Structural characterization of a novel branching pattern in the
lipopolysaccharide from nontypeable
Haemophilus influenzae
Martin Ma
˚
nsson
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, Novum, Huddinge, Sweden;
2
Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Structural analysis of the lipopolysaccharide (LPS) from
nontypeable Haemophilus influenzae strain 981 has been
achieved using NMR spectroscopy and ESI-MS on
O-deacylated LPS and core oligosaccharide (OS) material as
well as by ESI-MS
n
on permethylated dephosphorylated
OS. A heterogeneous glycoform population was identified,
resulting from the variable length of the OS branches
attached to the glucose residue in the common structural
element of H. influenzae LPS,
L
-a-

D
-Galp-(1fi4)-b-
D
-Glcp, or sequentially truncated ver-
sions thereof. This is the first time a branching sugar residue
has been reported in the outer-core region of H. influenzae
LPS. Additionally, a PEtn group was identified at O-3 of the
distal heptose residue in the inner-core.
Keywords: Haemophilus; lipopolysaccharide; phosphocho-
line; structural analysis; ESI-MS
n
.
Haemophilus influenzae is a Gram-negative pathogen that
routinely colonizes the human upper respiratory tract and
which can be found both in encapsulated (types a–f) and
unencapsulated (nontypeable) forms. While the incidence
of disease caused by H. influenzae type b (invasive diseases,
including meningitis and pneumonia) has been greatly
reduced in recent years as a result of the development of
conjugate vaccines, there exists no vaccine against nontype-
able H. influenzae (NTHi). NTHi strains are a common
cause of otitis media and respiratory tract infections [1] and
its lipopolysaccharide (LPS) molecule has been shown to be
important for colonization and bacterial persistence during
infection. H. influenzae LPS is composed of a membrane-
anchoring lipid A moiety linked by a single phosphorylated
3-deoxy-
D
-manno-oct-2-ulosonic acid (Kdo) residue to a
variable core oligosaccharide (OS) portion. The carbo-

D
-Glcp residue (either at O-2
[13] or O-3 [16]) or a b-
D
-Galp residue (either at O-2 [10] or
O-3 [19]). Analysis of LPS from lpsA mutants established in
a number of strain backgrounds supports a role for LpsA in
each of the alternative glycose substitutions of HepIII.
HepIII has also been found to be substituted by the
Correspondence to E. Schweda, University College of South
Stockholm, Clinical Research Centre, NOVUM, S-141 86 Huddinge,
Sweden. Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23,
E-mail:
Abbreviations: CE, capillary electrophoresis; PCho, phosphocholine;
PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine;
Hep, heptose;
D
,
D
-Hep,
D
-glycero-
D
-manno-heptose;
L
,
D
-Hep,
L
-glycero-

D
-Hep) [14] or
L
-glycero-
D
-
manno-heptose (
L
,
D
-Hep) [21]. In several strains, a disubsti-
tution-pattern of GlcI has been observed, including b-
D
-
Glcp (at O-4)/PCho (at O-6) [17], b-
D
-Galp (at O-4)/PCho
(at O-6) [20], Ac (at O-4)/PCho (at O-6) [18] and Ac (at
O-4)/
L
,
D
-Hep (at O-6) [21]. In each strain analysed, PCho
addition has been shown to be directed by the products of
the lic1 locus. Our recent studies have focussed on the
structural diversity of LPS expression, and the genetic basis
for that diversity, in a representative set consisting of 25
NTHi clinical isolates obtained from otitis media patients
[25]. In the present investigation we report on the structural
analysis of LPS from one of these isolates (NTHi strain

by centrifugation and the water-soluble part was repeatedly
chromatographed on a P-4 column, giving a major (OS-1,
8.5 mg) and a minor (OS-2, 3.7 mg) OS-containing
fraction.
Dephosphorylation of OS. Dephosphorylation of OS
material was performed with 48% aqueous HF, as
described previously [18].
Mass spectrometry
GLC-MS and ESI-MS were performed as described
previously [16,21]. ESI-MS
n
on permethylated dephos-
phorylated OS was performed using a Finnigan-MAT
LCQ ion trap mass spectrometer (Finnigan-MAT; San
Jose, CA, USA) in the positive ion mode. The samples
were dissolved in methanol/water (7 : 3) containing 1 m
M
NaOAc to a concentration of about 1 mgÆmL
)1
,andwere
injected into a running solvent of identical composition at
10 lLÆmin
)1
.
NMR spectroscopy
NMR spectra were obtained at 25 °C(OS)or20°C
[O-deacylated LPS (LPS-OH)] either on a Varian UNITY
600 MHz spectrometer or on a JEOL JNM-ECP500
spectrometer, using the previously described experiments
[16,18,21].

