Báo cáo khoa học: Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f Structural diversity observed in three strains ppt

Structural analysis of lipopolysaccharides from
Haemophilus
influenzae
serotype f
Structural diversity observed in three strains
Ha
˚
kan H. Yildirim
1
, Derek W. Hood
2
, E. Richard Moxon
2
and Elke K. H. Schweda
1
1
Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Sweden;
2
Molecular Infectious
Diseases Group, University of Oxford Department of Pediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Structural elucidation of the lipopolysaccharide (LPS) from
threeserotypefHaemophilus influenzae clinical isolates
RM6255, RM7290 and RM6252 has been achieved using
NMR spectroscopy techniques and ESI-MS on O-deacyl-
ated LPS and core oligosaccharide material (OS) as well as
ESI-MS
n
on permethylated dephosphorylated OS. This is
the first study to report structural details on LPS from sero-
type f strains. We found that the LPSs of all strains were

highly heterogeneous mixtures of glycoforms expressing the
common H. influenzae structural element
L
-a-
D
-Hepp-
(1fi2)-[PEtnfi6]-
L
-a-
D
-Hepp-(1fi3)-[b-
D
-Glcp-(1fi4)]-
L
-a-
D
-
Hepp-(1fi5)-[PPEtnfi4]-a-Kdo-(2fi6)-lipid A with variable
length of OS chains linked to each of the heptoses. The
terminal heptose (HepIII) in RM6255 is substituted at the
O-3 position by a b-
D
-Glcp residue whereas HepIII in strains
RM7290 and RM6252 is substituted at O-2 by the globoside
unit (a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D

-Glc) or trun-
cated versions thereof. The central heptose (HepII) is
substituted by an a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D
-
Glcp-(1fi4)-a-
D
-Glcp unit in RM7290 and RM6252 or
truncated versions thereof. Strain RM6255 does not express
galactose in its LPS and only shows a cellobiose unit elon-
gating from HepII (b-
D
-Glcp-(1fi4)-a-
D
-Glcp). ESI-MS
n
on
dephosphorylated and permethylated OS provided infor-
mation on the existence of additional minor isomeric
glycoforms.
Keywords: Haemophilus influenzae; lipopolysaccharide;
NMR; ESI-MS.
Haemophilus influenzae is an important cause of human
disease worldwide and exists in encapsulated (type a
through f) and unencapsulated (nontypeable) forms. Type
b capsular strains are associated with invasive bacteraemic

diseases, including meningitis and epiglottis, while acapsular
or nontypeable strains of H. influenzae (NTHi) are primary
pathogens in otitis media and lower respiratory tract
infections [1]. Lipopolysaccharide (LPS) is an essential
surface component of these pathogens and is implicated as a
major virulence factor. LPS of H. influenzae can mimic host
glycolipids and has a propensity for reversible switching of
terminal epitopes (phase variation) of the oligosaccharide
portion. Molecular structural studies of LPS from H. influ-
enzae type b, d and NTHi strains have shown that the
conserved part of LPS consists of a triheptosyl inner-core
moiety linked to lipid A via 3-deoxy-
D
-manno-oct-2-ulo-
sonic acid (Kdo). Each of the heptose residues (designated
HepI, HepII and HepIII) can provide a point for elongation
by oligosaccharide chains or for attachment of noncarbo-
hydrate substituents (Structure 1).
Correspondence to E. Schweda, University College of South Stock-
holm, Clinical Research Centre, NOVUM, S-141 86 Huddinge,
Sweden. Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23,
E-mail:
Abbreviations: Ac, acetate; AnKdo-ol, reduced anhydro Kdo; CE,
capillary electrophoresis; gHSQC, gradient heteronuclear single
quantum coherence; gHMQC, gradient selected heteronuclear mul-
tiple quantum coherence; gHMBC, gradient selected heteronuclear
multiple-bond correlation; GPC, gel permeation chromatography;
Hep, heptose;
L
,

D
-Hep,
L
-glycero-
D
-manno-heptose;Hex,hexose;
Hib, H. influenzae serotype b; Hif, H. influenzae serotype f; Kdo,
3-deoxy-
D
-manno-oct-2-ulosonic acid; lipid A-OH, O-deacylated
lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated lipopoly-
saccharide; PAD, pulsed amperometric detection; MS
n
, multiple step
tandem MS; Neu5Ac, N-acetyl neuraminic acid; NTHi, nontypeable
Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine;
PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine.
(Received 14 April 2003, revised 21 May 2003,
accepted 27 May 2003)
Eur. J. Biochem. 270, 3153–3167 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03693.x
To date, HepI has been found to be substituted by
glucose (R
1
¼ b-
D
-Glcp) in all strains investigated. This
glucose can provide a point for further chain extension.
There is no chain extension from HepII in the type
d-derived strain Rd [2], however, in three type b strains
and a number of NTHi strains HepII is substituted by a

glucose residue (R
2
¼ a-
D
-Glcp) which can provide a
point for further chain extension. HepIII is substituted
by galactose {R
3
¼ b-
D
-Galp-(1fi2) in type b strains [3];
R
3
¼ b-
D
-Galp-(1fi3), NTHi strain 176 [4]} or oligosac-
charides extending from glucose {R
3
¼ b-
D
-Glcp-(1fi2),
strain Rd, NTHi strain 1003 [5]; R
3
¼ b-
D
-Glcp-(1fi3),
NTHi strain 486 [6]}. In addition, N-acetyl neuraminic acid
has been found to be a common constituent of LPS in
H. influenzae [7]. Prominent noncarbohydrate substituents
are phosphate (P), pyrophosphoethanolamine (PPEtn),

phosphoethanolamine (PEtn), phosphocholine (PCho),
acetate (Ac) and glycine (Gly).
The oligosaccharide portion of H. influenzae LPS is
known to be subject to high-frequency phase variation of
terminal epitopes, which can lead to a very heterogeneous
population of LPS molecules within a single strain [8]. Phase
variation is thought to provide an adaptive mechanism
which is advantageous for survival of bacteria confronted
by the differing microenvironments and immune responses
of the host [9]. A genetic mechanism contributing to LPS
phase variation has been identified in five chromosomal loci;
lic1, lic2, lic3, lgtC and lex2 [10–12]. It has been demon-
strated that expression of PCho substituents in H. influen-
zae LPS is subject to phase variation mediated by the lic1
locus [13]. Genes comprising the lic2 locus have been shown
to be required for chain extension from HepII and, together
with lgtC, in the phase variable expression of the p
k
epitope [a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D
-Glcp-(1fi]
[12,14,15]. The lic3 locus has been shown to encode an
a-2,3-sialyltransferase that is responsible for addition of
sialic acid (N-acetylneuraminic acid or Neu5Ac) to terminal
lactose [16]. The phase-variable lex2 locus has been shown
to encode a glycosyltransferase important in further oligo-

saccharide extension from HepI in the inner core [17]. A
comprehensive study of LPS biosynthetic loci has been
undertaken in the type b strain Eagan (RM153) and in
strain Rd
–
(RM118) [18,19]. H. influenzae serotype f (Hif)
has its own structurally and serologically distinct capsular
polysaccharide distinguishing it from the other five sero-
types and the nontypeable strains [20].
Historically, Hif strains have been a rare cause of disease
in children and adults [21,22]. However, septic arthritis,
meningitis, sepsis, otitis media, pneumonia, cellulitis, and
bacteremia are diseases associated with Hif infections in
children who quite often have a predisposing medical
condition, including documented immunodeficiency [20,
23–25]. Countries engaged in global vaccination programs
against H. influenzae serotype b (Hib) experienced a rapid
decline in diseases caused by Hib strains. In the UK, Hib
infection decreased 98% in children younger than 5 years of
age [22], thus resulting in a greater proportion of infections
due to serotype f strains in the Hib postvaccination era [26].
The capsular polysaccharide of type f strains is known to be
composed of b-
D
-GalpNAc(1fi4)-a-
D
-GalpNAc(1fiPO
4
repeating units [27]. However, no data on LPS structures
are known. To gain further insight into the LPS structures

expressed by H. influenzae, we report detailed structural
analyses on three epidemiologically distinct type f strains. In
these strains we identify a novel combination of oligosac-
charide extensions not seen before in H. influenzae LPS.
Experimental procedures
Bacterial strains, growth conditions and LPS extraction
H. influenzae type f strains RM6255, RM7290 and RM6252
are epidemiologically distinct clinical isolates which are
representative of the limited genetic diversity observed
within type f isolates. RM6252 was isolated in 1966 from the
nasal pus of a 59-year-old male from Newcastle, England,
who suffered from subacute sinusitis. RM6255 was isolated
in 1967 from the sputum of a 72-year-old male from
Newcastle, England, who suffered from a postoperative
chest infection. RM7290 was isolated in 1974 from the
sputum of a 30-year-old female from Kuala Lumpur,
Malaysia, who suffered from respiratory infection and
malnutrition. Bacteria were grown in brain–heart infusion
broth supplemented with haemin (10 lgÆmL
)1
)andNAD
(2 lgÆmL
)1
). LPS was extracted from lyophilized bacteria
using phenol/chloroform/light petroleum as described pre-
viously but with the modification that the LPS was
precipitated with 6 vols diethyl ether/acetone (1 : 5, v/v)
[28]. LPS was purified by ultracentrifugation (82 000 g,
4 °C, 12 h). 11, 49 and 56 mg LPS was extracted from 1.6,
5.3, 3.4 g lyophilized bacteria from RM6255, RM7290 and

