, equatorial position; ND, not determined. Spectra were recorded for 2H2O solution at 30°C. Acetone (δH 2.225 and δC 31.05 ppm)
was used as internal reference. The 1JC-1,H-1 values obtained from a non-decoupled HSQC experiment, confirmed the α-pyranosyl configuration for
residues B, D, F, G, and X, and β-pyranosyl configuration for residues C, E, and Y.
a
The signals were not resolved.
b
4-deoxy-β-L-threo-hex-4-enopyranosyl.
Fig. 3. The fragments of NOESY (A) and HSQC-DEPT (B) spectra of the
affinity-purified decasaccharide isolated from Plesiomonas shigelloides
O54 LPS (strain CNCTC 113/92). The cross-peaks are labeled as
explained in the legend to Figures 1 and 2.
work-up and lability of its acyl side chains and the phosphate
group at O-1 during the acid hydrolysis (Wang and Cole,
1996). The ion at m/z 1661.4 corresponded to the monophosphorylated, hexaacylated lipid A molecule [M–P–H]−. Analysis of the complex pattern of this spectrum revealed several
542
species, differing by the number of fatty acids. The peaks at
lower m/z ratio represented monophosphorylated species
lacking the 12:0 (m/z 1558.9) and the 12:0(3-O-12:0) (m/z
1280.3) at O-3′. The peak at m/z 682.8 arose from deprotonation, followed by simple cleavage of the glycosidic bond
within the disaccharide backbone (Y1− ion), according to
Domon and Costello nomenclature (Domon and Costello,
1988), and represented the proximal GlcN residue phosphorylated at O-1 and acylated by 14:0(3-OH) at N-2 and
12:0(3-OH) at O-3 (Figure 5B, inset structure).
The substitution at O-4′ of distal GlcN (Y) by PEtn was
deduced from the mass difference between ions at m/z
1784.9 and 1661.4 (Δ 123 Da). The ion at m/z 1661.4 corresponded to the lipid A monophosphorylated at O-4′ and
devoid of phosphate group at O-1, which was lost during
mild acid hydrolysis of the LPS (Table III). The presence of
both α and β anomers of GlcN (residue X) was observed in
the NMR spectra of the isolated lipid A (data not shown),
confirming the lability of the phosphate group at O-1.
The structural information obtained from the negative
ion mode MALDI-TOF MS was further supported by the
positive ion mode (Figure 5C). MALDI-TOF MS analysis
performed at high laser power settings in positive ion mode
revealed the presence of sodium and potassium adducts of
the monophosphorylated lipid A (data not shown) and
Plesiomonas shigelloides O54 lipid A–inner core region
Table II. Selected inter-residue NOE and 3JH,C connectivities from the anomeric atoms of the fraction of the affinity-purified decasaccharide isolated
from Plesiomonas shigelloides O54 LPS (strain CNCTC 113/92)
Connectivities to
Residue
Atom H (ppm)
δC
δH
Inter-residue
Atom/residue
H-1/C-1
A
→4,5)-α-Kdo-(2→
B
→3,4)-L-α-D-Hepp-(1→
C
β-D-Galp-(1→
1.88 (H-3ax)
3.60*
H-6 of Z
2.12 (H-3eq)
3.60*
H-6 of Z
5.25
4.23
H-5 of A
74.9
4.23
C-4, H-4 of B
75.7
4.11
C-3, H-3 of B
80.2
4.19
C-2, H-2 of D
72.5
3.67*; 3.74
C-7, H-7a,b of D
77.9
4.23
C-3, H-3 of D
71.2
3.76; 4.27
C-6, H-6a,b of X
100.2
4.43
104.3
D
→2,3,7)-L-α-D-Hepp-(1→
E
β-D-Glcp-(1→
5.30
101.4
4.51
104.4
F
L-α-D-Hepp-(1→
G
α-ΔGalpAa-(1→
4.89
102.9
5.50
101.3
Y
→6)-β-D-GlcpN4P-(1→
4.81
100.7
ax, axial position; eq, equatorial position. The values marked with * represent NOE connectivities only.
