Báo cáo khoa học: Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide pdf

Novel globoside-like oligosaccharide expression patterns
in nontypeable Haemophilus influenzae lipopolysaccharide
Susanna L. Lundstro
¨
m
1
, Brigitte Twelkmeyer
1
, Malin K. Sagemark
1
, Jianjun Li
2
,
James C. Richards
2
, Derek W. Hood
3
, E. Richard Moxon
3
and Elke K. H. Schweda
1
1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Huddinge, Sweden
2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
3 Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK
Haemophilus influenzae is an important cause of
human disease worldwide and exists in encapsulated
(type a–f) and unencapsulated (nontypeable) forms.
Type b capsular strains are associated with invasive
bacteraemic diseases, including meningitis, epiglottitis,
cellulitis and pneumonia, whereas acapsular or non-

typeable strains of H. influenzae (NTHi) are primary
pathogens in otitis media and cause both acute and
chronic lower respiratory tract infections [1,2]. The
potential of H. influenzae to cause disease depends
Keywords
globoside; globotetraose; Haemophilus
influenzae; lipopolysaccharide; sialyllactose
Correspondence
E. Schweda, University College of South
Stockholm, Clinical Research Centre,
Novum, S-141 86 Huddinge, Sweden
Fax: +46 85 858 3820
Tel: +46 85 858 3823
E-mail:
(Received 29 May 2007, revised 18 July
2007, accepted 25 July 2007)
doi:10.1111/j.1742-4658.2007.06011.x
We report the novel pattern of lipopolysaccharide (LPS) expressed by two
disease-associated nontypeable Haemophilus influenzae strains, 1268 and
1200. The strains express the common structural motifs of H. influenzae;
globotetraose [b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-b-d-
Glcp] and its truncated versions globoside [a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-
b-d-Glcp] and lactose [b-d-Galp-(1fi4)-b-d-Glcp] linked to the terminal
heptose (HepIII) and the corresponding structures with an a-d-Glcp as the
reducing sugar linked to the middle heptose (HepII) in the same LPS mole-
cule. Previously these motifs had been found linked only to either the proxi-
mal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-a-d-
Hepp-(1fi2)-[PEtnfi6]-l-
a-d-Hepp-( 1fi3)-l-a-d-Hepp-(1fi5)-[PPEtnfi4]-
a-Kdo-(2fi6)-lipid A. This novel finding was obtained by structural studies

of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core
oligosaccharide material, as well as electrospray ionization-multiple-step
tandem mass spectrometry on permethylated dephosphorylated oligosaccha-
ride material. A lpsA mutant of strain 1268 expressed LPS of reduced
complexity that facilitated unambiguous structural determination. Using
capillary electrophoresis-ESI-MS ⁄ MS we identified sialylated glycoforms
that included sialyllactose as an extension from HepII, this is a further novel
finding for H. influenzae LPS. In addition, each LPS was found to carry
phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200
expressed identical LPS structures to 1268 with the difference that
strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS
has glycine at the same position.
Abbreviations
AnKdo-ol, reduced anhydro Kdo; CE, capillary electrophoresis; Hep,
L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine;
Kdo, 3-deoxy-
D-manno-oct-2-ulosonic acid; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated lipopolysaccharide;
MS
n
, multiple-step tandem mass spectrometry; Neu5Ac, N-acetyl neuraminic acid; NTHi, nontypeable Haemophilus influenzae; OS,
oligosaccharide; PCho, phosphocholine; PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine; tHep, terminal heptose.
4886 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
upon its surface-expressed carbohydrate antigens, cap-
sular polysaccharide [3] and lipopolysaccharide (LPS)
[4]. LPS is an essential and characteristic surface com-
ponent of H. influenzae. This bacterium has been
found to express short-chain LPS, lacking O-specific
polysaccharide chains and is often referred to as lipo-
oligosaccharide. Extensive structural studies of LPS
from H. influenzae by us and others have led to the

identification of a conserved glucose-substituted trihep-
tosyl inner-core moiety l-a-d-Hepp-(1fi2)-[PEtnfi6]-
l-a-d-Hepp-(1fi3)-[b-d-Glcp-(1fi4)]-l-a-d-Hepp linked
to lipid A via 3-deoxy-d-manno-oct-2-ulosonic acid
(Kdo) 4-phosphate. This inner-core unit provides the
template for attachment of oligosaccharides and non-
carbohydrate substituents [5]. The outer core region of
NTHi LPS mimics host glycolipids and the expression
of terminal epitopes is subject to high-frequency phase
variation, leading to a very heterogeneous population
of LPS molecules within a single strain. Phase varia-
tion is thought to provide an adaptive mechanism
which is advantageous for the survival of bacteria con-
fronted by the differing microenvironments and the
immune responses of the host. Several structures mim-
icking the globoside series of mammalian glycolipids
have been identified in NTHi LPS such as globotetraose
[b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-
b-d-Glcp-(1fi], globoside [a-d
-Galp-(1fi4)-b-d-Galp-
(1fi4)-b-d-Glcp], lactose [b-d-Galp-(1fi4)-b-d-Glcp]
and sialyllactose [a-Neu5Ac-(2fi3)-b-d-Galp-(1fi4)-b-
d-Glcp(1fi] [5]. Biosynthesis of these oligosaccharide
extensions has been shown to proceed in a stepwise
fashion [6]. It has also been shown that H. influenzae
can express sialyllacto -N-neotetraose [a-Neu5Ac-(2fi3)-
b-d-Galp-(1fi4)-b-d-GlcpNAc-(1fi3)-b-d-Galp-(1fi4)-
b-d-Glcp-(1fi] or the related structure, ( PEtnfi6)-a
-d-
GalpNAc-(1fi6)-b-d-Galp-(1fi4)-b-d-GlcpNAc-(1fi3)-

b-d-Galp-(1fi4)-b-d-Glcp-(1fi, both linked to l-gly-
cero-d-manno-heptose (Hep)I. Biosynthesis of these ter-
minal tetrasaccharide moieties has been found to
resemble that of the O-antigen repeating unit, with the
tetrasaccharide being added en bloc [7]. Noncarbo-
hydrate substituents such as pyrophosphoethanolamine
(PPEtn), phosphoethanolamine (PEtn), phosphocholine
(PCho), acetate (Ac) and glycine (Gly) are common in
NTHi LPS [5].
Our previous studies have focused on the conserva-
tion and variability of patterns of LPS expressed in a
representative set of NTHi clinical isolates obtained
from otitis media patients [8–16] and relating this to the
role of LPS in commensal and virulence behaviour.
Recently, we demonstrated that oligosaccharides
containing terminal sialic acid epitopes are essential
virulence determinants in experimental otitis media [17].
In this study, we present novel LPS structures
expressed in NTHi strains 1268 and 1200. The strains
were previously shown to be very closely related [18].
Herein, we demonstrate that the two strains, as pre-
dicted, have almost identical LPS structures, the only
difference being the presence of O-acetyl groups in
strain 1200. Both strains were found to express LPS
glycoforms containing globoside and globoside-like
epitopes extending simultaneously from HepIII and
HepII, respectively. These LPS glycoforms have not
previously been found in H. influenzae. In order to
unambiguously establish this, we made use of a geneti-
cally defined isogenic mutant strain, NTHi 1268lpsA,

which had oligosaccharide extensions from HepI and
HepII only. The mutant strain also allowed us to iden-
tify sialyllactose units substituting HepII. This is the
first time that sialyllactose has been detected in that
molecular environment. The presence of sialylated gly-
coforms likely contributes to the resistance of the
strain to killing by normal human serum.
Results
NTHi wild-type strains 1268 and 1200 and mutant
strain 1268lpsA
NTHi strains 1268 and 1200 are clinical isolates origi-
nating from the Finnish Otitis Media Study Group. The
strains have the same ribotype, and by multilocus
sequence typing had identical nucleotide sequences in
three of seven LPS alleles [18]. Because of the hetero-
geneous mixtures of LPS glycoforms typical of wild-type
NTHi strains, a lpsA mutant strain of 1268 was made to
facilitate the elucidation of its structure. It has previ-
ously been shown that the lpsA gene is responsible for
addition of a hexose (Hex) to the distal heptose (HepIII)
of the inner-core of the Hi LPS molecule [6]. By disrupt-
ing the lpsA gene, we sought to construct a mutant
(1268lpsA) lacking any chain elongation from HepIII,
but otherwise identical to the wild-type 1268 strain. The
two NTHi wild-type and the 1268lpsA mutant strains
were grown in liquid media, the bacteria harvested and
desiccated, the LPS was then isolated by extraction
using the phenol ⁄ chloroform ⁄ light petroleum method.
Characterization of LPS from NTHi strains 1268,
1200 and 1268lpsA