,
D
-Hep) or
L
-glycero-
D
-manno-heptose (
L
,
D
-Hep); R
2
,R
4
,
R
5
¼ H, glucose (Glc), galactose (Gal) or acetate (Ac)
6
;R
3
¼ HorGlc,
Y ¼ Gly, P or phosphoethanolamine (PEtn). Note that substitution
with Gly has also been reported at HepI, HepII and Kdo [24].
2980 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
sample indicated
D
-glucose (Glc),

under mild conditions, water-soluble O-deacylated LPS
(LPS-OH) was obtained. ESI-MS data (Table 1) indicated a
heterogeneous mixture of glycoforms consisting of two
subpopulations: a major subpopulation in which the
glycoform compositions comprised three heptoses and, to
agreatextent,PCho (Hep3-glycoforms); and a minor
subpopulation with compositions comprising four heptoses
but lacking PCho (Hep4-glycoforms). Quadruply charged
ions were observed at m/z 640.5/671.3 (minor), 680.8/
711.5 (major), 721.4/751.7 and 772.3/802.9, corresponding
to Hep3-glycoforms with the compositions PChoÆHex
2
Æ
Hep
3
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, PChoÆHex
3
ÆHep
3
Æ
PEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, PChoÆHex
4

charged ions at m/z 646.9/677.8, 687.7/718.4 (minor) and
728.1/758.8 (major) corresponded to Hep4-glycoforms
with the compositions Hex
2
ÆHep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid
A-OH, Hex
3
ÆHep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH and Hex
4
Æ
Hep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, respectively.
Characterization of OS

860.6, 941.8 (major) and 1022.5 (very minor) were consistent
with the respective compositions Hex
1)5
ÆHep
4
ÆPEtn
2
ÆAnK-
do-ol. In both OS fractions, minor peaks were observed
corresponding to the above-mentioned compositions but
additionally containing glycine or a phosphate group.
Minor peaks could also be observed corresponding to
glycoforms containing only one PEtn group or containing
one PEtn group and a phosphate group.
In order to obtain sequence and branching information,
the OS fractions were dephosphorylated and permethylated
and subjected to ESI-MS
n
[21,31]. Sodiated adduct ions
were observed in the MS spectra (positive mode) corres-
ponding to the compositions Hex
1)4
ÆHep
3
ÆAnKdo-ol, Hex-
NAc
1
ÆHex
3)4
ÆHep

ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
670.3 893.7 2684.6 2685.3 2 Hex
3
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
640.5 854.1 2565.6 2565.2 1 PChoÆHex
2
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
671.3 894.7 2688.2 2688.2 2 PChoÆHex
2
ÆHep
3
ÆPEtn

ÆKdoÆLipid A-OH
751.7 1003.2 3011.7 3012.5 4 PChoÆHex
4
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
772.3 1029.7 3092.6 3092.7 3 PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
802.9 1070.8 3215.5 3215.7 5 PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
3
ÆP

3
ÆHep
4
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
728.1 971.0 2916.2 2916.5 9 Hex
4
ÆHep
4
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
758.8 1012.2 3039.4 3039.6 17 Hex
4
ÆHep
4
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2981
monosaccharide sequence and branching for the different
glycoforms were obtained following collision-induced dis-
sociation (CID) of the glycosidic bonds [21,31]. Through the

1393.6. The assigned structure was confirmed by MS
3
experiments on selected product ions (data not shown). The
structures of the other Hep3-glycoforms were obtained in an
analogous manner (data not shown) and for all glycoforms
the hexoses were found to be members of a linear chain
attached to HepI (Table 3).
For the major Hep4-glycoform with the composition
Hex
4
ÆHep
4
ÆAnKdo-ol ([M + Na]
+
2124.0 Da), ions in the
MS
2
spectrum (precursor ion [M + 2Na]
2+
1073.5 Da)
(Fig. 4A) at m/z 1861.8 (loss of t-Hep) and 1613.8 (loss of
t-Hep–Hep) indicated the presence of a HepIII–HepII-
branch attached to HepI, as was also found in the Hep3-
glycoforms (Tables 3 and 4). An OS extension consisting
of four hexose residues and one heptose residue was
indicated to be linked to HepI from the occurrence of an
ion at m/z 1045.6 (counterpart at m/z 1101.6) correspond-
ing to loss of the entire Hex4Hep moiety. The fragmen-
tation pattern clearly indicated this OS extension not to be
arranged in a linear chain as fragment ions were found at