RM6252, respectively.
Chromatography
Gel permeation chromatography (GPC) was performed
using a Bio-Gel P4 column (2.5 · 80 cm) with pyridinium
acetate (0.1
M
, pH 5.3) as eluent and a differential refrac-
tometer as detector. GLC was carried out using a Hewlett-
Packard 5890 instrument with a DB-5 fused silica capillary
column (25 m · 0.25 mm; 0.25 lminternaldiameter)and
a temperature gradient of 160 °C(1min)fi 250 °Cat
3 °Cmin
)1
.
HPAEC was performed on a Dionex Series 4500i
chromatography system using a CarboPac PA1 column
(4 · 250 mm) and pulsed amperometric detection. Samples
were eluted using a linear gradient of 0–500 m
M
NaOAc in
0.1
M
NaOH over 20 min and a flow rate of 1 mLÆmin
)1
.
Preparation of oligosaccharides
O-Deacylation of LPS with hydrazine. O-Deacylation of
LPS was achieved as previously described [29]. Briefly, LPS
(1 mg) was mixed with anhydrous hydrazine (0.1 mL) and
stirred at 37 °C for 1 h. The reaction mixture was cooled

and cold acetone (1 mL) was added to destroy excess
hydrazine. The precipitated O-deacylated LPS (LPS-OH)
was centrifuged (48 200 g,20min),thepelletwaswashed
twice with cold acetone, once with diethyl ether, and then
dissolved in water followed by lyophilization.
Mild acid hydrolysis of LPS. Core oligosaccharide frac-
tions were obtained from LPS (10 mg RM6255, 22 mg
RM7290, 30 mg RM6252) following mild acid hydrolysis
3154 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(1% acetic acid, pH 3.1, 100 °C, 2 h). The insoluble lipid A
was separated from the hydrolysis mixtures by centrifuga-
tion. The reducing agent, borane-N-methylmorpholine
complex (1, 2 and 3 mg, respectively, for RM6255,
RM7290 and RM6252), was included in the hydrolysis
mixture. Following purification by GPC on the Biogel P4
column oligosaccharide (OS) fractions OS6255 (2.5 mg),
OS7290 (4.0 mg) and OS6252 (9.7 mg) were obtained and
investigated in this study.
Dephosphorylation. OS6255, OS7290 and OS6252 (1 mg
each) were incubated with 48% hydrogen fluoride (0.1 mL)
for 48 h at 4 °C. Then, the samples were placed into an ice
bath and HF was evaporated under a stream of nitrogen
gas. The samples, OS6255-P, OS7290-P and OS6252-P,
were dissolved in water and lyophilized.
Mass spectrometry. GLC-MS was carried out with a
Hewlett-Packard 5890 chromatograph connected to a
NERMAG R10-10H quadrupole mass spectrometer using
the same conditions for GLC as described above. ESI-MS
on LPS-OH and OS samples was recorded on a VG Quattro
triple quadrupole mass spectrometer (Micromass, Man-

chester, UK) in the negative ion mode. The samples were
dissolved in a mixture of water/acetonitrile (1 : 1, v/v).
Sample solutions were injected via a syringe pump into a
running solvent of H
2
O/CH
3
CN (1 : 1) at a flow rate of
10 lLÆmin
)1
. Multiple step tandem ESI-MS (ESI-MS
n
)
experiments were performed on a Finnigan LCQ iontrap
mass spectrometer (Finnigan-MAT, San Jose, CA, USA).
Samples were dissolved in 1 m
M
sodium acetate in 70%
MeOH/30% H
2
O. The applied flow rate was 10 lLÆmin
)1
.
All experiments were in the positive mode. Capillary
electrophoresis (CE)-ESI-MS
n
was carried out with a
Crystal model 310 CE instrument (ATI Unicam, Boston,
MA, USA) coupled to an API 3000 mass spectrometer
(Perkin-Elmer/Sciex, Concord, Canada) via a MicroIon-

spray interface as described previously [30].
Analytical methods
Sugars were identified by GLC-MS as their alditol acetates
as previously described [31]. Methylation was performed
with methyl iodide in dimethylsulfoxide in the presence of
lithium methylsulfinylmethanide [32]. The methylated com-
pounds were recovered using a SepPak C18 cartridge and
subjected to sugar analysis or ESI-MS
n
.Therelative
proportions of the various alditol acetates and partially
methylated alditol acetates obtained in sugar- and methyl-
ation analyses, discussed below, correspond to the detector
response of the GLC-MS. The absolute configuration of
glycoses was determined as previously described [33]. The
presence of glycine was determined by HPAEC following
treatment of LPS with 0.1
M
NaOH at 20–22 °C for 30 min.
N-acetylneuraminic acid was determined by treating LPS-
OH (0.2 mg) with 20 mU
1
of neuraminidase in 0.2 mL
10 m
M
NaOAc, pH 5.0, at 37 °C for 4 h. One unit will
liberate 1.0 lmol of Neu5Ac per min at pH 5.0 at 37 °C.
The reaction mixture was analyzed by HPAEC as previ-
ously described without further work-up [30]. The enzyme
cleaves terminal Neu5Ac residues linked a-2,3, a-2,6 or

a-2,8 to oligosaccharides. Fatty acids were identified as
described previously [34].
NMR spectroscopy
NMR spectra were recorded for OS samples in deuterium
oxide (D
2
O) at 30 °C. Spectra were acquired on JEOL
500 MHz and Varian UNITY 600 MHz spectrometers
using standard pulse sequences. The LPS-OH samples were
solubilized by adding perdeutero-EDTA (2 m
M
) and per-
deutero-SDS (10 mgÆmL
)1
)totheD
2
O solution. Chemical
shifts were reported in p.p.m., and externally referenced
to sodium 3-trimethylsilylpropanoate-d4 (d 0.00,
1
H) and
acetone (d 29.8,
13
C). COSY, TOCSY with a mixing time of
180 ms, gradient selected heteronuclear single quantum
coherence (gHSQC), gradient selected heteronuclear mul-
tiple quantum coherence (gHMQC), and gradient selected
heteronuclear multiple-bond correlation (gHMBC) experi-
ments were performed according to standard pulse
sequences. For interresidue correlation, two-dimensional

NOESY experiments with a mixing time of 250 ms were
used.
Results
Characterization of LPS
H. influenzae type f strains RM6255, RM7290 and RM6252
were grown in liquid culture and the LPS was isolated by
phenol/chloroform/light petroleum extraction [6].
Compositional analysis of the LPS samples for strains
RM7290 and RM6252 identified
D
-glucose (Glc),
D
-galactose (Gal), 2-amino-2-deoxyglucose (GlcN) and
L
-glycero-
D
-manno-heptose (Hep) as the constituent sugars
by GLC-MS of the derived alditol acetates and 2-butyl
Table 1. Sugar analysis data for LPS-OH and OS samples derived from H. influen zae strains RM6255, RM7290 and RM6252.
Relative detector response percentage
Sugar residue
a
RM6255 RM7290 RM6252
OS LPS-OH OS LPS-OH OS LPS-OH
Glc 81 64 59 43 51 40
Gal 32 23 37 33
Hep 19 20 9 21 11 14
GlcNAc 16 14 15
a
Sugars were identified by GLC-MS as their alditol acetates.

Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3155
glycoside derivatives on oligosaccharide material (Table 1).
Strain RM6255 showed the same sugars except for Gal.
In addition, glycine was detected as a substituent of each
LPS by HPAEC with pulsed amperometric detection
(HPAEC-PAD) as described earlier [30]. Neu5Ac can be
detected in H. influenzae LPS by a method involving
analysis by HPAEC-PAD of terminal N-acetyl neuraminic
2
acid residues released by neuraminidase treatment [7].
However, by applying this method in our study, N-acetyl
neuraminic acid was not detected in any strain. O-Deacyla-
tion of LPS by treatment with anhydrous hydrazine
under mild conditions afforded water-soluble material
(LPS-OH), which was subjected to ESI-MS. The ESI-MS
spectra of the samples (negative mode) revealed abundant
molecular peaks corresponding to triply and quadruply
deprotonated ions. The MS data (Table 2), pointed to
the presence of glycoforms in which each molecular
species contains the conserved PEtn-substituted triheptosyl
inner-core moiety attached via a phosphorylated Kdo
linked to the O-deacylated lipid A (structure 1). Two
populations of glycoforms were observed which differed by
123 Da (i.e. the mass of a PEtn group). As observed for other
H. influenzae strains, this would be consistent with either
phosphate or pyrophosphoethanolamine (PPEtn) substitu-
tion at the O-4 position of the Kdo residue [2,3,35].
Thus, for strain RM6255, abundant quadruply/triply
charged ions were observed at m/z 649.4/866.1 and 680.1/
907.0 indicating Hex4 glycoforms with the respective

compositions Hex
4
ÆHep
3
ÆPEtn
1,2
ÆP
1
ÆKdo
1
ÆLipidA-OH.
Signals corresponding to minor glycoforms with composi-
tions Hex
2
ÆHep
3
ÆPEtn
1,2
ÆP
1
ÆKdo
1
ÆLipidA-OH and Hex
3
Æ
Hep
3
ÆPEtn
1,2
ÆP

1
ÆKdo
1
ÆLipidA-OH were observed at m/z
568.2/758.2, 599.2/799.1, 608.5/811.7, and 639.5/853.5,
respectively. The ESI-MS spectrum of LPS-OH from
RM7290 revealed major quadruply/triply charged ions at
m/z 649.4/866.2, 730.5/974.3, and 761.3/1015.3 which indi-
cated the occurrence of glycoforms with compositions
Hex
4
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH, and Hex
6
ÆHep
3
Æ
PEtn
1,2
ÆP
1
ÆKdo
1

ÆLipidA-OH, respectively. Less abundant
quadruply charged ions at m/z 608.8, 639.6, 680.2, 690.0,
811.8 and 842.4 together with their triply charged counter-
parts corresponded to glycoforms with the following
compositions Hex
3
ÆHep
3
ÆPEtn
1,2
ÆP
1
ÆKdo
1
ÆLipidA-OH,
Hex
4
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH, Hex
5
ÆHep
3
ÆPEtn

1
Æ
P
1
ÆKdo
1
ÆLipidA-OH, and Hex
8
ÆHep
3
ÆPEtn
1,2
ÆP
1
ÆKdo
1
Æ
LipidA-OH, respectively.
The ESI-MS spectrum (negative mode) of LPS-OH from
strain RM6252 showed ions corresponding to the same
glycoforms as observed for strain RM7290. However,
additional ions corresponding to Hex7 glycoforms with
compositions Hex
7
ÆHep
3
ÆPEtn
1,2
ÆP
1

ÆKdo
1
ÆLipidA-OH were
observed at m/z 1028.2 and 1068.8. In general, the ions
corresponding to the higher glycoforms (Hex6 to Hex8)
were of greater abundance in the spectrum of RM6252 than
in the one of RM7290. From ESI-MS data it was also
evident that PCho was not a major substituent in the LPS
of the type f strains.
Characterization of the Kdo-lipid A-OH element
The structure of the lipid A-OH region in all H. influenzae
strains investigated so far has been found to consist of
a b-1,6-linked
D
-glucosamine disaccharide substituted by
N-linked 3-hydroxytetradecanoic acid at C-2 and C-2¢ and
phosphomonoester groups at C-1 and C-4¢ [2,3,5,6,34,36].
In this investigation, ESI-MS data (Table 2) and fatty
acid compositional analysis yielding 3-hydroxytetradeca-
noic acid indicated the presence of the same lipid A-OH
structure in Hif. In addition, in a
1
H–
31
P coupled
gHMQC experiment, a
31
Psignalatd )0.5 correlated
to
1

H d 5.48, which was assigned to the anomeric
1
Hshift
of the a-linked GlcN (GlcNI) in the lipid A. In the same
spectrum, a
31
Psignalatd 3.4correlatedtoa
1
Hsignalat
d 4.05 which was identified as the H-4 signal of the
b-GlcN residue (GlcNII).
Characterization of oligosaccharides
Mild acid hydrolysis of LPS with dilute aqueous acetic acid
afforded insoluble lipid A and core oligosaccharide, which
after purification by gel filtration resulted in oligosaccharide
samples OS6255, OS7290 and OS6252.
Sugar analysis of the OS samples shown in Table 1
revealed
D
-glucose (Glc),
L
-glycero-
D
-manno-heptose in
allthreestrainsaswellas
D
-galactose (Gal) in RM7290
and RM6252. The absence of 2-amino-2-deoxyglucose
(GlcN) in the OS samples confirmed this sugar to be part
of lipid A. After dephosphorylation of OS samples with

48% HF, methylation analysis of the resulting material
(OS6255-P) showed terminal Glc, 4-substituted Glc,
3-substituted-Hep, 2,3-disubstituted Hep and 3,4-disubsti-
tuted Hep as the major sugar components; linkage analysis
of OS7290-P and OS6252-P revealed terminal Glc, terminal
Gal, 4-substituted Glc, 4-substituted Gal, 3,4-disubstituted
Hep, 2,3-disubstituted Hep and 2-substituted Hep in the
relative proportions as shown in Table 3. The data was
consistent with triantennary structures, containing the
common inner-core element,
L
-a-
D
-Hepp-(1fi2)-
L
-a-
D
-
Hepp-(1fi3)-[b-
D
-Glcp-(1fi4)]-
L
-a-
D
-Hepp-(1fi5)-a-Kdop
of H. influenzae LPS. The presence of 3-substituted Hep in
OS6255-P indicated substitution at the O-3 position of
HepIII whereas in strains RM7290 and RM6252 substitu-
tion at O-2 of HepIII was indicated (structure 1). ESI-MS
on OS samples (Table 2) indicated OS6255 to be acetylated

and OS7290 and OS6252 to be both acetylated and
glycylilated. Thus in the ESI-MS spectrum of OS6255
(negative mode) major doubly charged ions were observed
at m/z 783.8 and 804.7 corresponding to glycoforms with
respective compositions Hex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol and
Ac
1
ÆHex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol. Minor doubly charged
ions at m/z 621.6, 642.8, 702.8, and 723.8 corresponded
to Hex
2
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol, Ac
1
ÆHex

2
ÆHep
3
ÆPEtn
1
Æ
AnKdo-ol, Hex
3
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol and Ac
1
ÆHex
3
Æ
Hep
3
ÆPEtn
1
ÆAnKdo-ol, respectively. The ESI-MS spectra
of OS7290 and OS6252 indicated 15 and 22 glycoforms,
respectively. The Hex6 glycoforms were the most predom-
inant ones in OS7290, comprising 64% of the OS molecules.
They were detected as doubly charged ions at m/z 946.1,
966.9, and 995.3 corresponding to the respective composi-
tions Hex
6
ÆHep

3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆAnKdo-ol, Ac
1
ÆHex
6
ÆHep
3
Æ
PEtn
1
ÆP
1
ÆKdo
1
ÆAnKdo-ol, and Ac
1
ÆGly
1
ÆHex
6
ÆHep
3
ÆPEtn
1