In earlier investigations it was found that the LPS of
NTHi strains 1268 and 1200 contained ester-linked
glycine and Neu5Ac, as shown by high-performance
anion-exchange chromatography with pulsed ampero-
metric detection following treatment of samples with
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4887
0.1 m NaOH and neuraminidase [19,20]. Furthermore,
the lipid A backbone of the respective LPS has been
described by Helander et al. [21,22].
LPS from all strains was treated with anhydrous
hydrazine under mild conditions to give the water-sol-
uble O-deacylated lipopolysaccharide (LPS-OH) which
was subjected to compositional and linkage analyses as
well as ESI-MS.
Compositional sugar analysis of LPS-OH from the
wild-type strain (1268) indicated d-glucose (Glc),
d-galactose (Gal), 2-amino-2-deoxy-d-glucose (GlcN),
2-amino-2-deoxy-d-galactose (GalN) and l-glycero-d-
manno-heptose (Hep) in a ratio of 32 : 28 : 21 : 9 : 10,
as identified by GLC-MS of their corresponding alditol
acetate and 2-butyl glycoside derivatives (Table S1).
Sugar analysis of LPS-OH from strain 1200 revealed
the presence of the same sugars as in 1268 in compara-
ble amounts (Table S1).
LPS-OH samples were dephosphorylated with 48%
hydrogen fluoride prior to methylation analysis. Mate-
rial from 1268 showed terminal Glc, terminal Gal,

4-substituted Gal, 4-substituted Glc, 3-substituted Gal,
terminal Hep, 2-substituted Hep, 3,4-substituted Hep,
terminal GalN, 2,3-substituted Hep, 4-substituted
GlcN and 6-substituted GlcN in the relative amounts
of 16 : 4 : 7 : 14 : 3 : 11 : 6 : 14 : 2 : 19 : 2 : 2. Meth-
ylation analysis on dephosphorylated LPS-OH from
NTHi strain 1200 revealed the presence of the same
sugars as 1268 in comparable amounts (Table S2). The
methylation analysis data were consistent with trian-
tennary structures in NTHi 1268 and 1200, 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 ESI-MS spectrum of LPS-OH from 1268 revealed
abundant molecular peaks corresponding to triply and
quadruply deprotonated ions (Table 1). The MS data
indicated the presence of heterogeneous mixtures of
glycoforms, consistent with each molecular species con-
taining the conserved PEtn substituted triheptosyl
inner-core moiety attached via a phosphorylated Kdo
linked to the O-deacylated lipid A (lipid A-OH). As a
characteristic feature, populations of glycoforms were
observed that differed by 123 Da (i.e. a PEtn group),
consistent with either phosphate or PPEtn substitution
at the O-4 position of the Kdo residue [23–25]. For clar-
ity, glycoforms containing five Hex with no N-acetyl-
hexosamine (HexNAc) residue are referred to as Hex5
glycoforms. Glycoforms containing five Hex including a
HexNAc residue are referred to as HexNAcHex4 glyco-
forms. In the ESI-MS spectrum (negative mode) major

quadruply charged ions were observed at m ⁄ z 609.4 and
640.2 corresponding to glycoforms with respective com-
positions PChoÆHex
2
ÆHep
3
ÆPEtnÆPÆKdoÆlipid A-OH
and PChoÆHex
2
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH. Ions
corresponding to HexNAc containing glycoforms with
respective compositions PChoÆHexNAcÆHex
4
ÆHep
3
Æ
PEtnÆPÆKdoÆlipid A-OH and PChoÆHexNAcÆHex
4
ÆHep
3
Æ
PEtn
2
ÆPÆKdoÆlipid A-OH, and PChoÆHexNAcÆHex
5
Æ

Hep
3
ÆPEtnÆPÆKdoÆlipid A-OH and PChoÆHexNAcÆHex
5
Æ
Hep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH were detected at m ⁄ z
741.3 ⁄ 772.1 and 781.8 ⁄ 812.8. Furthermore, glycoforms
with compositions PChoÆHex
5
ÆHep
3
ÆPEtnÆPÆKdoÆlipid
A-OH and PChoÆHex
5
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH
were indicated at m ⁄ z 731.1 and 761.7, respectively.
Peaks of low intensity corresponding to a minor Hex-
NAcHex4 glycoform without a PCho substituent were
also identified. The ESI-MS spectrum of LPS-OH from
strain 1200 showed the same ions as 1268 except for
those corresponding to HexNAcHex4 glycoforms with-
out a PCho substituent (Table 1).

ESI-MS data of LPS-OH from 1268lpsA showed less
heterogeneity with no indications of Hex5 or HexNAc-
Hex5 glycoforms. Ions corresponding to the HexNAc-
Hex4 glycoforms lacking PCho were moderately higher
in abundance in 1268lpsA than in 1268 (Table 1).
Ions corresponding to sialylated glycoforms were
not unambiguously identified in the full ESI-MS spec-
tra of LPS-OH samples due to extensive overlap with
those corresponding to major, nonsialylated glyco-
forms, and ⁄ or low abundance. However, their presence
was confirmed for LPS-OH of 1268lpsA in precursor
ion monitoring tandem mass spectrometry experiments
by scanning for loss of m ⁄ z 290 (Neu5Ac, negative ion
mode) or m ⁄ z 274 (Neu5Ac-H
2
O, positive mode) fol-
lowing capillary electrophoresis (CE)-ESI-MS ⁄ MS.
The data are shown in Fig. 1 and summarized in
Table S3. In the precursor negative-mode ion-scan
spectrum (Fig. 1A) quadruply and triply charged ions
corresponding to a complex mixture of sialylated gly-
coforms containing three to six hexose residues were
observed. The major ion at m ⁄ z 909.5 corresponded to
a Hex3 glycoform with the composition Neu5AcÆ Hex
3
Æ
Hep
3
ÆPEtnÆPÆKdoÆlipid A-OH. Particularly noteworthy
are HexNAc-containing glycoforms detected at m ⁄ z

1086.0, 1127.5, 1180.5, 1207.0 and 1249.5 having the
respective compositions, PChoÆNeu5AcÆHexNAcÆHex
4
Æ
Hep
3
ÆPEtnÆPÆKdoÆlipid A-OH, PChoÆNeu5AcÆHexNAcÆ
Hex
4
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH, PChoÆNeu5AcÆ
HexNAcÆHex
5
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH, PChoÆ
Neu5AcÆHexNAc
2
ÆHex
5
ÆHep
3
ÆPEtnÆ PÆKdoÆ lipid A-OH
and PChoÆNeu5AcÆHexNAc
2

ÆHex
5
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆ
lipid A-OH. In the precursor ion scan spectrum
obtained in the positive mode (Fig. 1B) ions corre-
sponding to sialylated Hex3 glycoforms were not
observed, whereas ions corresponding to sialylated
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.
4888 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
glycoforms containing HexNAc were readily detected
confirming their presence.
Characterization of oligosaccharides derived from
NTHi strains 1268, 1200 and 1268lpsA
Mild acid hydrolysis of LPS with dilute aqueous acetic
acid afforded insoluble lipid A and core oligosaccharide
material (OS), which after purification by gel-filtration
chromatography resulted in OS samples from the vari-
ous strains. Strains 1268 and 1200 gave leading fractions
of higher molecular mass referred to as 1268OS
and 1200OS which were investigated in detail.
Strain 1268lpsA gave lpsAOS.
Sugar analyses (Table S1) performed on 1268OS,
lpsAOS and 1200OS were consistent with the data
obtained on LPS-OH for both the wild-type and

mutant strains, revealing the presence of Glc, Gal,
Table 1. Negative ion ESI-MS data and proposed compositions for LPS-OH and OS of strains 1268, 1200 and 1268lpsA. Average mass units
were used to calculate molecular mass based on proposed compositions as follows: Hex, 162.14; HexNAc, 203.19; Hep, 192.17; Kdo,
220.18; P, 79.98; PEtn, 123.05; PCho, 165.13; AnKdo-ol, 222.20; Gly, 57.05; Ac, 42.04; lipid A-OH, 953.02. All LPS-OH and OS glycoforms
contain Hep
3
ÆPEtnÆPÆKdoÆlipid A-OH or Hep
3
ÆPEtnÆAnKdo-ol, respectively. nr, not rationalized.
Sample
Observed ions (m ⁄ z) Molecular mass (Da) Relative Abundance (%)
Proposed composition(M-4H)
4–
(M-3H)
3–
(M-2H)
2–
Obs Calc 1268 1268lpsA 1200
LPS-OH 609.4 812.8 2441.5 2442.2 17 36 16 PChoÆHex
2
640.2 854.1 2565.1 2565.2 30 45 34 PChoÆHex
2
ÆPEtn
680.9 2727.6 2727.3 6 PChoÆHex
3
ÆPEtn
700.1 933.7 2804.3 2804.5 2 5 HexNAcÆHex
4
731.1 974.6 2927.6 2927.5 2 3 HexNAcÆHex
4