Table 2. Negative ion ESI-MS data and proposed compositions for oligosaccharide (OS)-1 and (OS)-2 derived from the lipopolysaccharide (LPS) of
nontypeable Haemophilus influenzae (NTHi) strain 981. Average mass units were used for calculation of molecular mass values based on proposed
compositions as follows: Hex (hexose), 162.14; HexNAc (N-acetylhexosamine), 203.19; Hep (heptose), 192.17; AnKdo-ol (reduced anhydro Kdo),
222.20; PEtn (phosphoethanolamine), 123.05; and PCho (phosphocholine), 165.13. Relative abundance was estimated from the area of molecular
ion peak relative to the total area (expressed as a percentage). Minor peaks were observed corresponding to the proposed compositions but
additionally containing glycine or a phosphate group. Minor peaks could also be observed corresponding to glycoforms containing only one PEtn
group or containing one PEtn group and a phosphate group. ND, not detected.
Observed ions
(m/z) (M-2H)
2)
Molecular mass (Da) Relative abundance (%)
Proposed compositionObserved Calculated OS-1 OS-2
764.8 1531.6 1531.2 ND 9 Hex
3
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
684.9 1371.8 1372.1 3 3 PChoÆHex
1
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
766.0 1534.0 1534.2 5 5 PChoÆHex
2
ÆHep
3

ÆHex
4
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
698.4 1398.8 1399.1 ND 3 Hex
1
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
779.6 1561.2 1561.3 ND 36 Hex
2
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
860.6 1723.2 1723.4 ND 7 Hex
3
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
941.8 1885.6 1885.5 3 34 Hex
4

,
D
-Hep, 4,6-disubstituted-Glc (4,6-Glc),
4-
D
,
D
-Hep, 2-
L
,
D
-Hep and 3,4-
L
,
D
-Hep in the relative
proportions 24 : 5 : 31 : 2 : 9 : 3 : 3 : 10 : 13 together with
trace amounts of t-Glc and t-GalN. The methylation
analysis of intact OS-1 showed significantly decreasing
amounts of 2-
L
,
D
-Hep and t-
L
,
D
-Hep, which could be
derived from the presence of PEtn substituents at HepII and
HepIII, respectively (see below). Methylation analysis of

structural element Hex–Hep–Hex–(–) in the Hep4-glyco-
forms was probably Gal-(1fi4)-
D
,
D
-Hep-(1fi4,6)-Glc, and
this was confirmed and detailed by NMR spectroscopy (see
below).
Characterization of OS fractions and LPS-OH by NMR
The
1
H NMR resonances of OS fractions and LPS-OH
were assigned by
1
H–
1
H chemical shift correlation experi-
ments (DQF-COSY and TOCSY). Subspectra correspond-
ing 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. From the glycoform
compositions of the different OS fractions indicated by
ESI-MS (Table 2), the spin-systems could more easily be
identified as originating from either the Hep3- or the Hep4-
glycoform population. The
13
C NMR resonances of OS

Hex1 Hex2 Hex3 Hex4 HexNAc1Hex3 HexNAc1Hex4
Hex-Hep-AnKdo-ol Hex
2
-Hep-AnKdo-ol Hex
3
-Hep-AnKdo-ol Hex
4
-Hep-AnKdo-ol ND HexNAc-Hex
4
-Hep-AnKdo-ol
|| | | |
Hep Hep Hep Hep Hep
|| | | |
Hep Hep Hep Hep Hep
Fig. 2. Negative ion ESI-MS spectra of oligosaccharide (OS)-1 (A) and
OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981 showing doubly charged ions.
(A) The peak at m/z 684.9 corresponds to a glycoform with the com-
position, phosphocholine (PCho)ÆHex
1
ÆHep
3
Æphosphoethanolamine
(PEtn)
2
ÆAnKdo-ol. (B) The peak at m/z 698.4 corresponds to a gly-
coform with the composition Hex
1
ÆHep
4