Æ
P
1
ÆKdo
1
ÆAnKdo-ol. The most predominant doubly charged
ions in the ESI-MS spectrum of OS6252 at m/z 804.9 and
1129.2 corresponded to Hex4 and Hex8 glycoforms
3156 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
with compositions Ac
1
ÆHex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol and
Ac
1
ÆHex
8
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol, respectively.
ESI-MS/MS
Some information on the location of the acylation sites in
OS7290 and OS6252 was provided by ESI MS/MS

following on-line separation by CE in the positive mode
[30,36]. Thus, product ion spectra obtained from the
doubly charged ions at m/z 998 in OS7290 (composition:
Ac
1
ÆGly
1
ÆHex
6
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol) and m/z 1160 in
OS6252 (composition: Ac
1
ÆGly
1
ÆHex
8
ÆHep
3
ÆPEtn
1
Æ
AnKdo-ol) showed ions at m/z 204 and 235 corresponding
to AcÆHex and AcÆHep, respectively, indicating mixed
acetylation at either a hexose residue or a heptose. No ions
indicating glycylilation could be observed in the spectrum
of OS6252. However, the spectrum of OS7290 showed

ions at m/z 280 and 292 corresponding to GlyÆAnKdo-ol
and AcÆGlyÆHep, respectively, indicating glycylilation at
either Kdo or a heptose residue. No more detailed
information could be obtained from the spectra which
showed very random fragmentation patterns.
Table 2. Negative ion ESI-MS data and proposed compositions for LPS-OH and OS samples of H. influenzae f-type strains RM6255, RM7290 and
RM6252. Average mass units were used for calculation of molecular mass values based on proposed composition as follows: Hex, 162.14; Hep,
192.17; Kdo, 220.18; AnKdo-ol, 222.18; P, 79.98; PEtn, 123.05; Ac, 42.04; Gly, 57.05; and LipidA-OH 953.02. Relative abundance was estimated
from the area of molecular ion peak relative to the total area (expressed as percentage). Peaks representing less than 5% of the base peak intensity
are not included in the table.
Sample
Observed ions (m/z) Molecular mass (Da) Relative abundance (%)
(M-4H)
4–
(M-3H)
3–
(M-2H)
2–
Observed Calculated 6255 7290 6252 Proposed composition
LPS-OH –
a
758.2
b
2277.6 2277.0 2 Hex
2
ÆHep
3
ÆPEtn
1
ÆP

1
ÆKdo
1
ÆLipidA-OH
599.2
b
799.1
b
2400.6 2400.1 3 Hex
2
ÆHep
3
ÆPetn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH
608.9 811.5 2438.6 2439.2 6 6 6 Hex
3
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH

639.7 853.5 2563.2 2562.2 3 2 4 Hex
3
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH
649.4 866.1 2601.5 2601.4 37 16 16 Hex
4
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH
680.3 906.7 2724.2 2724.4 49 4 6 Hex
4
ÆHep
3
ÆPEtn
2
ÆP
1

ÆKdo
1
ÆLipidA-OH
689.9 920.1 2763.5 2763.4 5 11 Hex
5
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH
720.7 960.8 2886.1 2886.5 3 Hex
5
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH
730.4 973.5 2924.6 2925.6 37 8 Hex
6
ÆHep
3
ÆPEtn

1
ÆP
1
ÆKdo
1
ÆLipidA-OH
761.3 1015.1 3048.8 3048.6 23 7 Hex
6
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH
771.0 1028.3 3088.0 3087.7 10 Hex
7
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH
801.9 1068.8 3210.5 3210.8 6 Hex
7

ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1
ÆLipidA-OH
811.5 1082.2 3249.6 3249.9 12 Hex
8
ÆHep
3
ÆPEtn
1
ÆP
1
ÆKdo
1
ÆLipidA-OH
842.3 1123.1 3372.8 3372.9 2 13 Hex
8
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdo
1

ÆLipidA-OH
OS 621.6
b
1245.2 1246.0 6 Hex
2
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
642.8
b
1287.6 1288.0 3 Ac
1
ÆHex
2
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
702.8
b
1407.6 1408.2 5 Hex
3
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol

723.5 1449.0 1450.2 5 2 3 Ac
1
ÆHex
3
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
745.0 1492.0 1492.2 1 Ac
2
ÆHex
3
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
752.7 1507.0 1507.2 2 7 Ac
1
ÆGly
1
ÆHex
3
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
783.9 1569.8 1570.3 40 1 2 Hex

4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
805.1 1611.8 1612.3 42 8 11 Ac
1
ÆHex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
826.0 1653.0 1654.3 7 6 Ac
2
ÆHex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
833.6 1669.2 1669.4 2 1 Ac
1
ÆGly
1
ÆHex
4

ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
854.1 1710.2 1711.4 6 2 Ac
2
ÆGly
1
ÆHex
4
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
865.2 1732.8 1732.4 1 1 Hex
5
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
886.0 1774.0 1774.4 3 7 Ac
1
ÆHex
5
ÆHep
3
ÆPEtn

1
ÆAnKdo-ol
907.0 1815.8 1816.4 6 Ac
2
ÆHex
5
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
913.6 1829.2 1831.5 1 2 Ac
1
ÆGly
1
ÆHex
5
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
936.0 1873.6 1873.5 2 Ac
2
ÆGly
1
ÆHex
5
ÆHep
3

ÆPEtn
1
ÆAnKdo-ol
945.7 1893.8 1894.6 4 1 Hex
6
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
967.0 1936.8 1936.6 36 6 Ac
1
ÆHex
6
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
995.9 1993.8 1993.6 24 1 Ac
1
ÆGly
1
ÆHex
6
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol

1027.0 2055.8 2056.7 2 Hex
7
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
1048.1 2098.0 2098.7 9 Ac
1
ÆHex
7
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
1076.8 2156.8 2155.8 4 Ac
1
ÆGly
1
ÆHex
7
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
1108.0 2218.2 2218.9 2 Hex
8
ÆHep

3
ÆPEtn
1
ÆAnKdo-ol
1129.1 2260.4 2260.9 2 14 Ac
1
ÆHex
8
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
1157.6 2318.1 2317.9 1 9 Ac
1
ÆGly
1
ÆHex
8
ÆHep
3
ÆPEtn
1
ÆAnKdo-ol
a
Trace amounts of a signal at m/z 568.2 were detected.
b
The observed m/z-values correspond to spectra from RM6255. All other m/z-values
correspond to spectra from RM6252. Observed values in RM7290 and RM6255 varied ± 0.8 Da.
Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3157

Sequence analysis of OS6255-P, OS7290-P
and OS6522-P by MS
n
OS6255-P, OS7290-P and OS6252-P were permethylated
and analyzed by ESI-MS
n
in order to determine the
sequence and branching details of the various glycoforms.
This method was introduced by Reinhold et al. and has
been applied by us recently [36–38]. In the ESI-MS spectrum
of OS6255-P (positive mode), shown in Fig. 1A, four major
sodiated parent ions were observed at m/z 1264.3, 1468.1,
1672.4 and 1876.6 corresponding to the dephosphorylated
and permethylated Hex1 to Hex4 glycoforms (Hex
1)4
ÆHep
3
Æ
AnKdo-ol). These ions were further fragmented in MS
2
experiments, and some of the resulting product ions
underwent further fragmentation (MS
3
) to confirm the
existence of the proposed glycoforms shown in Table 4.
Two isomeric Hex1 glycoforms were identified by
performing MS
2
on parent ion m/z 1264.3. Two product
ions at m/z 797.4 and 737.3 corresponding to the neutral

losses of tHexÆHepIII and HepIÆAnKdo-ol, respectively,
confirmed the presence of a Hex1 glycoform in which
HepIII is substituted. The glycoform with a hexose elonga-
ting from HepI was confirmed by product ions m/z 1001.4
and 753.3, due to losses of t-HepIII and tHepIIIÆHepII,
respectively.
Performing MS
2
on ion m/z 1468.3, and subsequent
MS
3
on the resulting product ions determined four
isomeric Hex2 structures. The ion corresponding to the
loss of t-HepIII at m/z 1205.5 was further fragmented to
give the ion at m/z 957.5 corresponding to the loss of an
unsubstituted HepII. This confirmed the structure of the
glycoform in which a disaccharide moiety substitutes
HepI. Furthermore, in the same MS
3
spectrum an ion
was detected at m/z 753.5 due to the loss of a tHex–
HepII unit thus evidencing a structure with one hexose
residue substituted to both HepI and HepII. The ion at
m/z 548.9 corresponded to the loss of tHex–Hex–HepII.
Finally, a structure with one hexose residue each linked
to HepI and HepIII could be determined when the
product ion, m/z 1001.5, corresponding to the loss of a
terminal hexose linked to HepIII, was further fragmented
to give an ion at m/z 753.3 due to the loss of HepII.
Five isomeric Hex3 glycoforms were determined by

performing MS
2
on the parent ion m/z 1672.4 and
subsequent MS
3
on resulting product ions at m/z 1409.6
and 1205.6 due to losses of a terminal heptose and a
terminal Hex–Hep residue, respectively. When the ion at
m/z 1409.6 was further fragmented in a MS
3
experiment it
gave, inter alia, product ions at m/z 883.4 and 753.4 due to
Table 3. Methylation analysis data of the dephosphorylated OS samples derived from H. Influenzae type f strains RM6255, RM7290, and RM6252.
Methylated sugar
a
T
gm
b
Linkage assignment
Relative abundance
OS6255-P OS6252-P OS7290-P
2,3,4,6-Me
4
-Glc 1.00
D
-Glcp-(1- 54 10 15
2,3,4,6-Me
4
-Gal 1.05
D