ÆPEtn
721.3 2889.2 2889.5 7 PChoÆHex
4
ÆPEtn
731.1 975.2 2928.5 2928.6 3 4 PChoÆHex
5
741.3 988.8 2969.3 2969.6 5 5 PChoÆHexNAcÆHex
4
761.7 1015.9 3050.8 3051.6 12 8 PChoÆHex
5
ÆPEtn
772.1 1029.6 3092.1 3092.7 12 6 7 PChoÆHexNAcÆHex
4
ÆPEtn
781.8 1042.7 3131.2 3131.8 6 2 PChoÆHexNAcÆHex
5
812.8 1083.9 3255.0 3254.8 11 16 PChoÆHexNAcÆHex
5
ÆPEtn
OS 540.6 1083.2 1083.9 2 Hex
621.6 1245.2 1246.0 1 Hex
2
682.7 1367.4 – 13 nr
704.3 1410.6 1411.1 4 71 1 PChoÆHex
2
725.2 1452.4 1453.1 5 PChoÆAcÆHex
2
732.9 1467.8 1468.1 7 2 1 PChoÆGlyÆHex
2
746.3 1494.6 1495.2 4 PChoÆAc

2
ÆHex
2
754.2 1510.4 1510.2 1 PChoÆGlyÆAcÆHex
2
774.6 1551.2 1552.2 1 PChoÆGlyÆAc
2
ÆHex
2
783.8 1569.6 1570.3 3 Hex
4
785.6 1573.2 1573.2 3 PChoÆHex
3
806.9 1615.8 1615.3 2 PChoÆAcÆHex
3
866.5 1735.0 1735.4 4 1 2 PChoÆHex
4
885.5 1773.0 1773.5 3 HexNAcÆHex
4
887.5 1777.0 1777.4 4 PChoÆAcÆHex
4
895.0 1792.0 1792.4 1 PChoÆGlyÆHex
4
947.6 1897.2 1897.5 12 4 PChoÆHex
5
645.2 968.1 1938.3 1938.6 22 5 8 PChoÆHexNAcÆHex
4
968.3 1938.6 1939.5 2 PChoÆAcÆHex
5
976.0 1954.0 1954.6 2 5 PChoÆGlyÆHex

5
988.9 1979.8 1980.6 9 PChoÆAcÆHexNAcÆHex
4
988.9 1979.8 1981.6 2 PChoÆAc
2
ÆHex
5
664.5 996.3 1995.6 1995.6 8 6 PChoÆGlyÆHexNAcÆHex
4
1009.6 2021.2 2022.6 4 PChoÆAc
2
ÆHexNAcÆHex
4
1017.2 2036.4 2037.7 9 PChoÆGlyÆAcÆHexNAcÆHex
4
699.3 1049.2 2100.7 2100.7 33 12 PChoÆHexNAcÆHex
5
1069.9 2141.8 2142.7 15 PChoÆAcÆHexNAcÆHex
5
718.3 1077.5 2157.5 2157.8 7 PChoÆGlyÆHexNAcÆHex
5
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4889
Hep, GalN and GlcN. The considerable decrease in
GlcN in the OS samples confirmed this sugar to be
part of lipid A but also indicated traces of glycoforms
that contain GlcN.
1268OS, lpsAOS and 1200OS were dephosphorylated

with 48% hydrogen fluoride prior to methylation anal-
ysis. Methylation analysis (Table S2) on the resulting
material from 1268OS showed terminal Glc, terminal
Gal, 4-substituted Gal, 4-substituted Glc, 3-substituted
Gal, terminal Hep, 2-substituted Hep, 3,4-substituted
Hep, terminal GalN, 2,3-substituted Hep and 4-substi-
tuted GlcN. Methylation analysis performed on nonde-
phosphorylated 1268OS revealed the same sugars but
with a decrease in terminal Glc, 4-substituted Glc and
2,3-substituted Hep, which indicated phosphorylation
on those sugars (data not shown). Methylation analy-
sis on dephosphorylated lpsAOS gave the same sugar
derivatives as 1268OS but showing a significant
increase in terminal Hep and the absence of 2-substi-
tuted Hep. Moreover, a decrease of 4-substituted Gal,
4-substituted Glc and 3-substituted Gal was observed
in the methylation analysis of this OS sample. Methyl-
ation analysis data for 1200OS was comparable with
the data obtained for 1268OS (Table S2).
ESI-MS on OS samples (Table 1) indicated all
strains to be glycylated. In addition, OS samples from
NTHi 1200 showed ions corresponding to acetylated
glycoforms. ESI-MS on 1268OS and 1200OS revealed
major HexNAcHex4 and HexNAcHex5 glycoforms.
Lower molecular mass glycoforms were minor, in
agreement with OS samples being leading fractions
after GPC. Glycoforms, of which the O-glycylated
ones were of minor abundance, were evidenced as
doubly negatively charged ions as follows: PChoÆ
Hex

2
ÆHep
3
ÆPEtnÆAnKdo-ol and PChoÆGlyÆHex
2
ÆHep
3
Æ
PEtnÆAnKdo-ol (m ⁄ z 704.3 ⁄ 732.9), PChoÆHex
5
ÆHep
3
Æ
PEtnÆAnKdo-ol and PChoÆGlyÆHex
5
ÆHep
3
ÆPEtnÆ
AnKdo-ol (m ⁄ z 947.6 ⁄ 976.0), PChoÆHexNAcÆHex
4
Æ
Hep
3
ÆPEtnÆAnKdo-ol and PChoÆGlyÆHexNAcÆHex
4
Æ
Hep
3
ÆPEtnÆAnKdo-ol (m ⁄ z 968.1 ⁄ 996.3), and PChoÆ
HexNAcÆHex

5
ÆHep
3
ÆPEtnÆAnKdo-ol and PChoÆGlyÆ
HexNAcÆHex
5
ÆHep
3
ÆPEtnÆAnKdo-ol (m ⁄ z 1049.2 ⁄
1077.5). In addition, ions at m ⁄ z 866.5 ⁄ 895.0 indicated
the glycoforms PChoÆHex
4
ÆHep
3
ÆPEtnÆAnKdo-ol and
PChoÆGlyÆHex
4
ÆHep
3
ÆPEtnÆAnKdo-ol. The glycoforms
indicated in lpsAOS were in agreement with those
found in the equivalent LPS-OH and showed major
Hex2 glycoforms.
ESI-MS data of 1200OS revealed the presence of
glycoforms substituted by up to two acetate groups.
Information on the location of Ac was provided by ESI
multiple-step tandem mass spectrometry (MS
n
)inthe
positive-ion mode. The product ion spectrum obtained

from the molecular ion at m ⁄ z 1496.4 (composition:
PChoÆAc
2
ÆHex
2
ÆHep
3
ÆPEtnÆAnKdo-ol) (Fig. 2A) con-
tained, inter alia,theionatm ⁄ z 919.1 resulting from the
loss of Hex-HepI-AnKdo-ol. MS
3
performed on this ion
revealed a prominent ion at m ⁄ z 643.3 (composition:
PChoÆHexÆHepIIÆPEtn) (Fig. 2B) resulting from the loss
of a diacetylated heptose subunit indicative of HepIII
being substituted with two acetates. These experiments
also confirmed that PCho substituted the hexose linked
Fig. 1. CE-ESI-MS ⁄ MS spectra of LPS-OH
derived from NTHi 1268lpsA. The indicated
compositions include the PÆKdoÆlipid A-OH
element. (A) Precursor ion spectrum (nega-
tive mode) using m ⁄ z 290 as the fragment
ion for identification of sialylated compo-
nents in 1268lpsA. (B) Precursor ion spec-
trum (positive mode) using m ⁄ z 274 as the
fragment ion for identification of sialylated
components in 1268lpsA.
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.