4-deoxy-β-L-threo-hex-4-enopyranosyl.
a
Fig. 4. Structure of the affinity-purified decasaccharide of Plesiomonas shigelloides O54 LPS (strain CNCTC 113/92). The capital letters refer to carbohydrate residues. α-ΔGalpA, 4-deoxy-β-L-threo-hex-4-enopyranosyl.
oxonium ions (B1+) (Figure 5C, inset structures). The B1+
ions (m/z 1059.4, 831.6), arising from the cleavage of the
glycosidic linkage between the two GlcN residues, were
important for structure elucidation (Figure 5C). The oxonium ion at m/z 1059.4 corresponded to the phosphorylated, non-reducing tetraacylated GlcN (Y). On the basis of
chemical analyses of fatty acids, this GlcN was substituted
by 14:0(3-OH) at N-2′ and 12:0(3-OH) at O-3′, which could
be further substituted by ‘secondary’ fatty acids: 14:0 and
12:0 (Figure 5C, inset structures). The ion at m/z 831.6 represented phosphorylated, non-reducing triacylated GlcN
(Y) devoid of the 14:0 fatty acid (Δ 228 Da). The MS analyses of lipid A previously described by Sforza et al. (2004)
demonstrated that the fatty acid at N-2′ was preferentially
eliminated in the positive ion mode MS in comparison to
the negative ion mode experiments. Thus, the presence of
the ion at m/z 831.6 confirmed that 14:0 residue substituted the
amide-bound 14:0(3-OH) at N-2′ of lipid A. This provided the
evidence that the distal GlcN residue was substituted with
14:0(3-O-14:0) and 12:0(3-O-12:0) at positions N-2′ and O3′, respectively. The B1+ ion at m/z 1031.3 corresponded to
one of the minor forms of the lipid A containing a shorter
fatty acid (–28 Da) in comparison with the major form.
Such heterogeneity was not observed in the region of the ion
at m/z 831.6. The mass difference between ions at m/z 1031.3
and 831.6 suggested that in the minor form of lipid A the
14:0(3-OH) at N-2′ was substituted with the secondary 12:0
instead of the 14:0 (major form of lipid A). On the basis of
data obtained by chemical analysis and MALDI-TOF MS
investigation, we conclude that the prevailing structure of
543
J. Lukasiewicz et al.
Table III. Interpretation of ions (m/z) corresponding to the lipid A molecules,
observed in negative ion mode MALDI-TOF MS. Mass spectra were
acquired for the native LPS of Plesiomonas shigelloides O54 and lipid A
isolated by mild acid hydrolysis of LPS.
Fig. 5. (A) Selected low-mass region of the negative ion mode MALDITOF mass spectrum of the native Plesiomonas shigelloides O54 (strain
CNCTC 113/92) LPS. (B) Negative ion mode and (C) positive ion mode
MALDI-TOF mass spectra of lipid A isolated by mild acid hydrolysis of
P. shigelloides O54 LPS. Symbols * and # denote ions originating from
minor forms of lipid A substituted with shorter (−28 Da) and longer,
unsaturated (+26 Da) fatty acid, respectively.
Symbol M represents lipid A molecule consisting of the β-D-GlcpN4P(1→6)-α-D-GlcpN1P substituted with 14:0(3-OH), 12:0(3-OH),
14:0(3-O-14:0) and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2′ and O-3′,
respectively; Etn, ethanolamine.
(3-O-12:0) fatty acids at N-2, O-3, N-2′, and O-3′, respectively (Figure 5A, inset structure).