-Galp-(1- 14 23
2,3,6-Me
3
-Gal 1.16 )4)-
D
-Galp-(1- 13 6
2,3,6-Me
3
-Glc 1.18 )4)-
D
-Glcp-(1- 18 31 36
3,4,6,7-Me
4
-Hep 1.43 )2)-
L
,
D
-Hepp(1- 5 12
2,4,6,7-Me
4
-Hep 1.45 )3)-
L
,
D
-Hepp-(1- 10
2,6,7-Me
3
-Hep 1.50 )3,4)-
L
,

D
-Hepp-(1- 3 13 14
4,6,7-Me
3
-Hep 1.55 )2,3)-
L
,
D
-Hepp-(1- 13 13 8
a
2,3,4,6-Me
4
-Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-
D
-glucitol-1-d
1
, etc.
b
Retention times (T
gm
) are reported relative to
2,3,4,6-Me
4
-Glc.
Fig. 1. ESI-MS spectra (positive mode) of the permethylated (A)
OS6255-P (B) OS7290-P and (C) OS6252-P. The peaks marked by
asterisks
5
indicate glycoforms with mono-methylated phosphate groups
due to glycoforms resulting from incomplete dephosphorylation.

3158 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
losses of HepIÆAnKdo-ol and HexÆHexÆHepII elements,
respectively. Thus two structures with terminal HepIII were
identified: the first with a trisaccharide group linked to
HepII and the second with a disaccharide group linked to
HepII and a hexose linked to HepI. When the ion at m/z
1205.6 was further fragmented in a MS
3
experiment product
ions at m/z 957.6, 753.4, and 549.3 were observed defining
the losses of single substituted HepII, t-HexÆHepII, and
HexÆHexÆHepII. A single Hex4 glycoform was identified in
RM6255 comprising terminal hexose residues substituted to
Table 4. Structures of glycoforms from OS6255-P, OS7290-P and OS6252-P elucidated by ESI-MS
n
. Subscripts denoted by the letters a, b and c
indicated the number of hexose residues in the following structure:
Relative abundance
a
(%) Structure 6255 Structure 7290 Structure 6252 Relative abundance
b
6255 7290 6252 a b c a b c a b c 6255 7290 6252
Hex1 6.3 1 0 0 High
0 0 1 Low
Hex2 17.5 1.2 3.3 2 0 0 2 0 0 2 0 0 Trace Trace Trace
1 1 0 1 1 0 1 1 0 Low Low Medium
0 2 0 0 2 0 0 2 0 Low Low Low
1 0 1 1 0 1 1 0 1 Medium Low High
0 1 1 Low
0 0 2 Low

Hex3 23.3 2.4 12.4 1 2 0 1 2 0 1 2 0 Medium Low Low
2 0 1 1 1 1 1 1 1 Trace Medium Low
1 1 1 0 3 0 0 3 0 Medium Low Low
0 2 1 1 0 2 1 0 2 Low Medium Low
0 3 0 0 2 1 Low Low
2 0 1 Trace
2 1 0 Trace
Hex4 52.9 25.5 27.7 1 2 1 1 2 1 1 2 1 High Low Medium
1 3 0 1 3 0 High Medium
1 1 2 1 1 2 Low Low
0 4 0 Trace
0 3 1 Trace
3 0 1 Trace
1 0 3 Low
2 0 2 Trace
Hex5 6.1 16.5 1 4 0 1 4 0 Low Medium
1 3 1 1 3 1 Medium Low
0 3 2 0 3 2 Trace Trace
1 2 2 1 2 2 Low Low
2 3 0 1 1 3 Trace Trace
Hex6 60.6 15.7 1 3 2 1 3 2 High Medium
2 3 1 Trace
1 4 1 Medium
1 2 3 Trace
0 3 3 Trace
Hex7 1.2 10.3 1 3 3 1 3 3 Medium Medium
1 4 2 1 4 2 Medium Medium
0 4 3 Trace
Hex8 3.0 14.0 1 4 3 1 4 3 High High
a

Relative abundance for each glycoform. Calculated from the intensity of the molecular ion peak relative to the total intensity of all
molecular ion peaks in the MS spectrum expressed as percentage.
b
Relative abundance for structural isomers of each glycoform. Calculated
from the intensity of the product ions in the MS
2
spectrum and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace
(below 2%).
Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3159
both HepI and HepIII, and a HexÆHex unit substituted to
HepII. This structure is defined by a MS
2
experiment on
molecular ion m/z 1876.9 resulting in fragment ions m/z
1409.7 and 1145.5 due to the loss of tHexÆHepIII and
tHexÆHepÆAnKdo-ol, respectively. This structure is further
supported by the MS
3
experiment on m/z 1409.7 in which
the resulting fragment ions m/z 753.4 and 679.4 are due to
losses of tHexÆHexÆHepII and tHexÆHepIÆAnKdo-ol,
respectively.
In the ESI-MS spectra of permethylated OS7290-P and
OS6252-P (Figs 1B and C) seven sodiated parent ions
were observed corresponding to Hex2 to Hex8 glyco-
forms (Hex
1)8
ÆHep
3
ÆAnKdo-ol). In order to obtain

sequence and branching information, these molecular
ions were further fragmented in MS
2
experiments, and if
necessary MS
3
experiments were used. The fragment ions
produced in MS
2
and MS
3
experiments were analyzed in
analogy to OS6255-P described above and provided
evidence for 22 structures present in OS7290-P and 33
structures present in OS6252-P as shown in Table 4. It
was obvious that OS6252-P is a slightly more hetero-
geneous mixture of glycoforms than OS7290-P. The Hex4
and Hex6 glycoforms are the most abundant in OS7290-
P, while the abundance is almost equally distributed
between Hex3 and Hex8 glycoforms in OS6252-P. The
Hex6 glycoform is the most abundant in OS7290-P and
comprises only one structure while the same glycoform
has five isomers in OS6252-P. Thus, when the ion at m/z
2283.6 in OS7290-P was further fragmented in a MS
2
experiment (see Fig. 2A) it showed, inter alia,anionat
m/z 1613.8 due to the loss of a t-Hex–Hex–HepIII unit.
AMS
3
experiment on m/z 1613.8 revealed, inter alia,

the ion at m/z 884.3 due to the loss of a tHex–HepI–
AnKdo-ol unit, which gave evidence for the Hex6
glycoform in OS7290-P being substituted by a disacchar-
ide element at HepIII and a trisaccharide unit at HepII
(see Fig. 2B). Ions corresponding to this glycoform were
identified in the same manner in OS6252-P. However,
when the parent ion at m/z 2283.5 in OS6252-P was
fragmented further in a MS
2
experiment it revealed
additional ions at m/z 1817.8 and 1410.3 due to losses of
tHex–HepIII and tHex–Hex–Hex-HepIII units, respect-
ively (see Fig. 3A). The MS
3
experiment on m/z 1817.8
Fig. 2. The ESI-MS
n
analysis of permethyl-
ated OS7290-P. (A) Product ion spectrum of
m/z 2283.6 corresponding to the sodium
adduct of the Hex6 glycoform. (B) The MS
3
spectrum of the fragment ion m/z 1613.8
resulting from the MS
2
of m/z 2283.6. The
relevant fragmentation pathways are shown to
the right of each spectrum.
Fig. 3. The ESI-MS
n

analysis of permethylated OS6252-P. (A) Prod-
uct ion spectrum of m/z 2283.6 corresponding to the sodium adduct of
the Hex6 glycoform. The proposed fragmentations are shown beside
the spectrum. The number of hexoses, Hex
x
, linked to HepI and HepII
are determined by MS
3
.(B)MS
3
of the ions m/z 1410.3, 1613.9 and
1817.8 resulting from MS
2
of m/z 2283.5. The fragmentation pathways
are shown at the bottom of the spectra.
3160 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
showed ions at m/z 1088.6 and 957.9 due to losses of
tHex–HepI–AnKdo-ol and tHex–Hex–Hex–HepII, respect-
ively. This defined two Hex6 glycoforms having either a
trisaccharide or a tetrasaccharide unit linked to HepII
(see Fig. 3B). When the ion at m/z 1410.3 was further
fragmented in a MS
3
experiment it gave ions at m/z 884.4
and 679.9 due to losses of a HepI–AnKdo-ol and tHex–
HepI–AnKdo-ol units, respectively. This gave evidence
for two Hex6 glycoforms having either a disaccharide or
a trisaccharide unit linked to HepII (see Fig. 3B).
NMR spectroscopy on LPS-OH from RM6255
and O-deacylated OS7290 and OS6252

