Characterization of lipid A in Cronobacter sakazakii

Article abstract of DOI:10.1255/ejms.1074

We have characterized the structure of lipid A in the food-borne pathogen Cronobacter sakazakii BAA-894. Lipid A molecules were purified by mild acid hydrolysis, DEAE-cellulose anion exchange chromatography and preparative thin-layer chromatography. Its structure was analyzed by electrospray ionization/mass spectrometry. Two lipid A species have been found in C. sakazakii BAA-894. The majority has a structure of disaccharide of glucosamine, phosphorylated at the 1- and 4′-positions, and acylated with primary 3-hydroxymyristoyl chains at the 2-, 3-, 2′- and 3′-positions and secondary myristoyl residues at the 2′- and 3′-positions. The other contains a secondary lauroyl chain at the 2′-position in place of the myristoyl residue.

Full text of DOI:10.1255/ejms.1074

C. Zhang et al., Eur. J. Mass Spectrom. 16, 531–538 (2010)
531
received: 5 January 2010
n
revised: 27 March 2010 n Accepted: 28 March 2010 n Publication: 1 April 2010
EuroPEAN
JourNAL
of
MASS
SPEctroMEtry
Characterization of lipid A in Cronobacter
sakazakii
Chan Zhang, Yanyan Li, Guanjun Tao, Ye Li and Xiaoyuan Wang*
State Key Laboratory of food Science and technology, Jiangnan university, 1800 Lihu Avenue, Wuxi 214122, china.
E-mail: xiaoyuanwang@hotmail.com
We have characterized the structure of lipid A in the food-borne pathogen Cronobacter sakazakii BAA-894. Lipid A molecules were puri-
fied by mild acid hydrolysis, DEAE–cellulose anion exchange chromatography and preparative thin-layer chromatography. Its structure
was analyzed by electrospray ionization/mass spectrometry. Two lipid A species have been found in C. sakazakii BAA-894. The majority
has a structure of disaccharide of glucosamine, phosphorylated at the 1- and 4¢-positions, and acylated with primary 3-hydroxymyristoyl
chains at the 2-, 3-, 2¢- and 3¢-positions and secondary myristoyl residues at the 2¢- and 3¢-positions. The other contains a secondary
lauroyl chain at the 2¢-position in place of the myristoyl residue.
Keywords: lipopolysaccharide, lipid A, Cronobacter sakazakii, ESI/MS/MS
Introduction
Lipid A, also known as endotoxin, makes up the outer leaflet
of the outer membranes of most Gram-negative bacteria.1,2 It
is recognized by the tLr4/MD2 receptor of the innate immune
system,3,4 which triggers an inflammatory response accom-
panied by massive over-production of cytokines and leads to
Gram-negative septic shock and death.5
secondary myristoyl residues at the 2¢- and 3¢-positions. the
rest of the lipid A species contains a secondary lauroyl chain at
the 2¢-position in place of the myristoyl residue.
Materials and methods
Cronobacter sakazakii (C. sakazakii) is a Gram-negative path-
ogen associated with contaminated milk formula.6–8 It has adhe-
sive capacities to a number of in vitro cell lines9 and can infect
infants and cause necrotizing enterocolitis, severe meningitis
and sepsis.10,11 C. sakazakii can form biofilms to protect itself in a
thermal and desiccative environment.12 Lipid A from C. sakazakii
might play a significant role in bacterial infection.13 However, little
is known about the chemical structure of lipid A in C. sakazakii.
Materials and reagents
Glass-backed silica gel 60 thin-layer chromatography (tLc)
plates were from yantai Jiangyou Silica Development company.
chloroform, ammonium acetate, sodium acetate, pyridine,
methanol and 88% formic acid were obtained from Sinopharm
chemical reagent company. Luria broth, yeast extract and
tryptone were from oxoid. cellulose DE-52 was purchased
In this study, we have extracted and purified lipid A from C. from Whatman.
sakazakii BAA-894. the structure of lipid A was analyzed by
Purification and analysis of
usingelectrosprayionization/massspectrometry(ESI/MS). the
majority of lipid A species in C. sakazakii BAA-894 have a
structure of disaccharide of glucosamine, phosphorylated at
lipopolysaccharide from C. sakazakii BAA-894
and E. coli W3110
the 1- and 4¢-positions and acylated with primary 3-hydroxy- Lipopolysaccharide (LPS) was extracted from both C. sakazakii
myristoyl chains at the 2-, 3-, 2¢- and 3¢-positions and
BAA-894 and Escherichia coli (E. coli) W3110 according to the
ISSN: 1469-0667
doi: 10.1255/ejms.1074
© IM Publications LLP 2010
All rights reserved