HSQC spectrum. A crosspeak from the ester-linked glycine
substituent was observed at d 4.00/40.7 (in the HSQC
spectrum) as a result of correlation between the methylene
proton and its carbon. Several signals for methylene protons
of AnKdo-ol were observed in the DQF-COSY and
TOCSY spectra of OS-1 in the region d 2.26–1.67, as
observed and explained previously [10,16,21]. The mono-
saccharide sequence within the inner-core region, as
indicated by ESI-MS
n
(described above), was confirmed
and detailed from transglycosidic NOE connectivities
between the proton pairs HepIII H-1/HepII H-2, HepII
H-1/HepI H-3 (LPS-OH and OS-1) and HepI H-1/Kdo H-5
and H-7 (LPS-OH), evidencing the sequence as
L
-a-
D
-
Hepp-(1fi2)-
L
-a-
D
-Hepp-(1fi3)-
L
-a-
D
-Hepp-(1fi5)-a-Kdop.
Relatively large J
1,2

-Hex-Hep-AnKdo-ol HexNAc-Hex
3
-Hex-Hep-AnKdo-ol
|| | | | |
Hep Hep Hep Hep Hep Hep
|| | | | |
Hep Hep Hep Hep Hep Hep
Hep
b
|
Hex
2
-Hex-Hep-AnKdo-ol
|
Hep
|
Hep
a
Major isomer of the Hex3 glycoform (estimated from the intensity of the fragments in MS
2
experiments).
b
Minor isomer of the Hex3 glycoform (estimated from the intensity of the fragments in
MS
2
experiments).
Fig. 3. ESI-MS
2
analysis of permethylated dephosphorylated oligosac-
charide (OS) derived from the lipopolysaccharide (LPS) of nontypeable

found at d 4.41/3.69 (PCho), 4.15/3.29 (PEtnI) and 4.17/
3.29 (PEtnII).
1
H–
31
P NMR correlation studies (Fig. 6)
demonstrated PChotobelocatedatGlcIandthetwoPEtn
residues to be situated at HepII and HepIII, respectively.
Intense
31
P resonances from phosphodiesters were observed
at d 0.33, )0.50 and )0.62. Correlations between the former
signal and the signals from H-6 of HepII (d 4.55) and the
methylene proton pair of PEtnI (d 4.15) in the
1
H–
31
P
HMQC experiment evidenced substitution by PEtn at O-6
of HepII. The second PEtnresiduewasdemonstratedtobe
linked to O-3 of HepIII as the
31
P resonance at d )0.50
correlated to the signals from H-3 of HepIII (d 4.33) and the
methylene proton pair of PEtnII (d 4.17). Correlations
between the signal at d )0.62 and the signals from the H-6
protons of GlcI (d 4.30) and the methylene protons of PCho
(d 4.41) established the PCho substituent to be located at
O-6 of this residue.
The occurrence of interresidue NOE connectivities

truncations, which is consistent with the occurrence of small
amounts of t-Glc in the methylation analysis. Indeed, two
weak crosspeaks are observed in the COSY spectrum at d
4.57/3.50 and 4.56/3.38, which possibly arise from the two
b-
D
-Glcp residues appearing as terminal residues; however,
this could not be confirmed because of a significant overlap
of signals.
Structure of the core region of the Hep4-glycoforms. In
the
1
H NMR spectrum of OS-2 (Fig. 5B), anomeric
resonances of HepI–HepIII were observed at d 6.01–5.87
(HepII, not resolved), 5.17–5.03 (HepI, not resolved) and
5.14/5.10 (HepIII, not resolved). The chemical shift values
for the other resonances of HepI–HepIII (data not shown)
were similar to the corresponding values in the Hep3-
glycoforms. Transglycosidic NOE connectivities (data simi-
lar to that of the Hep3-glycoforms) confirmed the sequence
of the characteristic triheptosyl unit. Anomeric signals of the
fourth heptose were identified at d 5.06/4.94 (HepIV, not
resolved) and single intraresidue NOE between H-1 and H-2
confirmed its a-configuration. This residue could be
attributed to the 4-
D
,
D
-Hep identified in the methylation
analysis on the basis of chemical shift data (Table 6).