4890 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
to HepII and that PEtn substituted HepII. MS
3
experi-
ments on ions corresponding to monoacetylated
glycoforms revealed the same substitution pattern (data
not shown).
Sequence analysis on dephosphorylated and
permethylated oligosaccharide samples using
ESI-MS
n
Sequence and branching details of the various glyco-
forms in 1268OS, lpsAOS and 1200OS were obtained
using ESI-MS
n
in the positive mode on dephosphoryl-
ated and permethylated material [13,26]. Because of
the increased MS response obtained by permethylation
in combination with added sodium acetate, several gly-
coforms were observed in the MS spectra that were
not detected in underivatized samples. Thus, the ESI-
MS mass spectrum of 1268OS (positive mode)
(Fig. 3A) showed sodiated singly charged adduct ions
([M+Na]
+
) corresponding to the glycoforms Hex
2
Æ
Hep
3

ÆAnKdo-ol, Hex
3
ÆHep
3
ÆAnKdo-ol, Hex
4
ÆHep
3
Æ
AnKdo-ol and Hex
5
ÆHep
3
ÆAnKdo-ol (m ⁄ z 1467.9,
1672.4, 1875.8 and 2080.1), HexNAcÆ Hex
4
ÆHep
3
Æ
AnKdo-ol and HexNAcÆHex
5
ÆHep
3
ÆAnKdo-ol (m ⁄ z
2120.8 and 2325.3) and HexNAc
2
ÆHex
4
ÆHep
3

ÆAnKdo-ol
(m ⁄ z 2366.7). The HexNAc2Hex4 glycoform was not
detected in the underivatized samples due to low
abundance.
In order to obtain sequence and branching informa-
tion, these molecular ions were further fragmented in
MS
2
and MS
3
experiments. For most glycoforms the
presence of several isomeric compounds was revealed
by identifying product ions in MS
2
spectra (Table S4).
MS
3
experiments were used when necessary to confirm
structures.
Two isomeric Hex2 glycoforms were identified in
1268OS by fragmenting the molecular ion m ⁄ z 1467.9.
The resulting spectrum revealed ions at m ⁄ z 1206.1
(major) and 1002.0 (minor) corresponding to loss of
terminal (t)Hep and tHex-Hep. The ion at m ⁄ z 754.3
corresponded to the fragment tHex-HepI-AnKdo-ol.
Thus in the major Hex2 isomer terminal hexoses
substituted both HepI and HepII. In the minor Hex2
glycoform both HepI and HepIII were substituted with
terminal hexose residues. Performing MS
2

on the
ion m ⁄ z 1672.4 and subsequent MS
3
on the resulting
Fig. 3. ESI-MS
n
analysis of permethylated
OS of strain 1268. (A) Full-scan spectrum
(positive mode) on permethylated dephos-
phorylated 1268OS. (B) Product ion
spectrum of [M+Na]
+
m ⁄ z 2120.8 corre-
sponding to the HexNAcHex4 glycoform.
Proposed key fragments are indicated in the
structure. (C) MS
3
of the ion at m ⁄ z 1859.0
from MS
2
of m ⁄ z 2120.8. Proposed key
fragments are indicated in the structure.
Fig. 2. ESI-MS
n
analysis of OS derived from NTHi strain 1200.
(A) Product ion spectrum of [M+H]
+
m ⁄ z 1496.4 corresponding to
the PChoÆAc
2

ÆHex
2
ÆHep
3
ÆPEtnÆAnKdo-ol glycoform. The proposed
fragmentation is shown beside the spectrum. (B) MS
3
on fragment
ion m ⁄ z 919.1, corresponding to the loss of Hex-HepI-AnKdo-ol.
The proposed fragmentation is shown beside the spectrum.
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4891
product ions determined two major and two minor
Hex3 isomeric glycoforms. The ion corresponding to
the loss of tHepIII at m ⁄ z 1409.9 was further frag-
mented to give the ion at m ⁄ z 753.5 due to the loss of
a tHex-Hex-HepII unit, thus evidencing a structure
with one hexose residue substituted to HepI and a
disaccharide moiety substituting HepII. Furthermore,
in the same MS
3
spectrum a minor ion was detected at
m ⁄ z 957.3 corresponding to the loss of tHex-HepII.
This ion confirmed the structure of a glycoform in
which a disaccharide moiety substitutes HepI and one
hexose substitutes HepII. The ion at m ⁄ z 1206.0 corre-
sponded to the loss of tHex-HepIII from the molecular
ion. It was further fragmented to give the ion at m ⁄ z

753.4 which originated from the loss of a tHex-HepII
unit, thus indicating a structure with one hexose resi-
due substituting each heptose residue. Finally, a minor
structure with one hexose residue linked to HepI and
two hexoses linked to HepIII could be determined
when the product ion, m ⁄ z 1001.5, corresponding to
the loss of tHex-Hex-HepIII, was further fragmented
to give the ion at m ⁄ z 753.2 due to the loss of HepII.
Four isomeric Hex4 glycoforms were identified by per-
forming MS
2
on the parent ion at m ⁄ z 1875.8 and sub-
sequent MS
3
on the resulting product ions at m ⁄ z
1614.0, 1206.1 and 1001.7 due to the losses of a termi-
nal heptose, a terminal Hex-Hex-Hep residue and a
terminal Hex-Hex-Hex-Hep residue, respectively.
When the ion at m ⁄ z 1614.0 was further fragmented in
aMS
3
experiment it gave product ions at m⁄ z 883.5
and 753.4 due to losses of the epitopes tHex-HepI-
AnKdo-ol and tHex-Hex-Hex-HepII, respectively. Fur-
thermore, a product ion at m⁄ z 1161.2 indicated the
loss of tHex-HepII. Thus two structures with terminal
HepIII were identified: the first with one hexose linked
to HepI and a trisaccharide group linked to HepII and
the second containing elongation of a trisaccharide
group substituting HepI and one hexose on HepII.

When the ion at m ⁄ z 1206.1 was further fragmented in
MS
3
experiments a product ion at m ⁄ z 753.4 was
observed defining the loss of tHex-HepII, which indi-
cated a major glycoform containing a disaccharide unit
on HepIII and one hexose residue on each of HepI
and HepII. The product ion at m ⁄ z 1001.7 was further
fragmented to give the ion at m ⁄ z 753.3 due to the loss
of HepII, revealing a minor glycoform containing a tri-
saccharide unit on HepIII and with one hexose on
HepI. One isomeric Hex5 glycoform was observed by
fragmenting the molecular ion at m ⁄ z 2080.1. The iso-
mer was defined by the ions at m ⁄ z 1349.9 and 1206.2
corresponding to the loss of tHex-HepI-AnKdo-ol and
tHex-Hex-Hex-HepIII which indicated HepI to be
substituted by one hexose and HepIII to be elongated
by three hexoses. The structure was confirmed in MS
3
experiments on m ⁄ z 1206.2 where the product ion at
m ⁄ z 754.4 indicated the loss of tHex-HepII.
The molecular ion at m ⁄ z 2120.8 corresponded to a
glycoform with four hexoses and one hexosamine. One
single isomer (Fig. 3B,C) was identified by fragmenting
the molecular ion. In the resulting spectrum, fragment
ions were observed at m ⁄ z 1862.4, 1859.0 and 1390.7
resulting from the loss of tHexNAc, tHep and tHex-
HepI-AnKdo-ol, respectively. A MS
3
experiment on

m ⁄ z 1859.0, showing the loss of tHex-HepI-AnKdo-ol
(m ⁄ z 1129.4) confirmed that this glycoform contained
a tHexNAc-Hex-Hex-Hex elongation from HepII and
a single hexose substituting HepI. The molecular ion
at m ⁄ z 2325.3 corresponded to a HexNAcHex5 glyco-
form. When this ion was further fragmented it gave
ions at m ⁄ z 2065.5, 1594.8 and 1205.9 resulting from
the loss of tHexNAc, tHex-HepI-AnKdo-ol and tHex-
NAc-Hex-Hex-Hex-Hep, respectively. The ion at m ⁄ z
1205.9 was further fragmented to give the ion at m ⁄ z
754.1 due to the loss of a tHex-HepII unit, thus evi-
dencing a structure with one hexose residue substituted
to each of HepI and HepII, and a tetrasaccharide moi-
ety with terminal hexosamine substituting HepIII.
The molecular ion at m ⁄ z 2366.7 corresponded to
a glycoform with the composition HexNAc
2
ÆHex
4
Æ
Hep
3
ÆAnKdo-ol. The single isomer of this glycoform
was defined in the MS
2
spectrum by the ions at m ⁄ z
2108.0, 2105.0, 1903.5 and 1658.3 corresponding to the
loss of tHexNAc, tHep, tHexNAc-Hex and tHexNAc-
Hex-HexNAc. MS
3

performed on the ion at m ⁄ z
1658.3 indicated the loss of tHep (m ⁄ z 1396.1) and
the fragment ion of -Hex-Hex-HepI-AnKdo-ol (m ⁄ z
944.1). Thus, this glycoform contained a tHexNAc-
Hex-HexNAc-Hex-Hex- unit elongating from HepI
and with one hexose substituting HepII.
ESI-MS
n
data obtained from lpsAOS clearly indi-
cated the absence of glycoforms expressing chain
extension from HepIII. The major isoforms observed
were otherwise equivalent to those found in the wild-
type strain, except for an extra Hex1 glycoform (m ⁄ z
1264.1) containing one hexose substituent on HepI
determined from the fragment ion at m ⁄ z 753.2 indi-
cating the loss of tHepIII-HepII (Table S4).
Strain 1200 contained virtually the same glycoforms
as observed in strain 1268 except for those having
elongations from HepI. However, traces of three other
higher molecular mass forms; HexNAcÆHex
6
ÆHep
3
Æ
AnKdo-ol, HexNAcÆHex
7
ÆHep
3
ÆAnKdo-ol and Hex-
NAc