The complete structure of the P. shigelloides O54 LPS
P. shigelloides O54 lipid A is built of the disaccharide β-DGlcpN-(1→6)-α-D-GlcpN phosphorylated at O-1 and at O4′ and 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0
544
The data of structural analysis of lipid A–inner core region
were combined with those published for P. shigelloides O54
LPS core oligosaccharide (Niedziela et al., 2002) and biological repeating unit of the O-antigen (Czaja et al., 2000).
The complete structure of LPS isolated from P. shigelloides
O54 is presented here (Figure 6).
Plesiomonas shigelloides O54 lipid A–inner core region
Fig. 6. Complete structure of Plesiomonas shigelloides O54 lipopolysaccharide (strain CNCTC 113/92). D-β-D-Hepp, D-glycero-β-D-manno-Hepp; 6d-β-DHepp2OAc, 6-deoxy-β-D-manno-Hepp2OAc.
Biological activity
The cytokines (TNF-α and IL-6) and NO production by
J774A.1 cells upon stimulation with LPS of P. shigelloides
O54 and Escherichia coli O55 were compared. The LPS isolated from both bacteria demonstrated strong and dosedepended stimulating activities on the J774A.1 cells (Figure 7).
LPS of P. shigelloides O54 showed substantially stronger
effect in vitro on the TNF-α production for the lowest dose
(10 ng/ml) of LPS (p < 0.05). The differences in IL-6 production by J774A.1 cells upon stimulation with E. coli O55
and P. shigelloides O54 LPS were not statistically significant. The measurement of the total nitrite concentration
demonstrated that J774A.1 cells responded similar to stimulation with both lipopolysaccharides, with a stronger
effect for 1-μg dose of P. shigelloides O54 LPS (p < 0.05).
Endotoxicity of P. shigelloides O54 LPS in vivo was evaluated in actinomycin D-sensitized mice and was compared
to that of LPS preparations of E. coli O55 and E. coli O1.
The lethal effect of LPS administration was expressed as
LD50 and was 10 times stronger in the case of P. shigelloides O54 LPS in comparison with the LPS of E. coli O55
and O1. The LD50 values for LPS of P. shigelloides O54,
E. coli O55, and E. coli O1 were 0.3, 3, and 3 ng per mouse,
respectively.
Discussion
The pathogenicity of P. shigelloides is not yet fully
understood. Endotoxin, the main surface antigen of Gramnegative bacteria, was found as a constituent of cytotoxin
complex with anti-cholera toxin-reactive proteins exerted
by P. shigelloides, thus being a potential virulence factor
(Okawa et al., 2004).
LPSs of P. shigelloides are poorly characterized, and
until now, the structure of the P. shigelloides lipid A has not
been determined. This prompted us to resolve the structure
of entire LPS molecule by combining the data of structural
analysis of the lipid A and its linkage with intact Kdocontaining core region to the previously published structures
of the core oligosaccharide and O-specific biological repeating unit of P. shigelloides O54 LPS (strain CNCTC 113/92)
(Czaja et al., 2000; Niedziela et al., 2002). These data
showed that LPS of P. shigelloides O54 has the structure
shown in Figure 6.
The typical core oligosaccharide of LPS contains a Kdo
residue, which forms the linkage with lipid A via an acidlabile ketosidic bond. The presence of acid-labile constituents, such as diphosphate esters and one or two additional
Kdo residues in the inner and outer core of LPS, was also
reported (Holst, 2002). Resolving the complete structure of
LPS requires examination of acid-labile Kdo-containing
inner core region and its linkage with the lipid A carbohydrate backbone. The de-N,O-acylation of LPS, leading to
the isolation of the intact Kdo-containing inner core region,
is typically achieved using anhydrous hydrazine, followed
by aqueous 4 M KOH treatment, to cleave ester- and amidelinked fatty acids with subsequent isolation of phosphorylated oligosaccharides by high-performance anion-exchange
chromatography. This procedure, which is complementary
to mild acid hydrolysis, is necessary to explore the intact
Kdo-containing core region of LPS (Holst, 2000). Our preliminary structural studies of several P. shigelloides
lipopolysaccharides (unpublished data) have shown a lack
of phosphate groups and the presence of 4-substituted
GalA as characteristic features of the core oligosaccharide
of these bacteria. Thus NMR analysis of the intact Kdocontaining core region had to be preceded by isolation of