532
Structural Characterization of Lipid A in Cronobacter sakazakii
published methods14 with minor modifications. Briefly, the
bacterial cells were grown at 37°c in Luria broth, consisting
of 10gL−1 tryptone, 5gL−1 yeast extract, and 10gL−1 Nacl until
A600 reached 1.2. then 3mL of cell culture was centrifuged at
7000×g for 3min. the cell pellets were suspended in 100µL of
tAE buffer (2mmolL−1 EDtA, 40mmolL−1 tris acetate, pH8.5)
and mixed with 200µL of alkaline solution containing 3% (w/v)
sodium dodecyl sulfate (SDS), 0.6% (w/v) tris(hydroxymethyl)
aminomethane and 0.128 mol L−1 NaoH. the mixture was
heated at 60°c for 70min, mixed with 300µL of tris-saturated
spraying with a 10% ethanol solution of sulfuric acid, followed
by charring at 180°c.
to further purify lipid A, the above lipid A samples were
dissolved in 8 mL of chloroform / methanol / water (2 : 3 : 1,
v/v/v) and loaded on a 8mL DEAE-cellulose column.17 After
the run-through had been collected, the samples were eluted
stepwise with a solvent of chloroform/methanol and different
concentrations of ammonium acetate (2:3:1, v/v/v). the frac-
tions were converted to a two-phase Bligh–Dyer system by the
addition of chloroform and water. After being mixed thoroughly
phenol,and centrifuged at 15,700× g for 15 min. the super- and centrifuged, the lower phases were dried under a stream
natant aqueous phase was extracted with 300µL of tris-satu- of nitrogen, re-dissolved in chloroform/methanol (4:1, v/v)
rated phenol again to remove proteins and other contaminants. and analyzed by tLc.
After centrifugation, the supernatant was mixed with 200µL of
H2o and 50µL of 3molL−1 sodium acetate (pH5.2). LPS was
precipitated by adding 1.1mL of ethanol to the mixture. After
discarding the supernatant, the precipitate was dissolved in
200µL of solution containing 50mmolL−1 tris-Hcl (pH8.0) and
100mmolL−1 sodium acetate. then LPS was precipitated by
adding 400µL of ethanol to the mixture. the final precipitated
LPS was re-suspended in 10µL of H2o.
SDS–polyacrylamide gel electrophoresis (PAGE) and silver
stain of LPS were carried out according to the tsai and frasch
method.15 Briefly, 10 µL of LPS was mixed with an equal
volume of loading buffer, consisting of 0.1 mol L−1 tris-Hcl
(pH7.0), 4% SDS, 40% sucrose, 2% 2-mercaptoethanol and
0.02% bromophenol blue, and heated at 100 °c for 5min. the
the lipid A used for ESI/MS analysis was further purified
by preparative thin-layer chromatography. Briefly, the lipid A
samples purified by the DEAE-cellulose column were dissolved
in 400mL chloroform/methanol (4:1, v/v), and the samples
were applied to a silica gel 60 tLc plate and developed in chlo-
roform/pyridine/88% formic acid/water (50:50:16:5, v/v/v/v).
While the plates were drying, the lipid A bands could be seen
transiently as white zones. these bands were marked with a
pencil and scraped off after the plates were dry. the silica chips
were dissolved in 3.8mL single-phase Bligh–Dyer mixture and
incubated at room temperature for 5min. the mixture was then
