Báo cáo khoa học: Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae pptx

Identification and structural characterization of a sialylated
lacto-N-neotetraose structure in the lipopolysaccharide
of
Haemophilus influenzae
Andrew D. Cox
1
, Derek W. Hood
2
, Adele Martin
1
, Katherine M. Makepeace
2
, Mary E. Deadman
2
,
Jianjun Li
1
, Jean-Robert Brisson
1
, E. Richard Moxon
2
and James C. Richards
1
1
Institute for Biological Sciences, National Research Council, Ottawa, Canada;
2
University of Oxford Department of Paediatrics,
Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, UK
A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit
was identified and structurally characterized in the lipo-
polysaccharide (LPS) from the genome-sequenced strain

Road (RM118) of the human pathogen Haemophilus influ-
enzae grown in the presence of sialic acid. A combination of
molecular genetics, MS and NMR spectroscopy techniques
showed that this structural unit extended from the proximal
heptose residue of the inner core region of the LPS molecule.
The structure of the Sial-lNnT unit was identical to that
found in meningococcal LPS, but glycoforms containing
truncations of the Sial-lNnT unit, comprising fewer residues
than the complete oligosaccharide component, were not
detected. The finding of sialylated glycoforms that were
either fully extended or absent suggests a novel biosynthetic
feature for adding the terminal tetrasaccharide unit of the
Sial-lNnT to the glycose acceptor at the proximal inner core
heptose.
Keywords: Haemophilus influenzae; LPS; mass spectrometry;
NMR; sialic acid.
Haemophilus influenzae remains an important cause of
disease worldwide. Encapsulated strains can cause invasive,
bacteraemic infections such as septicemia and strains
lacking a capsule, so-called nontypeable strains (NTHi),
are a common cause of otitis media and acute lower
respiratory tract infections [1]. Lipopolysaccharide (LPS) is
critical to the integrity and functioning of the cell wall of
H. influenzae, and as a surface component is a target for
host immune responses. The structure of H. influenzae LPS
has been well established from several studies [2–8].
H. influenzae LPS is composed of a membrane-anchoring
lipid A moiety linked to a heterogeneous core oligosaccha-
ride via a phosphorylated 2-keto-3-deoxyoctulosonic acid
(Kdo) residue. The core oligosaccharide of H. influenzae

can be divided into inner and outer regions. The inner core
consists of a
L
-glycero-
D
-manno-heptose (Hep) trisaccharide
unit wherein the second heptose residue (Hep II) is substi-
tuted at the 6-position by a phosphoethanolamine (PEtn)
residue. Each of the Hep residues can provide a point for the
addition of hexose (Hex) residues each of which can be
further extended into oligosaccharide chains of the outer
core. The substitution pattern and degree of extension from
each Hep residue varies between and within strains. The
presence of nonglycose residues, including phosphate-
containing substituents and ester linked acetyl groups and
glycine molecules also contribute to the structural variability
of these molecules [9]. The LPS of H. influenzae lacks the
O-specific side chain characteristic of enteric bacteria.
Evidence from recent structural studies has confirmed the
presence and defined the position of sialic acid in the LPS of
H. influenzae [7,10–12]. Sialylation of LPS is a commonly
observed structural modification in Neisseria spp. and is
frequently found as the sialylated lacto-N-neotetraose
structure (Sial-lNnT) [13,14]. Indeed in neisserial species
sialylation of LPS renders the bacteria resistant to comple-
ment-mediated killing by normal human serum [15]. In the
gonococcus, LPS sialylation can occur only if an exogenous
supply of sialic acid is available [16]. In contrast, sialylated
meningococcal LPS glycoforms can be synthesized endo-
genously [17]. Sialylation of H. influenzae LPS appears to

depend upon the presence of an exogenous source of sialic
acid or its activated form CMP-sialic acid [11,18]. Sialyla-
tion of H. influenzae LPS was first indicated by alterations
in the reactivity of LPS with a mAb 3F11 that is specific for
the terminal lactosamine disaccharide of the lNnT group
following treatment with neuraminidase [19]. Consistent
with these findings, MS studies pointed to the presence of a
Sial-lNnT group [20]. However, the low amounts of
sialylated glycoforms present precluded definitive structural
characterization of the sialylated moiety. In a recent
structural study utilizing an exogenous source of sialic acid
in the growth medium we identified and localized another
sialylated species, namely sialyl-lactose (Sial-Lac) in
H. influenzae LPS [10,11].
The genetic control of the biosynthesis of H. influenzae
LPS inner and outer core oligosaccharides has been
investigated extensively. Most of the genes responsible for
Correspondence to A. D. Cox, Institute for Biological Sciences,
National Research Council, Ottawa, ON, Canada. K1A 0R6.
Fax: +44 613 952 9092, Tel.: +44 613 991 6172,
E-mail:
Abbreviations: NTHi, nontypeable strains of Haemophilus influenzae;
LPS, lipopolysaccharide; Kdo, 2-keto-3-deoxyoctulosonic acid;
Hep,
L
-glycero-
D
-manno-heptose; PEtn, phosphoethanolamine;
Hex, hexose; Sial-lNnT, sialylated lacto-N-neotetraose; Sial-Lac,
sialyl-lactose; BHI, brain heart infusion; ES-MS, electrospray;