545
J. Lukasiewicz et al.
2000
TNFα
[U/ml]
1500
1000
500
0
2000
IL-6
[U/ml]
1500
1000
500
0
50
NO
[μM]
40
30
20
10
0
1000
100
dose of LPS [ng/ml]
10
Fig. 7. TNF-α, IL-6, and NO production by J774A.1 cells stimulated with
the LPS of Plesiomonas shigelloides O54 (■), E. coli O55 (❏) (doses 1 μg/
ml, 100 ng/ml, and 10 ng/ml). Data are expressed as means ± SD of three
replicates.
the appropriate fragment from the mixture of polysaccharides and oligosaccharides resulting from the β-elimination
of a substituent at O-4 of uronic acid during deacylation of
LPS. Here we demonstrated that the relevant oligosaccharide could be separated by affinity chromatography. The
immobilized serotonin, which forms complexes with Kdo,
was used for the one-step isolation of the decasaccharide
possessing the intact Kdo-containing inner core region and
the lipid A carbohydrate backbone.
The lipid A of P. shigelloides O54 is a novel structure.
Primary-linked fatty acids are identical to those present in
Neisseria spp. lipid A (Kulshin et al., 1992), but contrary to
Neisseria spp., they are asymmetrically distributed (2 + 4).
Thus P. shigelloides O54 lipid A belongs to the group of
E. coli-type lipid A (Alexander and Zähringer, 2002). On
the basis of the phosphorylation of GlcN disaccharide at O-1
and O-4′ and the length of asymmetrically distributed acyl
546
groups limited to 12 or 14 carbons, the high cytokineinducing activity of P. shigelloides O54 LPS was expected
and confirmed by the experimental data.
Among components of LPS (O-specific polysaccharide,
core oligosaccharide, and lipid A), lipid A is known as the
immunostimulatory center. It determines endotoxicity of
LPS, which is modulated by core oligosaccharide and Ospecific polysaccharide (Alexander and Zähringer, 2002).
In contrast to the cytokine induction assay in vitro, there
was significant difference between P. shigelloides O54 LPS
and both E. coli O55 and E. coli O1 LPS in their lethal
effect evaluated in actinomycin D-sensitized mice. The
compared lipopolysaccharides were smooth, that is, contained the core oligosaccharide substituted with O-specific
polysaccharide and lipid A which allowed the full activity
of those endotoxins (asymmetric, hexaacylated with fatty
acids consisting of 12–14 carbons and bisphosphorylated
lipid A [Alexander and Zähringer, 2002]). The difference
between them in ‘general structure’ is the lack of phosphoryl groups and the presence of GalA in the core oligosaccharide for P. shigelloides O54. This difference did not
affect the activity of P. shigelloides O54 endotoxin in the
in vitro cytokine (TNFα and IL-6) and NO induction assays.
As suggested by Mueller et al., (2004), the active units of
endotoxins, amphiphilic molecules, are their aggregates.