The
1
H NMR spectra were assigned using chemical shift
correlation techniques (COSY, TOCSY, HMQC, HMBC
and HSQC experiments). The chemical shift data corres-
ponding to Hif 6255 is given in Table 5. NMR data
corresponding to Hif 7290 and 6252 were found to be
identical and are combined in Table 6. Subspectra corres-
ponding to the individual glycosyl residues were identified
on the basis of spin-connectivity pathways delineated in
the
1
H chemical shift correlation maps, the chemical shift
values, and the vicinal coupling constants. The chemical
shift data are consistent with each
D
-sugar residue being
present in the pyranosyl ring form. Further evidence for this
conclusion was obtained from NOE data [Table 7 and
Fig. 4 (RM6255) and Fig. 5 (RM6252)], which also served
to confirm the anomeric configurations of the linkages and
the monosaccharide sequence. The Hep ring systems were
identified on the basis of the small J
1,2
-values and their
a-configurations were confirmed by the occurrence of single
intraresidue NOE between the respective H-1 and H-2
resonances.
The structure of the Hex4 glycoform in strain RM6255
was determined by examining the

1
H-NMR spectrum of its
LPS-OH in detail. Anomeric
1
H/
13
C NMR resonances of
HepI, HepII and HepIII were identified at d 5.17/99.22,
5.78/97.6 and 5.08/100.1, respectively. Four anomeric
signals corresponding to Glc residues I to IV were observed
at d 4.57/101.7, 5.26/100.2, 4.54/102.4 and 4.54/99.3,
respectively. In addition, the anomeric signals of the
a-andb-linked GlcN residues of the lipid A part were
observed at d 5.48/93.3 and 4.62/101.6, respectively. Chem-
ical shift data are consistent with GlcI, III and IV being
terminal residues and GlcII being substituted in O-4 in
agreement with methylation analyses.
The occurrence of interresidue NOESY connectivities
between the proton pairs HepIII H-1/HepII H-2, HepII
H-1/HepI H-3, and HepI H-1/Kdo H-5 confirmed the
sequence of the heptose-containing trisaccharide unit in
the inner-core region and the point of attachment to Kdo
(structure 1). The occurrence of interresidue NOE between
H-1 of GlcI and H-4/H-6 of HepI confirmed the 1,4
linkage between GlcI and HepI. Interresidue NOE
connectivities between proton pairs GlcIII H-1/GlcII
H-4/H-6 and GlcII H-1/HepII H-2/H-3 established the
sequence of a disaccharide unit and its attachment point
to HepII as b-
D

-Glcp-(1fi4)-a-
D
-Glcp-(1fi3)-
L
-a-
D
-Hepp-
(1fi. Interresidue NOE between H-1 of GlcIV and H-3/
H-2 of HepIII gave evidence for the a b-
D
-Glcp-(1fi3)-
L
-
a-
D
-Hepp-(1fi unit.
Table 5.
1
Hand
13
C NMR chemical shifts for O-deacylated LPS of H. influenzae type f strain RM6255. Data was recorded in D
2
O containing 2 m
M
perdeutero-EDTA and 10 mgÆmL
)1
perdeutero-SDS at 30 °C.
3
J
H,H

-values for anomeric
1
H resonances are given in parenthesis. ND, Not
determined; NR, not resolved (small coupling).
Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6
A
/C-6 H-6
B
H-7
A
/C-7 H-7
B
GlcNI fi6)-a-
D
-GlcpN-(1fi 5.48 3.84 ND ND ND ND ND
93.3 53.40
GlcNII fi6)-b-
D
-GlcpN(1fi 4.62 3.88 ND 4.03 ND ND ND
101.6 55.6 72.0
HepI fi3,4)-
L
-a-
D
-Hepp(1fi I 5.17 (NR) 4.19 4.13 4.35 – 4.04 3.68 3.68
99.22 69.7 72.4 72.3 67.7 63.1
HepII fi2,3)-
L
-a-
D

-Hepp(1fi II 5.78 (NR) 4.28 4.08 ND 3.88 4.59 ND ND
97.6 78.1 78.3 ND 73.3
HepIII fi3)-
L
-a-
D
-Hepp(1fi III 5.08 (NR) 4.04 4.21 ND ND ND 3.73 3.86
100.1 67.6 76.5 63.1
GlcI b-
D
-Glcp (1fi IV 4.57 (8.0) 3.39 3.47 3.31 3.47 3.98 3.77
101.7 73.3 75.8 70.0 75.8 60.3
GlcII fi4)-a-
D
-Glcp(1fi V 5.26 (4.0) 3.59 3.86 3.70 3.89 3.97 3.89
100.0 71.2 71.3 78.6 70.8 59.4
GlcIII b-
D
-Glcp (1fi VI 4.54 (8.1) 3.41 3.52 3.44 3.52 3.95 3.78
102.4 72.5  75.0 69.2 75.5 60.3
GlcIV b-
D
-Glcp (1fi VII 4.54 (7.9) 3.42 3.44 3.41 3.44 3.93 3.76
99.3 72.5  75.0 69.3  75.0 60.3
Kdo fi4,5)-a-Kdop-(2fi 2.03/2.37 ND 4.29 ND 3.80
ND ND ND
PPEtn 4.14 3.28
61.4 39.4
PEtn 4.08 3.26
60.6 39.5

Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3161
The ESI-MS data of OS6255 (Table 2) indicated that the
most abundant glycoform in this strain was monoacetyl-
ated. The acetylation site was identified by NMR on
OS6255 (data not shown). It was observed that chemical
shift values for several HepIII resonances in OS6255
differed considerably from the corresponding values in
LPS-OH. Thus resonances for H-1/C-1, H-2/C-2 and
H-3/C-3 were observed at d 5.13/97.6, 5.16/68.9 and 4.47/
73.8, respectively. The chemical shift values were consistent
with acetylation at O-2 as the H-1 (+ 0.05 p.p.m), H-2
(+ 1.2), H-3 (+ 0.26 p.p.m) and C-2 (+ 1.3 p.p.m) signals
were shifted downfield, while C-1 () 2.5 p.p.m) and C-3
() 2.7 p.p.m) were shifted upfield [39]. From the combined
data it could thus be concluded that the Hex4 glycoform of
RM6255 has the structure 2.
Structural details of the oligosaccharide epitopes in
RM7290 and RM6252 were provided by
1
H-NMR and
13
C-NMR data of OS7290 and OS6252 (Table 6). Several
signals for methylene protons of AnKdo-ol were observed
in the COSY and TOCSY spectra in the region 2.25–
1.65 p.p.m. This is due to the fact that several anhydro-
forms of Kdo are formed during the hydrolysis by
elimination of phosphate or pyrophosphoethanolamine
from the C-4 position [40]. Anomeric resonances of HepI,
HepII and HepIII of OS7290 were identified at d 5.06–5.16,
5.68–5.72 and 5.08, respectively. As observed earlier, several

anomeric signals for HepI and HepII appear due to the
microheterogeneity caused by the anhydro-forms of Kdo
[40]. Subspectra corresponding to GlcI to IV, GalI and
GalII were identified in the two-dimensional COSY and
TOCSY spectra at d 4.54, 5.33, 4.58, 4.46, 4.47, and 4.44,
respectively. In addition, a minor spin system corresponding
to a-
D
-Galp was identified at d 4.96.Chemicalshiftdataare
consistent with GlcI and GalI being terminal residues in
Table 6.
1
Hand
13
C NMR chemical shifts for OS7290 and OS6252. DatawasrecordedinD
2
Oat30°C.
3
J
H,H
-values for anomeric
1
H resonances
are given in parenthesis. Signals corresponding to PCho methyl protons and carbons occurred at 3.20 and 53.6 p.p.m., respectively. Pairs of deoxy
protons of reduced, AnKdo were identified in the DQF-COSY at d 1.91–2.30.
3
J
H,H
-values for anomeric
1