ÆAnKdo-ol. The proposed structure is shown
in the inset in B. (B) MS
3
spectrum of the fragment ion at m/z 1613.8.
(C) MS
4
spectrum of the fragment ion at m/z 1191.6
7
.
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2985
intraresidue NOE, as described earlier (see above). The
residues with anomeric shifts of d 4.50, 4.54 and 4.57 could
be attributed to the 6-Glc (GlcI), 4,6-Glc (GlcI*) and 4-Glc
(GlcII) identified by methylation analysis, on the basis of the
chemical shift data and the large J
2,3
, J
3,4
and J
4,5
values
( 9 Hz). On the basis of low J
3,4
and J
4,5
values (<4 Hz)
and chemical shift data, the residues with anomeric
resonances at d 4.48, 4.95, 4.51/4.49 and 4.53 were attributed
to the t-Gal (GalI, GalII and GalIII) and 4-Gal (GalI*)
identified by linkage analysis.

-a-
D
-Hep
p-(1fi6)]-b-
D
-Glcp-(1fi4)-
L
-a-
D
-Hepp-(1fi. This monosac-
charide connectivity was also confirmed by transglycosidic
correlations in an HMBC experiment, where correlations
were seen between GalIII H-1/HepIV C-4, HepIV
Table 5.
1
Hand
13
C NMR chemical shifts for Hep3-glycoforms of oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981. Data were recorded in D
2
Oat25 °C. Signals corresponding to N-acetyl-
D
-galactosamine (GalNAc) and
phosphocholine (PCho) methyl protons and carbons occurred at 2.04/22.7 and 3.23/54.5 p.p.m., respectively. Pairs of deoxyprotons of AnKdo-ol
(reduced anhydro 3-deoxy-
D
-manno-oct-2-ulosonic acid) were identified in the DQF-COSY spectrum at 2.26–1.67 p.p.m.
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

L
-a-
D
-Hepp-(1fi 5.82–5.89
a
4.23–4.25
a
3.89–3.90
a
3.93 3.77 4.55 3.70 3.89
6
›
PEtn
98.4–98.7
a
79.6 69.8 66.7 71.8 75.0 63.0
HepIII
L
-a-
D
-Hepp-(1fi 5.22–5.24
a
4.24–4.26
a
4.33 3.87 – – – –
3
›
PEtn
101.6 70.2 76.6 65.8 – – –
GlcI fi4)-b-

fi3)-a-
D
-Galp-(1fi 4.91 3.89 3.95 4.25 4.38 3.68 –
100.9 68.1 79.2 69.5 70.8 –
GalNAc b-
D
-GalpNAc-(1fi 4.62 3.94 3.75 3.94 3.67
d
––
103.8 53.0 – 68.2 75.4 –
PEtnI 4.15 3.29
62.5 40.5
PEtnII 4.17 3.29
62.5 40.5
PCho 4.41 3.69
60.1 66.5
Gly 4.00
– 40.7
a
Several signals were observed for HepI, HepII and HepIII owing to heterogeneity in the AnKdo moiety.
b
H-4/H-6 of HepI were identified
at d 4.28/4.09–4.11 by NOE from GlcI.
c
–, not obtained owing to the complexity of the spectrum.
d
Tentative assignment from NOE data.
e
Residue marked with * denotes a further substituted analogue of the corresponding residue without *.
2986 M. Ma

A
/C-6 H-6
B
H-7
A
/C-7 H-7
B
GlcI fi6)-b-
D
-Glcp-(1fi 4.50 3.51 3.42 3.57 3.56 3.81 4.09
103.6 74.0 77.4 70.2 74.4 65.3
HepIV
a
fi4)-
D
-a-
D
-Hepp-(1fi 4.94 4.07 3.88 3.92 4.17 –
b
––
99.3 69.9 70.4 78.8 71.2 – –
5.06 4.05 3.80 3.90 4.28 – – –
100.6 69.2 70.4 80.2 71.4 – –
GalIII
a
b-
D
-Galp-(1fi 4.51 3.57 3.68 3.93 3.76
c
––