converted into a two-phase Bligh–Dyer system, mixed thor-
oughly, and centrifuged at 1000×g for 8min at room temperature.
the lower phases were then dried under a stream of nitrogen.
stacking and separating gels contain 4% and 15% acrylamide, finally, the tLc-purified samples were passed through a 6mL
respectively. Electrophoresis was performed at 12mA for the
stacking gel and 25mA for the separating gel. the LPS bands
in the gel were visualized by silver staining.
DEAE-cellulose column to remove residual silica chips.17
Alkaline hydrolysis of lipid A
to determine the positions of fatty acids in the lipid A mole-
cules, the purified lipid A was hydrolyzed with 25% ammo-
nium hydroxide at 50° for 6.5 h.18 the reaction mixtures were
neutralized with a few drops of concentrated hydrochloric
acid, and converted into a two-phase Bligh–Dyer system. After
intensive mixing, the lower phase was separated and dried
under a stream of nitrogen.
Isolation and purification of lipid A from C.
sakazakii BAA-894 and E. coli W3110
typically, 400 mL of LB broth was inoculated with overnight
cultured cells, starting at an initial A600 of 0.02. the cells
were grown to A600 of 1.2, harvested and washed twice with
phosphate-buffered saline. the cell pellet was re-suspended
in 114mL of a single-phase Bligh–Dyer mixture16 containing
cHcl3 / MeoH / H2o (1 : 2 : 0.8, v / v / v), incubated at room
temperature for 60 min. the insoluble debris was collected
by centrifugation at 1000 × g for 20 min and washed once
Electrospray ionization mass spectrometry
and tandem mass spectrometry analysis
All tandem (MS/MS) mass spectra were acquired on a Waters
with single-phase Bligh–Dyer mixture. to cleave the glyco- SyNAPt Q-tof mass spectrometer equipped with an electro-
sidic linkage between polysaccharide and lipid A in LPS, the
pellets were re-suspended in 27mL of 12.5mmolL−1 sodium
acetate (pH4.5), heated at 100°c for 30min, followed by sonic
irradiation in a bath apparatus.17 After being cooled to room
temperature, the suspension was converted into a two-phase
Bligh–Dyer system by addition of 30 mL of chloroform and
30mL of methanol. After mixed thoroughly and centrifuging,
the lower phase was pooled then dried under a stream of
nitrogen. the dried lipids were re-dissolved in chloroform
and methanol (4:1, v/v) mixture, spotted onto a silica gel 60
tLc plate, which was developed in the solvent chloroform /
pyridine/88% formic acid/water (50:50:16:5, v/v/v/v). After
being dried. the lipid A bands on the plate were visualized by
spray ionization (ESI) source. Lipid A samples were dissolved
in chloroform/methanol (2:1, v/v) and subjected to ESI/MS in
the negative ion mode. Argon gas was used as the collision
gas for MS/MS experiments. Data acquisition and analysis
were performed using MassLynx V4.1 software.
Results
Isolation and purification of lipolysaccharide
and lipid A from C. sakazakii BAA-894
We first purified LPS from C. sakazakii BAA-894 and analyzed
it by using SDS-PAGE [figure 1(a)]. the specific ladder