PCho, phosphocholine.
(Received 21 March 2002, revised 20 June 2002, accepted 2 July 2002)
Eur. J. Biochem. 269, 4009–4019 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03090.x
the biosynthesis of the glycose residues in the globotetraose-
containing Road (RM118) LPS glycoforms have been
recently reported [21]. Moreover, it is also known that
H. influenzae has the ability to modify its LPS structure by a
genetic mechanism known as phase variation [22]. We
recently identified one such phase variable gene lic3A as
encoding an a-2,3-sialyltransferase, which was found to be
responsible for the sialylation of a lactose group in the LPS
of H. influenzae strain RM118 [11]. In that study, we
observed that following insertional deletion of the lic3A
gene, LPS derived from the mutated strain still produced
sialylated glycoforms, suggesting the presence of alternate
sialylated structures. In this study we describe the identifi-
cation and structural analysis of a Sial-lNnT unit in
H. influenzae LPS.
MATERIALS AND METHODS
Bacterial strains and culture conditions
The H. influenzae strain RM118 (Rd) is derived from the
same source as the strain for which the complete genome
has been sequenced [23]. Isogenic strains RM118 lgtC and
the double mutant RM118 lgtC lic3A have been described
previously [11], as have strains RM118 lic2A, RM118 lpsA
and RM118 lgtF [21]. H. influenzae strains were grown
at 37 °C on brain heart infusion (BHI) agar (1% w/v)
supplemented with Levinthals reagent (10% v/v) and
when appropriate, Neu5Ac (25 lgÆmL
)1

), CMP-Neu5Ac
(50 lgÆmL
)1
), kanamycin (10 lgÆmL
)1
) or tetracycline
(4 lgÆmL
)1
).
Structural analysis and purification of LPS
LPS was prepared for structural analysis from cells
harvested after growth on batches of 20 BHI plates,
supplemented with Neu5Ac. LPS was extracted by the hot
phenol/water method [24] and O-deacylated as described
previously [3]. MS analyses were carried out as described
previously [8,25,26]. Electrospray (ES) MS was measured in
the negative ion mode on a VG Quattro triple quadrupole
mass spectrometer (Fisons Instruments) with an electro-
spray ion source.
Capillary electrophoresis (CE)-MS analysis was per-
formed on a crystal Model 310 CE instrument (ATI
Unicam, Boston, MA, USA) coupled to an API 3000 mass
spectrometer (MDS/Sciex, Concord, Canada) via a micro-
ionspray interface. A sheath solution (isopropanol/metha-
nol, 2 : 1, v/v) was delivered at a flow rate of 1 lLÆmin
)1
to
a low dead volume tee (250 lm internal diameter; Chro-
matographic Specialities, Brockville, Canada). All aqueous
solutions were filtered through a 0.45-lm filter (Millipore)

before use. An electrospray stainless steel needle (27 gauge)
was butted against the low dead volume tee and enabled the
delivery of the sheath solution to the end of the capillary
column. Separation was obtained on  90 cm length of bare
fused-silica capillary (192 lmo.d.· 50 lm internal diam-
eter; Polymicro Technologies, Phoenix, AZ, USA) using
30 m
M
morpholine in deionized water (negative ion mode),
pH 9.0, containing 5% methanol and 15 m
M
ammonium
acetate/ammonium hydroxide in deionized water (positive
ion mode), pH 9.0, containing 5% methanol. A voltage of
25 kV was typically applied at the injection. The outlet of
the capillary was tapered to  15 lm internal diameter
using a laser puller (Sutter Instruments, Novato, CA, USA).
Mass spectra were acquired with dwell times of 3.0 ms per
step of 1 m/z unit in full-mass scan mode. In the CE-ESMS,
30 nL sample was typically injected by using 300 mbar for a
duration of 0.1 min. For CE-ESMS/MS experiments
 60 nL sample was introduced using 300 mbar for
0.2min.TheMS/MSdatawereacquiredwithdwelltimes
of 2.0 ms per step of 1 m/z unit. Fragment ions formed by
collision activation of selected precursor ions with nitrogen
in the RF-only quadrupole collision cell, were mass
analysed by scanning the third quadrupole.
NMRexperimentswereperformedonVarianINOVA
600 and 500 NMR spectrometers using a 5-mm triple
resonance probe with Z gradient as described previously