– 69.0 – 69.3 – –
a
Two spin-systems were found for the residue probably as a result of differences in the chemical environments of the various glycoforms.
The first spin-system is attributed to the Hex2 glycoform, while the second spin-system is attributed to the Hex4 and Hex5 glycoforms.
b
–,
not obtained owing to the complexity of the spectrum.
c
Tentative assignment from NOE data.
d
Residue marked with * denotes a further
substituted analogue of the corresponding residue without *.
Fig. 5. The 600-MHz
1
H NMR spectra of oligosaccharide (OS)-1 (A)
and OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981 showing the anomeric
regions. (A) Anomeric resonances that are characteristic for the Hep3-
glycoforms are labelled. Also indicated is an ethylene proton signal
from phosphocholine (PCho) at d 4.41. (B) Anomeric resonances that
are characteristic for the Hep4-glycoforms are labelled.
Fig. 6. Part of the 500-MHz heteronuclear
1
H–
31
PHMQCspectrumof
oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of
nontypeable Haemophilus influenzae (NTHi) strain 981. Assignments
are labelled.
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2987

triheptosyl inner-core moiety. This is the first example of an
H. influenzae strain expressing globotetraose in this mole-
cular environment. However, recently the H. influenzae
type b strain, RM7004, was reported to express a trun-
cated epitope, the globoside trisaccharide (globotriose)
[a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D
-Glcp]inthesame
molecular environment [22]. Additionally, in that strain,
the globotriose epitope was expressed as a terminal unit of a
tetrasaccharide extension from HepII, the same tetrasac-
charide that previously had been found in the type b strain,
RM153, at that location [11]. A third molecular environ-
ment for the globotriose epitope has been reported for
H. influenzae strain RM118 (Rd) [13] and several NTHi
isolates [21], where globotriose/globotetraose are present as
terminal OS extensions at HepIII. The globotriose epitope is
found on many human cells (p
k
blood group antigen), and
in H. influenzae is thought to be important for virulence by
acting as a mimic of these host structures, thus allowing the
organism to evade some element of the host immune
response [5,32]. For strain RM118, the glycosyltransferases
involved in the assembly of its globotetraose side-chain
were recently identified [7]. The lpsA gene was shown to be

locus is consistent with the lack of OS extension from HepII
observed in strain 981 (data not shown) [33].
In the Hep3-glycoforms of strain 981, GlcI was, to a great
extent, also substituted by PCho at O-6, which provides a
disubstitution pattern of GlcI that previously has been
observed in the NTHi strains SB33 and 176 [17,19]. In the
Hep4-glycoforms, however, the disaccharide branch b-
D
-
Galp-(1fi4)-
D
-a-
D
-Hepp wasshowntobe1,6-linkedto
GlcI, which adds a new variant to the wide range of
alternative substitution patterns found at GlcI (see the
Introduction). This is the first time that GlcI has been
reported to be di-glycosyl-substituted (Scheme 1; R
1
¼ b-
D
-
Galp-(1fi4)-
D
-a-
D
-Hepp, R
2
¼ a-
D

a Glc or Gal residue), where the
D
,
D
-Hep residue is located
at the same position as in strain 981, but is substituted
differently. From our survey of LPS from a representative
set of 25 NTHi clinical isolates, we have found six isolates
(including strain 981) that express
D
,
D
-Hep in the outer-core
regions (M. Ma
˚
nsson and E. K. H. Schweda, unpublished
results). Notably, the
D
,
D
-Hep residue has, in all isolates,
been found to be 1,6-linked to GlcI, but shows, in turn, a
high degree of variability in its substitution pattern between
the isolates. Structural analysis shows that the
D
,
D
-Hep
residue can be terminal, monosubstituted (as in strain 981)
as well as di-glycosyl-substituted (M. Ma

-a-
D
-Hepp-(1fi6)-b-
D
-Glcp). Sialyl-lacto-
N-neotetraose can be found in both Neisseria meningitidis
and N. gonorrhoeae LPS, and, more recently, it was
described in H. influenzae strain RM118 [20].
Recently, another investigation performed by us showed
that for three NTHi isolates (1209, 1207 and 1233), the O-6
position of GlcI is either substituted by PCho or the
tetrasaccharide unit b-
D
-GalpNAc-(1fi3)-a-
D
-Galp-(1fi4)-
b-
D
-Galp-(1fi6)-
L
-a-
D
-Hepp (or sequentially truncated ver-
sions thereof) [21]. This alternative substitution-pattern at
O-6 of GlcI bears a strong resemblance to the case for strain
981, where alternative substitution by PCho/
D
,
D
-Hep is