2
ÆHex
7
ÆHep
3
ÆAnKdo-ol at m ⁄ z 2528.9, 2732.9 and
2976.9, respectively, were observed and investigated.
MS
2
of m ⁄ z 2528.9 gave fragment ions at m ⁄ z 2269.2,
1410.0 and 1799.5 corresponding to losses of
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.
4892 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
tHexNAc, tHexNAc-Hex-Hex-Hex-HepIII and tHex-
HepI-AnKdo-ol, indicating HepI to be substituted by
one hexose, HepII by two hexoses and HepIII by
a tHexNAc-Hex-Hex-Hex unit. The same spectrum
showed a second glycoform defined by the ion at m ⁄ z
1859.7 indicating that HepIII was elongated with two
hexoses. This was confirmed by MS
3
on the ion at
m ⁄ z 1799.5 giving the fragment ion at m ⁄ z 1128.9
corresponding to the loss of tHex-Hex-HepIII, thus
indicating HepII to be substituted by a tHexNAc-Hex-
Hex-Hex- unit. MS
2
of m ⁄ z 2732.9 gave fragment ions

at m ⁄ z 2474.4, 1613.7 and 2002.9 corresponding to the
losses of tHexNAc, tHexNAc-Hex-Hex-Hex-HepIII
and tHex-HepI-AnKdo-ol, respectively, revealing HepI
to be substituted by one hexose, HepII by three
hexoses and HepIII by the tHexNAc-Hex-Hex-Hex
unit. MS
2
of m ⁄ z 2976.9 gave the fragment ions m ⁄ z
2718.4 and 1858.5, indicating the loss of tHexNAc and
tHexNAc-Hex-Hex-Hex-HepIII. The ion at m ⁄ z 1858.5
was further fragmented which gave the fragment ions
at m ⁄ z 1599.7 and 1129.0 corresponding to the losses
of tHexNAc and tHex-HepI-AnKdo-ol. This indicated
the HexNAc2Hex7 isomer to be substituted by one
hexose at HepI and tHexNAc-Hex-Hex-Hex units
substituting both HepII and HepIII.
Characterization of lpsAOS, 1268OS and 1200OS
by NMR
Major structures were elucidated by detailed
1
H,
13
C
and
31
P NMR analyses.
1
H and
13
C NMR resonances

were assigned using gradient chemical shift correlation
techniques (COSY, TOCSY and HMQC experiments).
The chemical shift data corresponding to 1268OS,
lpsAOS and 1200OS are given in Table 2. Prior to
NMR analyses the samples were treated with 1 m NH
3
to remove O-acyl groups. Subspectra corresponding 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 mono-
saccharide sequences of the major glycoforms were
confirmed from transglycosidic NOE connectivities
between anomeric and aglyconic protons on adjacent
residues (Table S5). The chemical shift data are consis-
tent with each sugar residue being present in the pyr-
anosyl ring form. Further evidence for this conclusion
was obtained from NOE data which also served to
confirm the anomeric configurations of the linkages
and the monosaccharide sequence. NOESY spectra of
1268OS, lpsAOS and 1200OS revealed inter-residue
NOE connectivities between the anomeric protons of
HepIII to HepII H-1 ⁄ H-2, HepII to HepI H-3, HepI
to Kdo H-5 ⁄ H-7 and GlcIV to HepI H-4 ⁄ H-6, which
confirmed the sequence of the conserved triheptosyl
inner core unit. Several signals for methylene protons
of An Kdo-ol were observed in the COSY and TOCSY
spectra in the region d 1.87–2.18. 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 [27].
1
H–
31
P correlation experiments indicated PEtn
(d
P
0.01) to be linked to O-6 of HepII.
Structure of the Hex2, Hex4 and HexNAcHex4
glycoforms in lpsAOS
Sequence analysis of lpsAOS by ESI-MS
n
revealed a
predominant Hex2 glycoform having a triheptosyl
inner-core from which chain elongation by hexoses
only appeared from HepI and HepII (Table S4). In
addition, glycoforms having further extensions from
HepII by HexNAc-Hex-Hex-Hex or truncated versions
thereof were detected. In the
1
H NMR spectrum of
lpsAOS, anomeric resonances corresponding to the
triheptosyl moiety (HepI–HepIII) were identified at
d 5.05–5.16, 5.83 and 5.03, respectively. Subspectra cor-
responding to the hexose residues were identified in the
2D COSY and TOCSY (Fig. 4A) spectra at d 5.28
(Glc residue V), 4.97 (Gal residue VII), 4.92 (Gal resi-
due VII), 4.66 (GalNAc residue VIII), 4.57 ⁄ 4.64 (Gal
residue VI) and 4.54 (Glc residue IV), respectively. The

chemical shift data were consistent with VII (d
H-1
4.97)
and VIII being terminal residues. The terminal and
4-substituted forms of residue V could be distinguished
by different H-2 and H-4 shifts (d
H-2
3.54 ⁄ 3.59 and
d
H-4
3.50 ⁄ 3.80), which was also confirmed in COSY
and
1
H)
13
C HMQC experiments (d
C-4
69.8 ⁄ 76.3). The
high H-6
A ⁄ B
shifts of V (d 4.11 ⁄ 4.18) indicated this
position to be substituted with a PCho subunit, which
was confirmed in
1
H–
31
P correlation experiments
showing a
31
P resonance at d )0.05 correlating to

H-6
A ⁄ B
of V and the methylene protons of PCho at
d 4.35. The spin systems at d 4.57 and 4.64 could both
be assigned to residue VI indicating the anomeric
proton of this residue to be sensitive to changes in
molecular environment due to the microheterogeneity
of the sample. Because the oligosaccharide contains
Hex4 glycoforms with and without PCho, we assume
that the proton at d 4.57 corresponds to glycoforms
substituted by PCho and the one at d 4.64 to those
that do not. Inter-residue NOE between the proton
pairs of V H-1 ⁄ II H-3 confirmed these residues to be
linked to position O-3 in HepII. Inter-residue NOE
were observed between the VII H-1 ⁄ VI H-4 confirming
a a-d-Galp-(1fi4)-b-d-Galp unit within the extension
from HepII. Inter-residue NOE from VI H-1 and
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4893
Table 2.
1
H and
13
C chemical shifts for 1268OS, lpsAOS and 1200OS. Prior to NMR analyses the samples were O-deacylated. Spectra were
recorded in D
2
Oat25°C. Chemical shift values compared between the three strains could vary by up to ± 0.01 p.p.m. Signals originating
from the Hex5 and HexNAcHex5 glycoforms were not observed in the lpsA mutant. Signals corresponding to PCho methyl protons and car-

bons occurred at d 3.23 and 54.7, respectively. Pairs of deoxy protons of reduced AnKdo-ol were identified in COSY and TOCSY spectra at d
1.87–2.18. Signals corresponding to GalNAc methyl
1
H and
13
C occurred at d 2.05 and 23.02, respectively.
a
Observed from intense NOE signals.
b
–, not determined.
c
Observed in TOCSY of strain 1268 only.
d
Observed as intra-residue NOE from
H-1 of d 4.64 only.
e
An extra terminal b-hexose was observed in strain 1200 and 1268 (weak) at d
H-1,C-1
4.46, 102.7; d
H-2,C-2
3.54,72.9;
d
H-3,C-3
3.69,72.7; d
H-4
3.54 and d
H-5
3.74, respectively. Also, intra-residue NOE signals from the anomeric proton to H-3 and H-5 were
observed. No inter-residue NOE connections could be detected.
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro

¨
m et al.
4894 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
V H-3 were not observed, probably due to low abun-
dance of the corresponding glycoforms. However,
NMR data combined with data from methylation and
tandem MS analysis corroborate the sequence of the
extending glycose unit from HepII as [b-d-GalNAcp-
(1fi3)-a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-a-d-Glcp-(1fi].
The HexNAcHex4 glycoform in lpsAOS is shown in
Fig. 5. Also indicated are the truncated Hex4 and
Hex2 glycoforms.
Structure of the Hex5 and HexNAcHex5 glycoforms in
1268OS and 1200OS
Sequence analysis of 1268OS by ESI-MS
n
revealed in
addition to HexNAcHex4 glycoforms, abundant Hex-
NAcHex5 glycoforms having the structure observed
for lpsAOS and also those with chain elongation from
HepIII (Table S4). In the
1
H NMR spectrum of
1268OS (Fig. S1A), anomeric resonances correspond-
ing to the triheptosyl moiety (HepI–HepIII) were iden-
tified at d 5.05–5.16, 5.71 and 5.13, respectively. Spin
systems corresponding to the hexose residues were
identified in the COSY and TOCSY spectra. The
occurrence of inter-residue NOESY connectivities
between protons on contiguous residues in 1268OS

confirmed an identical structural element as shown in
Fig. 5. In addition, in the COSY and TOCSY spectra
of 1268OS anomeric signals at d 4.43 and 4.52 could
be attributed to fi4)-b-d-Glcp (IX) and fi4)-b-d-Galp
(X) residues, respectively. Additional spin systems cor-
responding to terminal GalNAc and Gal residues indi-
cated by methylation analysis were not observed. It
was reasonable to assume that these overlapped with
the resonances of the corresponding sugars extending
from HepII. Thus resonances at d 4.92 and 4.66 were
assigned to correspond to residues XI and XII, respec-
tively. Inter-residue NOE between X H-1 ⁄ IX H-4
and IX H-1 ⁄ III H-1,2 (Fig. 6A) gave evidence for the
fi4)-b-d-Gal p-(1fi4)-b-d-Glcp-(1fi2)-l-a-d-HepIIIp-(1fi
unit. Because inter-residue NOE between XII H-1 ⁄
XI H-3 and XI H-1 ⁄ X H-4 was observed we propose
that a globotetraose unit is the full extension from
HepIII in 1268OS. The HexNAcHex5 glycoform in
1268OS and 1200OS is shown in Fig. 7 as well as the
truncated Hex5 glycoform.
Fig. 4. Selected region of phase sensitive TOCSY spectra (mixing
time 180 ms) of (A) lpsAOS and (B) 1200OS. Cross-peaks of impor-
tance are labelled. See Table 2 for an explanation of the roman
numerals. (A) Signals corresponding to structures with full exten-
sion from HepII (Fig. 5) are indicated. (B) Signals corresponding to
structures with full extension from HepIII (Fig. 7) are indicated.
IV
β-
D-Glcp-(1→4)-
L

-α-D
-HepIp-(1→5)-AnKdo-ol
PCho 3
Hex4 Hex2 ↓↑
6 1
β-
D
-GalNAc
p-(1→3)-
α
-
D-Galp-(1→4)-β-
D
-Galp
-(1
→
4
)-
α
-
D
-Glc
p-(1→3)-
L
-α
-
D
-HepII
p
6←

PEtn
2
VIII VII VI V ↑
1
L
-α-D-HepIIIp
Fig. 5. Structure proposed for the HexNAcHex4 glycoform in lpsAOS. Also indicated are the truncated Hex4 and Hex2 glycoforms.
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4895
In agreement with ESI-MS data, NMR spectra of
1200OS revealed minor signals originating from an
acetyl methyl group at d
H-2
2.15 ⁄ 2.19 and d
C-2
21.0.
O-Acetyl groups were of very minor abundance and
could not be assigned by NMR experiments. NMR
experiments performed on O-deacylated 1200OS gave
virtually the same spectra as those obtained for
1268OS with only small variations in intensity between
the two strains. This can clearly be seen in the Fig. S1,
where the
1
H NMR spectra of 1268OS (A) and
the anomeric region of 1200OS (B) are compared.
Figure 4B shows part of the TOCSY spectrum of
1200OS in which signals corresponding to structures

with full extension from HepIII (shown in Fig. 7) are
indicated to distinguish them from signals originating
from the also present HexNAcHex4 glycoform. Of
notice is that XI and XII can not be differentiated
from VII and VIII. In addition, signals originating
Fig. 6. Selected region of 2D gradient NOESY spectra (mixing time 250 ms) of (A) 1200OS, (B) lpsAOS and (C) 1268OS. The structural rep-
resentations show full extensions from (D) HepII and (E) HepIII. Cross-peaks of significant importance are labelled. See Table 2 for an expla-
nation of the roman numerals.
IV
β
-
D
-Glc
p
-(1
→4)-
L-α-D-HepIp-(1
→
5)-AnKdo-ol
PCho 3
↓↑
6 1
α-
D-Glcp-(1→3)-L-α-D-HepIIp6←PEtn
V
2
Hex5 ↑
1
β-
D-GalNAcp-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→2)-L-α-D-HepIIIp

XII XI X IX
Fig. 7. Structure proposed for the HexNAcHex5 glycoform in 1268OS and 1200OS. Also indicated is the truncated Hex5 glycoform.
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.
4896 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
from IX and X are present compared to the TOCSY
spectrum of lpsAOS (Fig. 4A). Parts of NOESY spec-
tra of 1200OS, lpsAOS and 1268OS are compared in
Fig. 6. The same inter-residue signals as described for
1268OS are detected in 1200OS (Fig. 6A,C). The inter-
residue signal of VII-1 ⁄ VI-4 evidencing digalactoside
in the extension from HepII is indicated in the
spectrum from lpsAOS (Fig. 6B). The same signal is
indicated in 1200OS (Fig. 6A), however, here it is
overlapping with XI-1⁄ X-4 from the HexNAcHex5
glycoform.
Discussion
As a part of our ongoing studies on the role of
H. influenzae LPS in disease pathogenesis and in elicit-
ing protective immune responses, we have undertaken
a systematic analysis of LPS from a genetically diverse
set of NTHi strains obtained from children with otitis
media in Finland. Two of these strains, NTHi 1268
and 1200, are closely related clinical isolates that share
the same ribotype and are identical for four of seven
LPS gene sequences after multi locus sequence typing
of LPS biosynthesis genes [18]. From this information
we would expect the strains to express similar LPS
structures and this has now been confirmed by our

detailed structural analysis. LPS from both strains con-
tains the triheptosyl inner-core moiety l-a- d-Hepp-
(1fi2)-[PEtnfi6]-l-a-d-Hepp-(1fi3)-l-a-d-Hepp linked
to lipid A via Kdo 4-phosphate, that is common to all
H. influenzae LPS. As observed for every H. influenzae
strain investigated to date, the proximal heptose
(HepI) is substituted by b-d-Glcp (GlcI) at O-4. For
NTHi strain 1268, trace amounts of glycoforms were
identified in which GlcI can be further substituted by
tHex-Hex or tHexNAc-Hex-HexNAc-Hex units evi-
denced by LC-MS
n
on permethylated OS material.
The identity of these structures could not be estab-
lished using analytical methods because of limited
material. However, immunoblotting studies have indi-
cated low levels of glycoform populations containing
[(PEtnfi6)-a-d-GalpNAc-(1fi6)-b-d-Galp-(1fi4)-b-d-
GlcpNAc-(1fi3)-b-d-Galp-(1fi4] in NTHi strains 1268
and 1200 (DW Hood et al. unpublished). The biosyn-
thesis of this terminal tetrasaccharide unit is by a
mechanism that resembles that of O-antigen repeat
units and is mediated by genes in the hmg locus [7].
The tetrasaccharide unit is added en bloc, not stepwise,
to O-4 of GlcI and therefore does not provide infor-
mation to help identify the Hex-Hex unit extending
from GlcI in other minor glycoforms. Previously,
b-d-Galp-(1fi4)-b-d-Glcp and a-d-Galp-(1fi4)-b-d-
Galp have been identified as extensions from GlcI in
some NTHi LPS [13,15]. The gene involved in adding