[25]. Measurements were made at 25 °C at concentrations of
 2mgÆmL
)1
in D
2
O, subsequent to several lyophilizations
with D
2
O. For the proton chemical shift reference, the
HDO resonance was set at 4.78 p.p.m. at 25 °Crelativeto
the methyl resonance of external acetone at 2.225 p.p.m. All
of the NMR data was acquired using Varian sequences
provided with the VNMR 6.1B software. The same
program was used for processing.
The proton spectrum was acquired with a sweep width of
10 p.p.m., 1024 transients, water presaturation during the
relaxation delay of 1.5 s and acquisition time of 1.7 s. It was
processed with a low-pass digital filter for solvent suppres-
sion and zero filling for a final resolution of 0.37 Hz per
point. Homonuclear two-dimensional experiments, COSY
(16 h), TOCSY (7 h) and NOESY (7 h) were acquired using
the following parameters: water presaturation during the
relaxation delay of 1.0 s, spectral width of 7 p.p.m. (COSY;
10 p.p.m.), acquisition time in t
2
of 0.15 s (COSY; 0.20 s)
and 200 increments (COSY; 128) with 52 (TOCSY), 40
(NOESY) or 360 (COSY) scans per increment. The sign
discrimination in F
1

was achieved by the States method. The
mixing time for the NOESY and TOCSY experiments was
0.4 and 0.08 s, respectively. The phase-sensitive spectra were
processedwithforwardlinearpredictionint
1
, unshifted
Gaussian window functions, and zero filling to 1024*1024
complex points for a resolution of 2.9 Hz per point in both
dimensions. The one-dimensional NOESY experiment was
acquired with a sweep width of 16 p.p.m., water presatura-
tion during the relaxation delay of 1 s, acquisition time of 1 s,
a mixing time of 800 ms, selective pulse with 100 Hz
bandwidth, and 2048 transients for a duration of 2 h. Using
similar acquisition parameters, the one-dimensional NO-
ESY-TOCSY experiment [27] was acquired with a NOE
mixing time of 800 ms, spin lock time of 80 ms, and 12288
transients for a duration of 10 h. The one-dimensional
TOCSY experiment was acquired with a sweep width of
16 p.p.m., water presaturation during the relaxation delay of
1 s, acquisition time of 1.7 s, mixing times of 80 ms, selective
pulse with 30 Hz bandwidth, and 5120 transients for a
duration of 4 h. Using similar acquisition parameters, the
one-dimensional TOCSY-NOESY experiment [27] was
acquired with a NOE mixing time of 800 ms, spin lock time
of 80 ms, and 20480 transients for a duration of 21 h.
Electrophoretic analysis of LPS
LPSwaspreparedandanalysedbytricine-SDS/PAGEas
described previously [10].
4010 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Analytical methods and methylation analysis

Sugars were determined as their alditol acetates and
partially methylated alditol acetate derivatives by GLC-
MS as described previously [8].
RESULTS
In a previous study on LPS from H. influenzae strain
RM118 lgtC we had identified a sialylated lactosyl
(Sial-Lac) extension from the distal heptose residue
(Hep III) of the inner core [11]. Interestingly, following
mutation of the sialyltransferase gene (lic3A) responsible for
addition of Neu5Ac to the lactose moiety in this strain,
creating the double mutant (lgtC lic3A), sialylated glyco-
forms were still observed. In SDS/PAGE analysis sialylated
species of strain RM118 lgtC lic3A LPS were found as the
LPS constituents with the lowest mobility, identified by
virtue of an alteration in their migration pattern following
treatment with neuraminidase, an enzyme that specifi-
cally cleaves sialic acid residues (Fig. 1; lanes 1 and 2).
H. influenzae LPS typically migrates as a complex series of
bands in SDS/PAGE, each band corresponding to gain or
loss of a glycose moiety due to the phase variable nature of
H. influenzae LPS synthesis [3,21,22]. In LPS from RM118
lgtC lic3A it was interesting to note that the band
corresponding to the sialylated LPS glycoform separated
from those of fast migrating nonsialylated glycoforms by
several glycose units. ES-MS analysis of the O-deacylated
LPS from the RM118 lgtC lic3A double-mutant strain
revealed ions with an expected composition consistent with
the presence of a sialic acid residue (Fig. 2; Table 1). It
has previously been established that sialic acid residues are
not removed by the hydrazinolysis treatment used to