,
D
-Hep during in vivo growth and pathogenesis
are not understood. It may be that C-reactive protein binds
and mediates killing efficiently of organisms expressing the
PCho-containing glycoforms and that OS elongation pro-
tects the organism from killing mediated by naturally
acquired antibody and complement.
In several H. influenzae strains, the core region has been
found to be substituted by a phosphate group or a PEtn
group in addition to the common PEtn group at HepII and
the pyrophosphoethanolamine (PPEtn) group at the Kdo
residue. In NTHi strain 2019 [9], MS/MS analysis showed
the PEtn substituent to be located at HepIII; however, for
the majority of strains, the molecular environment has not
yet been established [12,14,40]. The present investigation
locates the additional PEtn group to O-3 of HepIII. In the
type b strain, RM153, a phosphate group was identified at
O-4 of HepIII [11]. MS analysis has indicated the presence
of a phosphate group in several other strains [12–14], but,
owing to the low abundance of the phosphate-containing
glycoforms, its location has not been determined. For NTHi
strain 981, capillary electrophoresis (CE)-ESI-MS/MS
experiments indicated the location of the phosphate
substituent to be variable and dependent upon whether
one or two PEtn groups substituted the triheptosyl unit.
When only one PEtn group was present (at HepII), the
phosphate group was indicated to be linked to HepIII (data
not shown). However, when two PEtn substituents were
present (at HepII and HepIII), the phosphate group

Study Group for the provision of strains used in this study. The
Swedish NMR centre (Go
¨
teborg, Sweden) is acknowledged for
providing access to their 600 MHz facilities. Mary Deadman and
Gaynor Randle are acknowledged for culturing H. influenzae.
Dr Jianjun Li is acknowledged for CE-ESI-MS/MS experiments.
References
1. Murphy, T.F. & Apicella, M.A. (1987) Nontypeable Haemophilus
influenzae: a review of clinical aspects, surface antigens, and the
human immune response to infection. Rev. Infect. Dis. 9, 1–15.
2. Mandrell, R.E., Griffiss, J.M. & Macher, B.A. (1988) Lipooligo-
saccharides (LOS) of Neisseria gonorrhoeae and Neisseria
meningitidis have components that are immunochemically similar
to precursors of human blood group antigens. Carbohydrate
sequence specificity of the mouse monoclonal antibodies that
recognize crossreacting antigens on LOS and human erythrocytes.
J. Exp. Med. 168, 107–126.
3. Mandrell, R.E., McLaughlin, R., Kwaik, Y.A., Lesse, A.,
Yamasaki, R., Gibson, B., Spinola, S.M. & Apicella, M.A. (1992)
Lipooligosaccharides (LOS) of some Haemophilus species mimic
human glycosphingolipids, and some LOS are sialylated. Infect.
Immun. 60, 1322–1328.
4. Kimura, A. & Hansen, E.J. (1986) Antigenic and phenotypic
variations of Haemophilus influenzae type b lipopolysaccharide
and their relationship to virulence. Infect. Immun. 51, 69–79.
5. Weiser, J.N. & Pan, N. (1998) Adaption of Haemophilus influenzae
to acquired and innate humoral immunity based on phase vari-
ation of lipopolysaccharide. Mol. Microbiol. 30, 767–775.
6. Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A.,

Richards, J.C. (1997) Structure of the variable and conser-
ved lipopolysaccharide oligosaccharide epitopes expressed by
Haemophilus influenzae serotype b strain Eagan. Biochemistry 36,
2091–2103.
12. Risberg, A., Schweda, E.K.H. & Jansson, P E. (1997) Structural
studies of the cell-envelope oligosaccharide from the lipopoly-
saccharide of Haemophilus influenzae strain RM.118-28. Eur. J.
Biochem. 243, 701–707.
13. Risberg, A., Masoud, H., Martin, A., Richards, J.C., Moxon,
E.R. & Schweda, E.K.H. (1999) Structural analysis of the lipo-
polysaccharide oligosaccharide epitopes expressed by a capsule-
deficient strain of Haemophilus influenzae Rd. Eur. J. Biochem.
261, 171–180.
14. Rahman, M.M., Gu, X X., Tsai, C M., Kolli, V.S.K. &
Carlson, R.W. (1999) The structural heterogeneity of the lipoo-
ligosaccharide (LOS) expressed by pathogenic non-typeable
Haemophilus influenzae strain NTHi 9274. Glycobiology 9,
1371–1380.
15. Schweda, E.K.H., Brisson, J R., Alvelius, G., Martin, A., Weiser,
J.N., Hood, D.W., Moxon, E.R. & Richards, J.C. (2000) Char-
acterization of the phosphocholine substituted oligosaccharide in
lipopolysaccharides of type b Haemophilus influenzae. Eur. J.
Biochem. 267, 3902–3913.
16. Ma
˚
nsson, M., Bauer, S.H.J., Hood, D.W., Richards, J.C., Mox-
on, E.R. & Schweda, E.K.H. (2001) A new structural type for
Haemophilus influenzae lipopolysaccharide. Structural analysis of
the lipopolysaccharide from nontypeable Haemophilus influenzae
strain 486. Eur. J. Biochem. 268, 2148–2159.