the first hexose to GlcI is lex2B [28]. Allelic variants of
lex2B determine whether this hexose is either a b-d-
Glcp or b-d-Gal
p and sequence analysis of lex2B in
both strains 1200 and 1268 indicates that the gene
would direct the addition of a b-d-Glcp (DW Hood
et al. unpublished). HepIII in NTHi strains 1268 and
1200 was shown to be substituted at O-2 by globo-
tetraose [b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4)-b-d-
Galp-(1fi4)-b-d-Glcp-(1fi] or truncated versions
thereof. The gene required for initiation of oligosac-
charide extensions from HepIII has been identified as
lpsA [6]. Allelic variants of this gene can direct the
addition of either a b-d-Glcp or b-d-Galp in either a
(1fi2)- or (1fi3)-linkage and in agreement with our
structural observations the sequence for lpsA for
strains 1200 and 1268 would predict a b-d-Glcp to be
added in (1fi2)-linkage to HepIII [29]. We created a
lpsA mutant of NTHi strain 1268 which was not
expected to have chain extension from HepIII but
otherwise would express extensions from HepI and
HepII as in the parent strain. Structural analysis of
LPS from the lpsA mutant strain enabled us to unam-
biguously determine the extension from HepII and also
confirmed the function of the lpsA gene in the NTHi
1268 strain background.
In all H. influenzae strains investigated to date (both
typeable and NT
Hi) that have chain extension from
HepII, the initial substitution is always at O-3 by

a-d-Glcp. The a-d-Glcp residue has been found to be
terminal [8] or to be further elongated at O-4 by
globotriose [a-d-Galp-(1fi4)-b-d-Galp-(1fi4)-b-d-
Glcp-(1fi] [25,30] or truncated analogues. In our
study, elongation from the a-d-Glcp is initiated by
b-d-Galp which in turn can be further extended by
[b-d-GalpNAc-(1fi3)-a-d-Galp-(1fi4]. Only in one
other strain has the a-d-Glcp been found substituted
by b-d-Galp [31]. For NTHi strains 1268 and 1200
both HepIII and HepII can be substituted by globotet-
raose ⁄ globotetraose-like structures and their truncated
variants. This is the first report of this extension from
HepII and thus strains 1200 and 1268 are representa-
tive of this LPS structural motif in H. influenzae. One
glycoform containing full extension from both HepII
and HepIII was identified in minor amounts in
strain 1200 during permethylation analysis. The pro-
posed structure of this form is seen in Fig. 8.
From the structural detail obtained we can make
some observations on the inclusion of epitopes in the
LPS of strains 1200 and 1268 that are known to be rel-
evant to the virulence of H. influenzae
. The digalacto-
side, [a-d-Galp-(1fi4)-b-d-Galp-(1fi], structure has
been shown to confer resistance to killing by naturally
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4897
acquired antibody and complement present in human

serum [32]. This is most likely due to molecular
mimicry whereby this epitope, which is also expressed
on the surface of some host cells, allows the bacterium
to camouflage itself from the human immune defence
system. Expression of two digalactoside extensions,
rather than one, in isogenic strains has been shown to
be associated with increased virulence in an in vivo
model of H. influenzae infection [33].
PCho substitution is a common feature of H. influ-
enzae LPS that contributes to the ability of NTHi to
colonize and persist within the human respiratory
tract, at least in part by mediating bacterial adherence
to and invasion of the host epithelia [34–36]. A major-
ity of H. influenzae strains, including NTHi strains and
strain Rd, have been shown to carry PCho at O-6 of
GlcI [13,23], in other strains, including H. influenzae
type b strains, RM7004 and Eagan, PCho is found at
O-6 of a terminal b-d-Galp residue at HepIII [16,37]
and, in a third structural variant, PCho has been
found at O-6 of the a-d-Glcp residue at HepII [8].
NTHi strains 1268 and 1200 can be added to the short
list of strains having PCho at O-6 of a-d-Glcp at
HepII. Allelic variants of the lic1D gene (encoding the
PCho transferase) direct the addition of PCho to these
three positions in the LPS molecule. Sequence analysis
has confirmed that strain 1200 contains the same allelic
variant of lic1D as is found in other NTHi strains
where PCho is added to the a-d-Glcp residue at HepII
(A Cody, DW Hood & ER Moxon, John Radcliffe
Hospital, Oxford, UK, unpublished results).

Sialic acid is a common and important structural
feature of H. influenzae LPS. It has been shown that,
when sialic acid is present in the LPS, it helps the bac-
teria to resist the killing effect of normal human serum
but has little effect on their attachment to, or invasion
of, cultured human cells or neutrophils [38]. Recently,
we demonstrated that host-derived Neu5Ac is incorpo-
rated in H. influenzae LPS in vivo and that it is an
essential virulence factor in experimental otitis media
in the chinchilla [17]. Four genes have been described
as encoding sialyltransferases in H. influenzae, lic3A,
lic3B, lsgB and siaA [39–41]. Lic3A
encodes the phase
variable a-2,3-sialyltransferase sialylating terminal lac-
tose extending from HepIII, lic3B can add mono- or
disialic acid to the same position, and lsgB and siaA
are involved in sialylation of the lacto-N-neotetraose
units, described above, extending from HepI. The level
of sialylation in H. influenzae LPS is generally low and
detailed structural analysis of sialylated glycoforms has
only been possible following growth of strains under
appropriate conditions that maximize sialylated glyco-
form expression [8,9]. Genetic analysis has confirmed
that NTHi strains 1268 and 1200 contain each of the
sialyltransferase genes, lic3A, lsgB and siaA, but not
lic3B. Previously, sialylation of these strains was
demonstrated by us in a survey of 24 NTHi strains
made using high-performance anion-exchange chroma-
tography with pulsed amperometric detection in combi-
nation with neuraminidase treatment [19]. A more

detailed characterization of novel sialylated glycoforms
in NTHi 1268 could be done here by using precursor
ion scan CE-ESI-MS ⁄ MS. We found that 1268lpsA
expresses sialylated glycoforms ranging from Hex3 to
HexNAc2Hex5 species. The most important of these
was the glycoform with the composition Neu5AcÆ
PChoÆHexNAc
2
ÆHex
5
ÆHep
3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH.
In this glycoform we propose [(PEtnfi6)-a-d-Galp-
NAc-(1fi6)-b-d-Galp-(1fi4)-b-d-GlcpNAc-(1fi3)-b-d-
Galp-(1fi4)-b-d-Glcp-(1fi] to substitute HepI with
sialyllactose substituting HepII. Sialyllactose has not
previously been found to extend from HepII. In the
glycoforms with compositions Neu5AcÆPChoÆHexNAcÆ
Hex
5
ÆHep
3
ÆPEtnÆPÆKdoÆlipid A-OH and Neu5AcÆPChoÆ
PPEtn
↓
4
β-

D-Glcp-(1→4)-L
-α-
D-Hepp-(1→5)- α−Kdop-(2→6)-Lipid A
PCho 3
↓↑
6
1
β-
D-GalNAcp-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-α-D-Glcp-(1→3)-L-α-D-Hepp6←PEtn
2
↑
1
β-
D-GalNAcp-(1→3)-α-D-Galp-(1→4)-β-D
-Gal
p-(1→4
)-
β
-
D-Glcp-(1→
2)-
L-α-D-Hepp
Fig. 8. Proposed structure for the glycoform of NTHi strains 1268 and 1200 having full extension from both HepII and HepIII.
LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.
4898 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
HexNAcÆHex
5
ÆHep

3
ÆPEtn
2
ÆPÆKdoÆlipid A-OH, sialyl-
lacto-N-neotetraose presumably substitutes HepI. Sialy-
lated Hex3 glycoforms can comprise those having
sialyllactose extending from either HepII or HepI. The
latter scenario is unlikely since the lex2B sequence
indicates the first hexose linked to GlcI to be a b-d-Glcp
(see above). It would be reasonable to assume that lic3A
is the sialyltransferase involved in the biosynthesis of
sialyllactose in NTHi strain 1268.
The main difference between strains 1268 and 1200
was observed at HepIII where strain 1268 can be
substituted by a glycine subunit whereas strain 1200 is
substituted with up to two acetates. It has recently been
demonstrated that the phase variable gene oafA is
responsible for O-acetylation of HepIII in NTHi strains
and that O-acetylation of LPS decreases the killing
effect of normal human serum on the bacterium [42].
The oafA gene is present in both isolates 1200 and 1268
but interestingly neither gene contains the tetranucleo-
tide repeat tract necessary to allow the gene to be phase
variably expressed. The alternative substitutions at
HepIII for these strains might reflect a competition for
the addition of glycine or O-acetyl groups during LPS
biosynthesis, which is different between strains.
Experimental procedures
Bacterial strains used and construction
of mutant strain