H resonances are given in parenthesis.
NR, Not resolved (small coupling); ND, not determined.
Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6
A
/C-6 H-6
B
H-7
A
/C-7 H-7
B
Hex6 glycoform
HepI fi3,4)-
L
-a-
D
-Hepp(1fi I 5.06–5.16 (NR) 4.01 4.06 4.15 ND 4.31 ND
96.2 69.7 76.8 74.2 72.8
HepII fi2,3)-
L
-a-
D
-Hepp(1fi II 5.68–5.72 (NR) 4.29 4.12 ND 3.73 4.56 3.79 3.90
97.9 77.8 77.4 71.2 73.9 61.7
HepIII fi2)-
L
-a-
D
-Hepp(1fi III 5.08 (NR) 4.01 4.08 ND ND ND ND
98.7 77.8 69.1
GlcI b-

D
-Glcp (1fi IV 4.54 (8.5) 3.35 3.47 3.41 3.47 3.77 3.96
102.2 73.3 76.0 72.0 76.0 60.6
GlcII fi4)-a-
D
-Glcp(1fi V 5.33 (4.8) 3.62 3.87 3.71 3.89 3.97 3.90
99.6 71.1 71.0 78.0 71.0 59.6
GlcIII fi4)-b-
D
-Glcp (1fi VI 4.58 (8.0) 3.39 3.67 3.71 3.71 3.84 4.00
102.0 72.0 74.4 78.0 71.0 59.6
GalI b-
D
-Galp(1fi VII 4.47 (8.4) 3.55 3.68 3.94 3.70 ND
102.5 76.5 72.0 68.0 74.9
GlcIV fi4)-b-
D
-Glcp (1fi VIII 4.46 (8.1) 3.42 3.70 3.69 3.69 3.85 4.00
101.5 69.6 74.3 78.0 70.9 59.6
GalII b-
D
-Galp(1fi IX 4.44 (8.4) 3.57 3.70 3.95 3.70 ND
102.5 76.5 72.0 68.0 74.9
PEtn 4.12 3.24
62.3 40.0
Hex8 glycoform
GalI fi4)-b-
D
-Galp(1fi VII 4.54 (7.8) 3.59 3.76 4.07 3.81 ND
102.3 70.5 72.3 76.8 75.5

GalIII a-
D
-Galp(1fi X 4.98 (3.9) 3.84 3.90 4.05 4.37 3.72 3.72
99.8 68.0 68.7 72.2 70.5 60.0 ND
GalII fi4)-b-
D
-Galp(1fi IX 4.52 (7.8) 3.61 3.76 4.07 3.81 ND
102.3 70.5 72.3 76.8 75.5
GalV a-
D
-Galp(1fi XI 4.96 (3.9) 3.86 3.92 4.05 4.37 3.72 3.72
99.8 68.0 68.8 72.2 70.6 60.0 ND
3162 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
agreement with methylation analyses. Interresidue NOE
connectivities between proton pairs GlcI H-1/HepI H-4,6,
HepIII H-1/HepII H-2 and HepII H-1/HepI H-3 confirmed
the presence of the common inner-core triheptosyl moiety of
H. influenzae substituted with a glucose residue at HepI.
Interresidue NOE between GlcIII H-1/GlcII H-4 and H-6,
and GlcII H-1/HepII H-3/H-2 confirmed the presence of the
structural element fi4)-b-
D
-Glcp-(1fi4)-a-
D
-Glcp-(1fi3)-
L
-
a-
D
-Hepp-(1fi. This was corroborated by cross-peaks in the

HMBC spectrum between GlcIII H-1/GlcII C-4 and GlcII
H-1/HepII C-3. In addition, transglycosidic NOEs between
GlcIV H-1/HepIII H-2/H-1 confirmed the presence of fi4)-
b-
D
-Glcp substituting HepIII at O-2. In the HMBC
spectrum, a cross-peak between GlcIV H-1/HepIII C-3
was in agreement with this. Furthermore, NOE cross-peaks
were detected between GalI/GalII H-1 and H-4 and
H-6
a,b
of GlcIII/GlcIV, evidence that these residues
were substituted by b-
D
-Galp residues see structure 3.
This was also confirmed by a cross-peak between GalI/
GalII H-1 and GlcIII/GlcIV C-4 in the HMBC spec-
trum. GalI and GalII are interchangeable since it was
not possible to differentiate which b-Glc residue they
were substituted to, as both b-Glc residues have
overlapping H-4 resonances.
From the combined data on RM7290, the Hex6 structure
in H. influenzae RM7290 is proposed, see structure 3.
A minor spin system corresponding to terminal a-
D
-
Galp was identified at d 4.97. The a-
D
-Galp residues had
interresidue NOE connectivities to b-

D
-Galp H-4. This
finding indicated the existence of a a-
D
-Galp-(1fi4)-b-
D
-
Galp-(1fi moeity present in RM7290, see structure 4.
1
H-NMR and
13
C-NMR data of O-deacylated OS6252
were found to be identical to those observed for OS7290.
In particular, RM6252 had the same NOE and HMBC
interactions observed in RM7290 and therefore struc-
tures 3 and 4 also represent structures found in RM6252.
As indicated by ESI-MS RM6252 had a higher propor-
tion of Hex7 and Hex8 glycoforms. Thus it was not
surprising that some signals in the NMR spectra were
more pronounced than in RM7290. In particular, the
anomeric
1
Hand
13
C signals in gHMQC for a-
D
-Galp
were stronger in RM6252. Moreover, clearly separated
HMBC cross-peaks between H-1 of GalIII/GalIV and
C-4 of GalI/GalII could be observed, and the NOE

interaction between H-1 of GalIII/GalIV and H-4 of
GalI/GalII was more intense. It was thus concluded that
structure 4 represents the Hex8 glycoform in both
RM7290 and RM6252. It is assumed that minor glyco-
forms observed by ESI-MS
n
on permethylated OS sam-
ples (see Table 4) represent truncated forms of this
glycoform.
Strain RM6252 reacted with a monoclonal antibody,
TEPC 15, which has high affinity for phosphocholine [15].
This strain was also the only one for which a characteristic
singlet corresponding to the methyl groups of PCho could be
observed in the
1
H NMR spectrum at d 3.22. The corres-
ponding
13
C signal was observed in the HMQC spectrum at
d 53.5 and spin-systems for ethylene protons from this
residue were observed at d 4.38 and 3.74 in the COSY
spectrum. From NMR data it was estimated that this strain
expressed about 8.6% of this substituent and due to this low
amount the location of this residue could not be determined.
Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3163
Discussion
We report the first structural details for the LPS from
H. influenzae type f strains. Previous analyses on LPS from
H. influenzae strains expressing a capsule have been limited
to type b and a single strain derived from a type d isolate.