22. Masoud, H., Martin, A., Thibault, P., Moxon, E.R. & Richards,
J.C. (2003) Structure of extended lipopolysaccharide glycoforms
containing two globotriose units in Haemophilus influenzae sero-
type b strain RM7004. Biochemistry 42, 4463–4475.
23. Yildirim, H.H., Hood, D.W., Moxon, E.R. & Schweda, E.K.H.
(2003) Structural analysis of lipopolysaccharides from Haemo-
philus influenzae serotype f. Structural diversity observed in three
strains. Eur. J. Biochem. 270, doi:10.1046/j.1432-1033.2003.03693.x.
24. Li, J., Bauer, S.H.J., Ma
˚
nsson, M., Moxon, E.R., Richards, J.C. &
Schweda, E.K.H. (2001) Glycine is a common substituent of the
inner-core in Haemophilus influenzae lipopolysaccharide. Glyco-
biology 11, 1009–1015.
25. Cody, A.J., Field, D., Feil, E.J., Stringer, S., Deadman, M.E.,
Tsolaki, A.G., Gratz, B., Bouchet, V., Goldstein, R., Hood, D.W.
& Moxon, E.R. (2003) High rates of recombination in otitis media
isolates of non-typeable Haemophilus influenzae. Infect. Genet.
Evol. 3, 57–66.
2990 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
26. Holst, O., Brade, L., Kosma, P. & Brade, H. (1991) Structure,
serological specificity, and synthesis of artificial glycoconjugates
representing the genus-specific lipopolysaccharide epitope of
Chlamydia spp. J. Bacteriol. 173, 1862–1866.
27. Sawardeker, J.S., Sloneker, J.H. & Jeanes, A. (1965) Quantitative
determination of monosaccharides as their alditol acetates by gas
liquid chromatography. Anal. Chem. 37, 1602–1604.
28. Gerwig, G.J., Kamerling, J.P. & Vliegenthart, J.F. (1979) Determi-

34. Jarosik, G.P. & Hansen, E.J. (1994) Identification of a new locus
involved in expression of Haemophilus influenzae type b
lipooligosaccharide. Infect. Immun. 62, 4861–4867.
35. Reference withdrawn.
36. Schweda, E.K.H., Jonasson, J.A. & Jansson, P E. (1995) Struc-
tural studies of lipooligosaccharides from Haemophilus ducreyi
ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence
for intrastrain heterogeneity with the production of mutually
exclusive sialylated or elongated glycoforms. J. Bacteriol. 177,
5316–5321.
37. Weiser, J.N., Shchepetov, M. & Chong, S.T.H. (1997) Decoration
of lipopolysaccharide with phosphorylcholine: a phase-variable
characteristic of Haemophilus influenzae. Infect. Immun. 65, 943–
950.
38. Weiser, J.N., Pan, N., McGowan, K.L., Musher, D., Martin, A. &
Richards, J. (1998) Phosphorylcholine on the lipopolysaccharide
of Haemophilus influenzae contributes to persistence in the
respiratory tract and sensitivity to serum killing mediated by
C-reactive protein. J. Exp. Med. 187, 631–640.
39. Lysenko,E.,Richards,J.C.,Cox,A.D.,Stewart,A.,Martin,A.,
Kapoor, M. & Weiser, J.N. (2000) The position of phosphor-
ylcholine on the lipopolysaccharide of Haemophilus influenzae
affects binding and sensitivity to C-reactive protein-mediated
killing. Mol. Microbiol. 35, 234–245.
40. Hood, D.W., Makepeace, K., Deadman, M.E., Rest, R.F., Thi-
bault, P., Martin, A., Richards, J.C. & Moxon, E.R. (1999) Sialic
acid in the lipopolysaccharide of Haemophilus influenzae:strain
distribution, influence on serum resistance and structural char-
acterization. Mol. Microbiol. 33, 679–692.
41. Hood,D.W.,Cox,A.D.,Gilbert,M.,Makepeace,K.,Walsh,S.,