C. Zhang et al., Eur. J. Mass Spectrom. 16, 531–538 (2010)
533
SDS-PAGE
TLC
(b)
(a)
LipidA
Figure 2. ESI/MS of purified lipid A from C. sakazakii BAA-894.
The most prominent peaks at m/z 1824.65 and 1796.62 are
molecular ions of lipid A species A1 and A2, respectively.
Origin
they differ by 28Da, that is, two methylene units. the peaks
at m/z 1598.41 and m/z 1570.37 might be derived from the
molecular ions by loss of a 3-hydromyristoyl group, respec-
tively. these data suggest that there are two lipid A species
in C. sakazakii. the two lipid A species are designated A1 and
A2, respectively. the chemical structures of A1 and A2 are
proposed in figure 3, in which the 28 Da difference is predicted
on the secondary fatty acids at 2¢-position. the exact mass of
A1 is 1825.25 and of A2 is 1797.22. fatty acid structural hetero-
geneity of lipid A was also observed in E. coli, but there was
only one major species showing a peak at m/z 1796.60 in the
1
2
1
2
1: E. coli W3110
2: C. sakazakii BAA-894
Figure 1. Isolation of LPS and lipid A from E. coli W3110 (lane 1)
and C. sakazakii BAA-894 (lane 2). (a) The silver-stained SDS-
PAGE of LPS. (b) Thin-layer chromatography of purified lipid A.
patterns were observed on the SDS-PAGE for C. sakazakii
LPS, but not the control sample E. coli LPS, suggesting that C. spectrum (data not shown).
sakazakii LPS contains o-antigens. Indeed, there are genes
ESI/MS analysis of lipid A species hydrolyzed
encoding enzymes for the biosynthesis of o-antigens in E.
sakazkaii.19 the chemical structures of o-polysaccharide in
C. sakazakii species have also been studied recently.20,21 the
control E. coli LPS did not show the ladder patterns on the
SDS-PAGE, because the W3110 strain does not synthesize
o-antigens.22
by ammonium hydroxide
to confirm that the structure difference between A1 and A2 is
on the secondary fatty acids at 2¢-position, the purified lipid
A was hydrolyzed with ammonium hydroxide. the hydrolyzed
lipid A was purified and analyzed by using ESI/MS.
Lipid A is the hydrophobic anchor of LPS. to isolate lipid
Alkaline hydrolysis combined with MS have been used
A in C. sakazakii, cells were first treated with the single- to characterize the structure of lipid A from different
phase Bligh–Dyer solvent system to remove soluble
proteins and phospholipids. the insoluble debris, which
contains LPS, was hydrolyzed at pH 4.5 to detach the
polysaccharide of LPS. then the hydrophobic lipid A was
recovered by extracting with chloroform. the extracted lipid
A was purified by a DEAE–cellulose column and eluted with
cHcl3 / MeoH / 240 mmol L−1 NH4Ac (2 : 3 : 1, v / v / v). the lipid
A fraction was further purified by using preparative tLc. As
shown in figure 1(b), the purified lipid A from C. sakazakii
BAA-894 migrated at the same rate on tLc as the control
sample E. coli lipid A.
bacteria.23 Hydrolysis of lipid A with 25% ammonium
hydroxide would cleave the o-linked fatty acid chains at the
3- and 3¢-positions as shown in figure 4(a). therefore, we
hydrolyzed A1 and A2 with 25% ammonium hydroxide, using
E. coli lipid A as a control. E. coli lipid A was completely
deacylated at the 3- and 3¢-positions, resulting in a hydro-
lyzed lipid A as judged by the major peak at m/z 1133.57 and
the minor peak at m/z 1161.60 in its spectrum [figure 4(b)].
Hydrolyzed C. sakazakii lipid A species A1 and A2 showed a
pair of peaks at m/z 1161.59 and m/z 1133.57 in the spec-
trum [figure 4(c)], consistent with the exact masses of the
hydrolyzed A1 and A2 as shown in figure 4(a). this suggests
that the difference between A1 and A2 is on the secondary
fatty acids at 2¢-position. the peaks at m/z 1035.60 in figure
4(b) and m/z 1063.63 in figure 4(c) could be derived from
their molecular ions from the loss of a phosphate group,
Negative ion ESI/MS analysis of lipid A from
C. sakazakii BAA-894
Lipid A purified from C. sakazakii BAA-894 was analyzed by ESI/
MS in negative ion mode (figure 2). the peaks at m/z 1824.65
and m/z 1796.62 are interpreted as [M–H]− molecular ions, respectively. this analysis suggests that the difference