Population analysis of a large number of H. influenzae
strains by Musser and colleagues using multilocus enzyme
electrophoresis typing indicated that type f isolates form a
relatively tight group of strains that is distinct from the type
b and type d isolates [41]. H. influenzae type f strains
RM6255, RM7290 and RM6252 are three epidemiologi-
cally distinct isolates that were selected to be representative
ofthediversitywithinthetypefcluster.
Previous studies of LPS from H. influenzae have
resulted in a structural model consisting of a conserved
Fig. 4. Selected region of the two-dimensional
NOESY spectrum (mixing time 250 ms) of
LPS-OH derived from LPS of RM6255.
Cross-peaks of significant importance are
labelled.
Table 7. NOE data for H. Influenzae type f strains RM6255, RM7290 and RM6252. Cross-peaks detected in NOESY spectrum were acquired with
250 ms spin lock time.
Anomeric proton
Observed proton
Intraresidue NOE Interresidue NOE
RM6255
HepI I H-2 Kdo H-5 and H-7
HepII II H-2 HepI H-3, HepIII H-1, H-5 and H-7
HepIII III H-2 HepII H-1 and H-2, GlcI H-4
GlcI IV H-3 and H-5 HepI H-4 and H-6
GlcII V H-2 HepII H-3 (weak signals to H-2 and H-4)
GlcIII VI H-3 and H-5 GlcII H-4 and H-6
GlcIV VII H-3 and H-5 HepIII H-2 and H-3
RM7290 and RM6252Hex6 glycoform
HepI I H-2 Kdo H-5

HepII II H-2 HepI H-3, HepIII H-1
HepIII III H-2 HepII H-1 and H-2, GlcIV H-1
GlcI IV H-3 and H-5 HepI H-4 and H-6
GlcII V H-2 HepII H-3 (and weak signal to H-2)
GlcIII VI H-3 and H-5 GlcII H-4 and H-6
GalI VII H-3 and H-5 GlcIV or GlcIII H-4 and H-6
GlcIV VIII H-3 and H-5 HepIII H-1 and H-2
GalII IX H-3 and H-5 GlcIV or GlcIII H-4 and H-6
RM7290 and RM6252 Hex8 glycoform
GalI VII H-3 and H-5 GlcIV or GlcIII H-4 and H-6
GalII IX H-3 and H-5 GlcIV or GlcIII H-4 and H-6
GalIII X H-2 GalI or GalII H-4
GalIV XI H-2 GalI or GalII H-4
3164 H. H. Yildirim et al. (Eur. J. Biochem. 270) Ó FEBS 2003
L
-glycero-
D
-manno-heptose-containing trisaccharide inner
core attached via a phosphorylated Kdo unit to the lipid A
moiety (structure 1). The results of the present study confirm
the presence of this structural element in strains RM6255,
RM7290 and RM6252. All strains express heterogeneous
mixtures of LPS molecules in which extension can occur
from each of three inner-core heptose residues. HepI is
found to be substituted by glucose (R
1
¼ b-
D
-Glcp). As
observed for type b strains (Eagan, RM7004) HepII is

substituted by an a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D
-
Glcp-(1fi4)-a-
D
-Glcp unit in RM7290 and RM6252 or
truncated versions thereof. Strain RM6255 does not express
galactose in its LPS, thus it only shows a cellobiose unit
elongating from HepII (R
2
¼ b-
D
-Glcp-(1fi4)-a-
D
-Glcp).
All strains show substitution at HepIII by a b-
D
-Glcp
residue. However, in strain RM6255 it is linked to O-3
(R
3
¼ b-
D
-Glcp-(1fi3)whereasintheothertwostrainsitis
linkedtoO-2(R
3

¼ b-
D
-Glcp-(1fi2) of HepIII. Strain
RM6255 does not show further elongation from this
residue. Strains RM7290 and RM6252 are more extended
and express the globoside unit (a-
D
-Galp-(1fi4)-b-
D
-Galp-
(1fi4)-b-
D
-Glc) or truncated versions thereof from HepIII.
Thus, the investigated Hif strains were found to express
oligosaccharide epitopes common for H. influenzae LPS.
However, chain extension by globoside units from both
HepII and HepIII in the same glycoform has not been
observed previously.
The availability of the complete genome sequence of
H. influenzae strain Rd has facilitated a comprehensive
study of LPS biosynthetic loci in the type b strain Eagan
(RM153) and in a capsule deficient strain Rd
–
(RM118),
derived from a type d isolate [7,12]. Gene functions have
been identified that are responsible for most of the steps in
the biosynthesis of the oligosaccharide portion of the LPS
molecules. The genes lpsA, lic2A and lgtC encode
glycosyltransferase enzymes involved in the addition of
the globoside epitope to O-2 of HepIII in strain Rd

–
.
LpsA is responsible for adding the first glycose residue,
lic2A is responsible for adding b-
D
-Galp in a 1,4 linkage to
the b-
D
-Glcp residue, and lgtC is responsible for adding
a-
D
-Galp in a 1,4 linkage to the b-
D
-Galp residue. The
lic2A and lgtC homologues were also found in strain
Eagan and were shown to be responsible for linking the
same sugars but as part of the Galp-(1fi4)-b-
D
-Galp-
(1fi4)-b-
D
-Glcp-(1fi4)-a-
D
-Glcp extension from HepII in
that strain. The digalactoside expressed in the LPS of
H. influenzae is proposed to play some role in evasion
of the host immune response by behaving as a mimic of
epitopes found on host glycolipids. PCR and/or Southern
blot analyses performed on strains RM6255 and RM6252
revealed that lpsA, lic2A and lgtC are each present in both

strains (data not shown). These data indicate that strain
RM6255 apparently retains a genetic capacity to add
galactose residues (lic2A and lgtC) to either the HepII or
HepIII extensions observed. An explanation for the lack
of galactose addition observed in this strain might be that
the lic2A gene has been altered by mutation or simply that
at the time of growth to prepare the LPS for analysis,
the number of tetranucleotide repeats within this phase
variable gene was inappropriate for expression of the
galactosyl transferase.
A globoside unit extended from HepIII in strains
RM6252 and RM7290 is the same as in the LPS of strain
Rd except that in strain Rd the structure can be capped
by a GalNAc residue to form globotetraose. The gene
required to add this terminal GalNAc, lgtA,isabsentin
each of the three strains included in the present study
(data not shown).
The lex2 gene, which is responsible for adding a b-
D
-
Glcp residue to the b-
D
-Glcp-(1fi4)-HepI unit, was found
in strain RM6252 whereas RM6255 was devoid of this
gene (data not shown). None of the strains expressed high
levels of chain extension from HepI. However, ESI-MS
n
on permethylated and dephosphorylated OS samples
indicated minor glycoforms with disaccharide units elon-
gating from HepI in all three strains. RM6255, which does

not contain lex2 might then express a b-
D
-Galp-(1fi4)-
b-
D
-Glcp-HepI unit as observed in H. influenzae 2019
whereas the other strains may express b-
D
-Glcp-(1fi4)-
b-
D
-Glcp units [42].
Modification of LPS with Neu5Ac is widespread
among H. influenzae strains [7]. However, levels of
Fig. 5. Selected region of the two-dimensional
NOESY spectrum (mixing time 250 ms) of OS
derived from LPS of RM6252. Cross-peaks
that are characteristic for the Hex6 and Hex8
glycoforms are labelled.
Ó FEBS 2003 Structural diversity of LPS in H. influenzae (Eur. J. Biochem. 270) 3165
sialylation vary and it has been shown that sialic acid
expression in H. influenzae LPS is dependent on growth
conditions. In general, the amounts of sialic acid are low
in material obtained after liquid growth. A phase variable
gene, lic3A,encodesthea-2,3-sialyltransferase that adds
Neu5Ac to a terminal lactose unit from HepIII. Although
each strain was found to express the lic3A gene (D. Hood,
unpublished results) none of the type f strains were found
to contain detectable amounts of Neu5Ac in the LPS.
PCho is a prominent substituent in H. influenzae LPS.

Levels of expression have been shown to be high in NTHi
and strain Rd, however, type b strains express PCho only to
a minor extent [15]. The presence of PCho has been shown
to be important in colonization and virulence [43,44]. The
expression and phase variation of the PCho epitope on
H. influenzae LPS require the four genes of the lic1 locus.
The three strains in our study contained the lic1 locus,
however, PCho was detected only in the LPS from strain
RM6252 in very low levels. PCho expression in type f
strains during in vitro growth was lower than was found in
type b strains. Analysis of large numbers of individual
colonies of strain RM6255 and RM7290 by immunoblot-
ting with monoclonal antibody TEPC 15 confirmed that
PCho was variably expressed in each strain, albeit at a very
low frequency (data not shown). The LPS of type f strains is
substituted by acetates and glycine as has been observed for
other strains. The position of one O-acetyl group was found
to be on HepIII in RM6255. The biological implication of
these substituents has not been determined.
Acknowledgements
The Swedish NMR centre in (Go
¨
teborg, Sweden) is acknowledged for
providing access to their 600 MHz facilities. Mary Deadman is
acknowledged for culturing of H. influenzae strains. Dr Jianjun Li is
acknowledged for CE-ESI-MS/MS experiments.
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