Thus the LPSs of E. coli and P. shigelloides O54 form
aggregates which are biologically active and exhibit similar
effects in vitro. The lethal effect of such aggregates formed
by P. shigelloides O54 LPS, administered in vivo, was 10
times stronger in comparison with that of E. coli O55 and
O1 LPS. The observed discrepancy could originate from
differences in the complexity of in vivo and in vitro experiments. Injection of LPS in vivo induces the production of
various endogenous inflammatory mediators by effector
cells and triggers the systemic regulatory mechanisms
important to prevent endotoxin-induced pathogenic proinflammatory response and the onset of sepsis (Rietschel
et al., 1996). Moreover, endotoxin clearance and neutralization mechanisms involve interaction between LPS and
serum proteins (BPI and LBP) (Weiss, 2003; Hamann et al.,
2005), antimicrobial peptides (Levy, 2000), and plasma
lipoproteins (Brandenburg et al., 2002). Studies on the
interaction between LPS and the integral outer membrane
protein FhuA identified a conserved structural motif
responsible for LPS recognition among LPS-binding proteins
such as BPI, lactoferrin, lysozyme, and Limulus anti-LPS
factor (Ferguson et al., 2000). In the case of FhuA–LPS
and SMAP-29-LPS complexes (Tack et al., 2002), positively charged groups present in proteins interact with
phosphorylated carbohydrate backbone of lipid A, Hep,
Kdo, and negatively charged phosphate groups of the inner
core oligosaccharide.
As most of the biophysical studies on the interactions of LPS
with protein/peptide (Ferguson et al., 2000; Gutsmann et al.,
2000; Gutsmann et al., 2001) and lipoprotein (Brandenburg
et al., 2002) were limited to the isolated lipid A or R-forms
of E. coli LPS, the influence of non-typical structures of the
core oligosaccharide on these interactions cannot be excluded.
The presence of GalA and absence of phosphate residues in
the core oligosaccharide for S-type P. shigelloides O54 LPS
could play role in dissagregation, less effective neutralization,
Plesiomonas shigelloides O54 lipid A–inner core region
and clearance mechanisms resulting in higher toxicity of
this LPS in vivo.
Materials and Methods
Bacteria
P. shigelloides serovar O54:H2 (strain CNCTC 113/92) was
obtained from the Collection of the National Institute of
Public Health, Prague, Czech Republic. Bacteria were
grown and harvested as described previously (Petersson
et al., 1997). E. coli O1 was obtained from the Polish
Collection of Microorganisms (PCM), Institute of Immunology and Experimental Therapy, Wroclaw, Poland.
Cell lines
The mouse macrophage-like cell line J774A.1 was obtained
from the German Collection of Microorganisms and Cell
Cultures (Braunschweig, Germany). TNFα-sensitive WEHI
164.13 mouse fibrosarcoma cells were donated by Prof. M.
Zimecki (Institute of Immunology and Experimental Therapy) and cultured as previously described (Lugowski et al.,
1996). The mouse IL-6-dependent B-cell hybridoma 7TD1
cell line, provided by Prof. S. Szymaniec (Institute of Immunology and Experimental Therapy), was cultured using
Iscove’s medium containing 10% fetal calf serum, supplemented with 1.5 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 0.1 mM hypoxanthine, and 0.016 mM thymidine.
Animals
The BALB/c mice (females, 6–8 weeks of age) were housed
at the animal facility of the Institute of Immunology and
Experimental Therapy. The experiments were approved by
the Local Ethical Commission for Animal Experimentation.
ammonium acetate and equilibrated with water. DeN,O-acylated LPS fraction (8 mg) was dissolved in water
and loaded on the column. Fractions (40 ml) were eluted
by water and a discontinuous ammonium acetate gradient (0.025 M, 0.05 M, 0.1 M, 0.25 M, and 1 M), freezedried, and checked by NMR spectroscopy. The yields of
the fractions eluted were 1.5 mg (water), 2.0 mg (0.025 M),
3.0 mg (0.05 M), 0.1 mg (0.1 M), 0.2 mg (0.25 M), and
0.8 mg (1 M).