534
Structural Characterization of Lipid A in Cronobacter sakazakii
Figure 3. Proposed chemical structure of lipid A species purified from C. sakazakii BAA-894.
(a)
(b)
(c)
Figure 4. (a) Proposed hydrolysis reaction of the lipid A species purified from C. sakazakii BAA-894. (b) ESI/MS of the hydrolyzed lipid A
from E. coli W3110. (c) ESI/MS of the hydrolyzed lipid A from C. sakazakii BAA-894.

C. Zhang et al., Eur. J. Mass Spectrom. 16, 531–538 (2010)
535
between A1 and A2 could only be on the fatty acid chains at
the 2- or 2¢-positions of lipid A (figure 3).
(a)
ESI/MS/MS analysis of hydrolyzed lipid A
ESI/MS/MS has been widely used for analysis of lipid A struc-
ture in recent years.24–27 to further confirm the exact location
of the difference between A1 and A2 at the 2- or 2¢-posi-
tions in lipid A, molecular ions of the hydrolyzed A1 and A2
at m/z 1161.59 and m/z 1133.57 were analyzed by ESI/MS/
MS (figure 5). Peaks at m/z 1143.59 [figure 5(a)] and m/z
1115.56 [figure 5(b)] were derived from the parent ions by
loss of a water molecule; they generated the fragment ions
at m/z 1063.62 [figure 5(a)] and m/z 1035.60 [figure 5(b)] by
further cleavage of a phosphate, respectively. the peaks at
m/z 933.39 observed in both spectra of hydrolyzed A1 and
A2 could be derived from the parent ions by loss of a water
molecule and the secondary fatty acid chain at the 2¢-position
[figure 5(c)], suggesting that the difference in A1 and A2
originates from the secondary fatty acid at the 2¢-position, as
proposed in figure 3.
(b)
Discussion
(c)
Infection by the food-borne pathogen C. sakazakii can cause
severe disease,28 but little is known at the molecular level of
its virulence. As a major component on the surface of Gram-
negative bacteria, lipid A is the active component of LPS29
and is closely related to the infection ability of bacteria.30–34
recently, several genes have been found to modify the struc-
ture of lipid A in bacteria.35–38 thus, the elucidation of the
biochemical structure of lipid A in C. sakazakii is necessary for
research into its virulence.
Lipid A biosynthesis has been well studied in E. coli. the
pathway is mediated by nine enzymes and biosynthesis
occurs in the cytoplasm and on the inner surface of the
inner membrane.39 the initial building block of lipid A is
uDP-GlcNAc. the first three enzymes, LpxA, Lpxc and LpxD,
add two 3-hydroxymyristoyl chains to the 2- and 3-positions
of the uDP-GlcNAc to form uDP-diacyl-GlcN which can be
further hydrolyzed by LpxH to form lipid X. Next, enzyme LpxB
condenses lipid X and its precursor uDP-diacyl-GlcN to form
the disaccharide of glucosamine, with four 3-hydroxymyristoyl
chains at the 2-, 3-, 2¢- and 3¢-positions. At a later stage of lipid
A biosynthesis, enzyme LpxL adds a secondary lauroyl residue
at the 2¢-position and enzyme LpxM adds a myristoyl residue
to the 3¢-position. Here, we have characterized the structure
of lipid A in C. sakazakii BAA-894. there are two lipid A species,
A1 and A2, found in C. sakazakii. the molecular weight differ-
ence between A1 and A2 is 28 Da, which is a two-carbon differ-
ence for the chain length of the secondary fatty acid at the
2¢-position of lipid A, as judged by hydrolysis. LpxL was first
found in E. coli40 and then in other bacteria.41,42 It can affect the
chemical structure and toxicity of lipid A and determine the
cell membrane integrity and permeability.43–46 the ortholog
of LpxL also exists in C. sakazakii BAA-894 and shares 79%
Figure 5. Negative ion ESI/MS/MS analysis of ions in the
spectra of hydrolyzed lipid A species from C. sakazakii lipid
A. (a) The spectrum of ion at m/z 1161.59 in Figure 4(c). (b)
The spectrum ion at m/z 1133.60 in Figure 4(c). (c) Proposed
chemical structures of fragment ions produced in ESI/MS/MS
spectra.

536
Structural Characterization of Lipid A in Cronobacter sakazakii
identity to E. coli LpxL. It would be interesting to study whether
LpxL in C. sakazakii is related to its infection ability.
sakazakii: an emerging bacterial pathogen with
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mechanisms of clinically relevant microorganisms”,
Clin. Microbiol. Rev. 15, 167–193 (2002). doi: 10.1128/
cMr.15.2.167-193.2002
Acknowldgements
funding was provided by grants from the National Natural
Science foundation of china (NSfc30770114, NSfc30870074),
the Program of State Key Laboratory of food Science and
technology (SKLf-MB-200801), the 111 project (111-2-06)
and the Basic research Programs of Jiangsu Province
(BK2009003).
14. N. Kido, M. ohta and N. Kato, “Detection of
lipopolysaccharides by ethidium bromide staining after
sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis”, J. Bacteriol. 172, 1145–1147 (1990).
15. c.M. tsai and c.E. frasch, “A sensitive silver stain for
detecting lipopolysaccharides in polyacrylamide gels”,
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16. E.G. Bligh and W.J. Dyer, “A rapid method of total lipid
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911–917 (1959). doi: 10.1139/y59-099
17. Z. Zhou, S. Lin, r.J. cotter and c.r. raetz, “Lipid A
modifications characteristic of Salmonella typhimurium
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