NTHi strains 1268 and 1200 are distinct clinical isolates
taken from the middle ear of patients with otitis media and
are obtained from the Finnish Otitis Media Study Group
[18]. Strain 1268lpsA was constructed by transformation
[43] of isolate 1268 with plasmid p11
2,
containing a cloned
lpsA gene interrupted by a kanamycin resistance cassette, as
described previously [44]. The mutant strain was confirmed
after growth on brain–heart infusion–kanamycin
(10 lgÆmL
)1
) by PCR and Southern analyses, as described
previously [44].
Cultivation and preparation of LPS
Bacteria were grown in 1 L batch cultures of brain–heart
infusion broth supplement with haemin (10 lgÆmL
)1
) and
nicotinamide adenine dinucleotide (2 lgÆmL
)1
). Bacteria
were harvested then LPS was extracted from lyophilized bac-
teria using phenol ⁄ chloroform ⁄ light petroleum as described
by Galanos et al. [45], with the modification that the LPS
was precipitated with diethyl ether ⁄ acetone (1 : 5, v ⁄ v;
6 vol). LPS was purified with ultracentrifugation (75 000 g,
4 °C, 16 h) using a Beckman L-80 ultracentrifuge and Ti70
rotor (Beckman Coulter Inc., Fullerton, CA).
Preparation of oligosaccharides

O-Deacylation of LPS with hydrazine
LPS was O-deacylated with anhydrous hydrazine under mild
conditions as described previously [46]. LPS (2 mg) was
treated with anhydrous hydrazine (110 lL) and stirred at
40 °C for 1 h. The reaction mixture was cooled (0 °C) and
surplus hydrazine destroyed by addition of cold acetone
(1 mL). The product from the reaction was obtained by
centrifugation (13 200 g, 3 min) using a microcentrifuge
(Denver Instrument, Denver, CO). The precipitated product
was washed twice with cold acetone and finally lyophilized
in water.
Mild acid hydrolysis of LPS
Reduced core OS material was obtained after mild acid
hydrolysis of LPS, in 1% acetic acid, pH 3.1, 100 °C, 2 h,
in the presence of borane-N-methylmorpholine complex.
The insoluble lipid A part was separated from the OS by
centrifugation (6 100 g,4°C, 10 min) using a Sorvall Evo-
lution RC, Kendro Laboratory Products (Newtown, CT)
and SA300 rotor (Du Pont, Newtown, CT). The superna-
tant was lyophilized and purified by gel-permeation chro-
matography. 50 mg LPS of 1268 and 1200 were used to
obtain OS fractions, 1268OS (6.8 mg), 1268OS2 (4.0 mg),
1200OS (5.6 mg), 1200OS2 (5.4 mg) and 1200OS3 (3.2 mg).
The 1268lpsA provided one OS fraction, lpsAOS (2.0 mg)
from 10 mg of LPS.
Dephosphorylation of OS
OS (1 mg) was dissolved in cold 48% aqueous hydrofluoric
acid (100 lL) in a polypropylene tube and stored for 48 h
at 4 °C. Aqueous hydrofluoric acid was evaporated on ice
[13].

Deacylation of OS
OS material was dissolved in 1 m NH
3
solution and stirred
for 24 h at 21 °C.
Analytical methods
Sugars were identified as their alditol acetates as described
previously [47]. Permethylation was accomplished on acety-
lated LPS-OH and OS, or on dephosphorylated OS. Acety-
lation was obtained by treatment of the material with acetic
anhydride (200 lL) and 4-dimethylaminopyridine (2–3 mg)
at 21 °C for 4–5 h [8]. After incubation with dimethyl sul-
phoxide (48 h at 21 °C) methylation was performed with
methyl iodide in the presence of lithium methylsulfinylmeth-
anide [48]. The methylated compounds were recovered on a
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4899
SepPak C
18
cartridge and subjected to sugar analysis or to
ESI-MS
n
. The relative proportions of the various alditol
acetates and partially methylated alditol acetates obtained
in sugar and methylation analyses correspond to the detec-
tor response of the GLC-MS. Absolute configuration of the
hexoses was determined by the method devised by Gerwig
et al. [49].

Chromatography
Gel-permeation chromatography was performed on a Bio-
GelP-4 column (2.5 · 80 cm, fraction range 800–4000 Da)
or a Bio-Gel G-15 column (2.5 · 80 cm, fraction range
£ 1.500 Da) with pyridinium acetate (0.1 m, pH 5.3) as
eluent and a differential refractometer as detector. GLC
was carried out on a Hewlett-Packard 5890 instrument with
a DB-5 fused silica capillary column (30 m · 25 mm ·
0.25 lm) and a temperature gradient of 130 °C (1 min) to
250 °C (1 min) at 3 °CÆmin
)1
[8]. HPLC was performed on
a 2690 Waters HPLC system (Waters, Milford, MA) using
a microbore C-18 column [Phenomenex LUNA 5l C18(2)]
at 21 °C. A mobile phase gradient was used having a con-
stant concentration of 1% HOAc and 0.1 mm sodium ace-
tate, changing gradually over time methanol (A) and water
(B) at a flow rate of 0.08 mLÆmin
)1
. The gradient was as
follows: 50% A at 0 min, 100% A at 50 min, 100% A at
60 min and 50% A at 70 min.
Mass spectrometry
GLC-MS was carried out with a Hewlett-Packard 6890
chromatograph (using the same type of column and gradient
system described above) connected to a Micromass quad-
rupole mass spectrometer. ESI-MS was performed with a
VG Quattro Mass Spectrometer (Micromass, Manchester,
UK) in negative ion mode. LPS-OH and OS samples
were dissolved in water ⁄ acetonitrile (1 : 1, v ⁄ v) to a con-

centration of 1 mgÆmL
)1
. Sample solutions were loop
injected into running solvent of water ⁄ acetonitrile (1 : 1,
v ⁄ v) at a flow rate of 10 lLÆmin
)1
. ESI-MS
n
on 1200OS
(positive mode) was performed on a Finnigan LCQ ion-trap
mass spectrometer (Finnigan-MAT, San Jose, CA) using a
running solvent of 1% acetic acid in acetonitrile ⁄ water (1:1,
v ⁄ v) and a flow rate of 5 lLÆmin
)1
. ESI-MS
n
experiments
on permethylated OS samples were performed in the posi-
tive ion mode [M+Na]
+
on the same instrument. Samples
were dissolved in 1 mm NaOAc in methanol ⁄ water (7 : 1,
v ⁄ v) at a flow rate of 10 lLÆmin
)1
and loop injected or
introduced by HPLC (parameters described above).
CE-ESI-MS ⁄ MS was carried out with a Crystal model
310 CE instrument (ATI, Unicam, Boston, MA) coupled to
an API 3000 mass spectrometer (Perkin-Elmer ⁄ Sciex, Con-
cord, Ontario, Canada) via a MicroIonspray interface as

described previously [20].
NMR spectrometry
NMR spectra of OS were recorded in deuterium oxide at
25 °C on a JEOL JNM-ECP500. Chemical shifts were re-
ported in p.p.m., referenced to internal sodium 3-trimethyl-
silylpropanoate-d4 (d 0.00
1
H), external acetone (d 30.1
13
C) and external phosphoric acid (d 0.00
31
P).
1
H,
1
H- COSY, TOCSY with mixing times of 50 and 180 ms
and gradient selected HMQC were performed according to
standard pulse sequences. For inter-residue correlation, 2D
NOESY experiments with a mixing time of 200 and 250 ms
were used.
Acknowledgements
We thank the members of the Finnish Otitis Media
Study group at the National Health Institute in
Finland for the provision of NTHi nasopharyngeal iso-
lates, and Richard Goldstein for providing the ribo-
type of strains 1200 and 1268. This project was
supported by a grant from the Swedish Research
Council (EKHS). DWH and ERM were supported by
the Medical Research Council, UK.
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LPS structure of NTHi strains 1268 and 1200 S. L. Lundstro
¨
m et al.
4902 FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS
Supplementary material
The following supplementary material is available
online:
Fig. S1.
1
H NMR spectra of (A) 1268OS and (B)
1200OS recorded in D
2
Oat25°C.
Table S1. Sugar analysis data of LPS-OH and OS
derived from strains 1268, 1200 and 1268 lpsA.
Table S2. Methylation analysis data performed on
dephosphorylated LPS-OH and OS derived from strain
1268, strain 1200 and dephosphorylated OS obtained
from 1268lpsA.
Table S3. Negative- and positive-ion ESI-MS data
with proposed compositions for sialylated LPS-OH
based on precursor ion scan (m ⁄ z 290 and 274, respec-
tively) of NTHi strain 1268lpsA.
Table S4. Glycoform structures obtained from strains
1268OS, 1200OS and lpsAOS, elucidated by ESI-MS
n
and HPLC-ESI-MS

n
on permethylated dephosphoryl-
ated OS.
Table S5. Combined NOE data of 1268OS, 1200OS
and lpsAOS.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
S. L. Lundstro
¨
m et al. LPS structure of NTHi strains 1268 and 1200
FEBS Journal 274 (2007) 4886–4903 ª 2007 The Authors Journal compilation ª 2007 FEBS 4903