Analytical procedures
To determine the absolute configuration of GlcN residues
in the lipid A carbohydrate backbone, lipid A (5 mg) was
hydrolyzed using 4 M HCl (12 h, 100°C). The solvent was
evaporated, and the sample was subjected to chloroform/
water extraction (1:2, v/v). The water-soluble part was lyophilized and then dephosphorylated with aqueous 48% HF
(72 h, 4°C). The absolute configuration was determined
as previously described (Gerwig et al., 1978, 1979) using
(−)-2-butanol for the formation of 2-butyl glycosides. The
trimethylsilylated butyl glycosides were then identified by
comparison with the standards produced from D-GlcN (Sigma,
St. Louis, MO) and (−)-2-butanol and (+)-2-butanol
(Fluka, Buchs, Switzerland) on GC-MS. Amide- and esterbound fatty acids were analyzed separately (Wollenweber
and Rietschel, 1990) by GC-MS performed with a HewlettPackard 5971A system, using an HP-1 fused-silica capillary
column (0.2 mm × 12 m), the temperature program 100–
270°C at 8°C/min. 3-Hydroxy fatty acids were converted to
(S)-3-methoxy-phenylethylamide derivatives and analyzed by
GC-MS using the HP-5 column (0.25 mm × 30 m) with a
temperature program, 150–270°C at 8°C/min, to determine
their absolute configurations (Gradowska and Larsson, 1994).
MALDI-TOF MS analysis
LPS (200 mg) was de-N,O-acylated by mild hydrazinolysis
followed by 4 M KOH treatment (Holst, 2000), and the
reaction mixture was neutralized with HClO3. The precipitated salt was removed by centrifugation for 30 min at 4°C
(4000 × g), and the supernatant was further desalted using a
Bio-Gel P-2 column (28 mg).
MALDI-TOF mass spectrometry, in positive and negative
ion modes, was run on a Kratos Kompact-SEQ instrument. LPS was suspended in water (10 mg/ml) and desodiated with Dowex 50 W×8 (H+). 2,5-Dihydroxybenzoic
acid (10 mg/ml in 0.01 M citric acid) was used as matrix
(Therisod et al., 2001). Norharmane—9H-Pyrido[3,4b]indole (1% in acetonitrile:water; 1:1, v/v)—was used as
matrix for the analysis of lipid A in linear, negative ion
mode. To obtain positive ion mass spectra of lipid A, sample was dissolved in CHCl3/isopropyl alcohol/water
(5:3:0.25, v/v/v) and desodiated as previously described
(Gudlavalleti and Forsberg, 2003). 2,4,6-Trihydroxyacetophenone (25 mg/ml in acetonitrile:water, 1:1, v/v) was
used as matrix. Lipid A was suspended in water and
extracted with chloroform:methanol (3:1, v/v) prior to all
MALDI-TOF MS analysis.
Purification of de-N,O-acylated LPS
NMR spectroscopy
The de-N,O-acylated LPS (8 mg) was purified using
affinity chromatography on a serotonin–Sepharose 4B
column. Briefly, Sepharose 4B was activated with BrCN
(Cuatrecasas, 1970), and serotonin (200 mg) was coupled
to the activated Sepharose 4B (10 ml) by incubating the
mixture in 0.1 M sodium carbonate (pH 9.0) for 24 h at
4°C. The column (1.5 × 10 cm) was washed with 1 M
All spectra were obtained for 2H2O solutions at 30°C on a
Bruker DRX 600 instrument, using acetone (δH 2.225 ppm
and δC 31.05 ppm) as internal reference and in the 31P NMR
experiments 80% phosphoric acid (δP 0.0 ppm) as external
reference. In the clean-TOCSY experiments, the mixing
times used were 30, 60, and 100 ms. The delay time in
HMBC was 60 ms, and the mixing time in NOESY was 200
Lipopolysaccharide and lipid A isolation
LPS was isolated from lyophilized bacteria by the phenol/
water extraction and purified as previously described
(Westphal and Jann, 1965). Lipid A was obtained from
LPS as a water-insoluble fraction by treatment with 1.5%
acetic acid (45 min, at 100°C) followed by centrifugation
(40,000 × g, 20 min).
De-N,O-acylation of LPS
547
J. Lukasiewicz et al.
ms. Data were acquired using standard Bruker software
and assigned with the SPARKY program (Goddard and
Kneller, 2001).
LD50 determination
Groups of mice (4 mice per LPS dose) were observed
throughout the quarantine period and experiments. BALB/
c mice (females, 6–8 weeks of age) were injected i.v. with a
sterile solution of increasing doses of LPS (0.1, 1, 10, and
100 ng/mouse) in PBS (125 μl), containing 0.02% actinomycin D. LD50 values were determined by the method of Reed
and Muench (Reed and Muench, 1938). The lethality endpoint was determined after 48 h.
TNF-a, IL-6, and NO release in J774A.1 cells
Incubation of J774A.1 cells with LPS was done as previously
described (Lukasiewicz et al., 2003). LPS of E. coli O55
(Sigma) and E. coli O1 (prepared and purified as previously
described [Petersson et al., 1997]) were used as standard enterobacterial endotoxins. The supernatants were collected to
determine TNF-α (after 4 h), IL-6 (after 24 h), and total concentration of nitrite and nitrate as stable metabolites of NO
(after 48 h) and stored at –75°C until determination. TNF-α
concentration was determined in the cytotoxic assay using
WEHI 164.13 bioassay (Espevik and Nissen-Meyer, 1986) as
described previously (Lugowski et al., 1996). Recombinant
mouse TNF-α (Pharmingen, San Jose, CA) was used as standard. IL-6 content was assayed using the mouse IL-6-dependent B-cell hybridoma 7TD1 cell line (Snick et al., 1986).
Briefly, 2–3 × 103 7TD1 cells/well of a 96-well flat-bottom
microtiter plate were cultured for 3 days with a series of culture supernatants dilutions in a final volume of 100 μl, in
humidified atmosphere at 37°C with 5% CO2 (Iscove’s
medium, 1.5 mM L-glutamine, 0.05 mM 2-mercaptoethanol,
0.1 mM hypoxanthine, 0.016 mM thymidine, and 10% fetal
bovine serum). Recombinant mouse IL-6 (R & D Systems,
Minneapolis, MN) was used as standard. Viability of the cells
was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] colorimetric assay (Mossman,
1983) with modification described previously (Hansen et al.,
1989). Amounts of the released cytokines were expressed in
U/ml, defined as the concentration of TNF-α reducing cell
survival by 50% (1U = 4 pg/ml) and concentration of IL-6
producing half-maximal growth of the cells (1U = 5.8 pg/ml).
Total nitrite concentration was determined using the Griess
reaction (Green et al., 1982; Schmidt, 1995) as described previously (Lukasiewicz et al., 2003). Assays were performed
using triplicate cultures. Data are expressed as means ± standard deviation, and significance of differences between them
was analyzed using a one-way analysis of variance.
Acknowledgments
Scholarship for J.L. from the Foundation for Polish Science
is acknowledged. Preliminary results of these studies were
presented at 11th European Carbohydrate Symposium,
Lisbon 2001. This work was supported by research grant 3
P04A 091 22 from The State Committee for Scientific
Research (KBN).
548
Conflict of Interest Statement
None declared.
Abbreviations
COSY, correlated spectroscopy; DEPT, distortionless
enhancement by polarization transfer; GC, gas chromatography; GlcN, glucosamine; L-α-D-Hep, L-glycero-αD-manno-heptose; HMBC, heteronuclear multiple bond
correlation; HSQC, heteronuclear single quantum coherence; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; IL-6;
interleukin-6; LPS, lipopolysaccharide; MALDI-TOF
MS, matrix-assisted laser desorption/ionization time-offlight mass spectrometry; NO, nitric oxide; NOESY,
nuclear Overhauser effect spectroscopy; P; phosphate;
TNFα, tumor necrosis factor alpha; TOCSY, total correlation spectroscopy.
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