O-deacylate LPS [11]. The molecular mass of the major
sialylated glycoforms in the lgtC lic3A double mutant
(3381.6 and 3546.0 Da) indicated the presence of an
additional 527 atomic mass units, corresponding to an
N-acetyl-hexosamine residue and two hexose residues over
the Sial-Lac glycoform in the O-deacylated LPS from the
Fig. 1. SDS/PAGE analysis of LPS from strains RM118 lgtC lic3A,
RM118 lgtC, RM118 (wt), RM118 lic2A,RM118lpsA and RM118
lgtF before and after treatment with neuraminidase (as indicated by – and
+, respectively). The sialylated tetrasaccharide-containing glycoforms
are indicated by an asterisk. All strains were grown on media con-
taining Neu5Ac except for the wild-type (wt) strain RM118.
Fig. 2. Triply charged region of the ES-MS spectrum from the O-deacylated LPS obtained from strain RM118 lgtC lic3A grown on BHI plates
supplemented with Neu5Ac. The structure of the inner core glycoforms is shown and ions arising from the inner core and from the addition of the
sialylated tetrasaccharide are indicated.
Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4011
lgtC single mutant (Table 1) [11]. The two major sialylated
glycoforms observed differed by 165 atomic mass units,
consistent with the gain or loss of a phosphocholine (PCho)
residue. The PCho group is appended to the glucose residue
at the proximal heptose residue (Hep I) in the parent strain
[5] and lgtC mutant [21]. CE-MS analysis was carried out on
Table 1. Negative ion ES-MS data and proposed compositions of O-deacylated LPS from H. infl uenza e strains RM118, RM118 lgtC,RM118
lgtC lic3A, RM118 lic2A and RM118 lpsA when grown on media containing sialic acid. Average mass units were used for calculation of molecular
weight based on proposed composition as follows: lipid A, 953.00; Hex, 162.15; HexNAc, 203.19; Hep, 192.17; Kdo-P, 300.16; PEtn, 123.05; PCho,
165.05; Sial, 291.05.
Strain [M-3H]
3–
[M-2H]
2–

Observed
molecular ion
Calculated
molecular ion
Relative
intensity
Proposed
composition
RM118 lgtC 853.2 1280.3 2562.6 2562.2 0.50 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
867.2 1300.9 2604.2 2604.2 0.85 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A
908.3 1362.6 2727.5 2727.3 1.00 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
964.5 1446.6 2895.8 2895.3 0.35 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P,
Lipid A
1005.2 1508.3 3018.6 3018.3 0.70 Sial, PCho, 3Hex, 3Hep, 2PEtn, Kdo-P,
Lipid A
1126.0 – 3381.0 3380.8 0.25 (Sial, 2Hex, HexNAc), 3Hex, 3Hep,
2PEtn, Kdo-P, Lipid A
1181.4 – 3547.2 3545.9 0.20 (Sial, 2Hex, HexNAc), PCho, 3Hex,
3Hep, 2PEtn, Kdo-P, Lipid A
RM118 lgtC 867.3 1301.2 2604.7 2604.2 1.00 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A
lic3A 908.3 1362.6 2727.5 2727.3 0.90 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
1126.2 – 3381.6 3380.8 0.20 (Sial, 2Hex, HexNAc), 3Hex, 3Hep,
2PEtn, Kdo-P, Lipid A
1181.0 – 3546.0 3545.9 0.20 (Sial, 2Hex, HexNAc), PCho, 3Hex,
3Hep, 2PEtn, Kdo-P, Lipid A
RM118 812.2 – 2439.6 2439.2 0.15 3Hex, 3Hep, PEtn, Kdo-P, Lipid A
866.9 – 2603.7 2604.2 0.50 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A
920.9 – 2765.7 2766.3 0.80 PCho, 4Hex, 3Hep, PEtn, Kdo-P, Lipid A
962.0 – 2889.0 2889.3 0.45 PCho, 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
964.0 – 2895.0 2895.3 1.00 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P,

Lipid A
989.0 – 2970.0 2969.5 0.60 PCho, 4Hex, HexNAc, 3Hep, PEtn,
Kdo-P, Lipid A
1005.0 – 3018.0 3018.3 0.70 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P,
Lipid A
1030.0 – 3093.0 3092.6 0.25 PCho, 4Hex, HexNAc, 3Hep, 2PEtn,
Kdo-P, Lipid A
1179.7 – 3542.1 3542.9 0.20 (Sial, 2Hex, HexNAc), 4Hex, 3Hep,
2PEtn, Kdo-P, Lipid A
1235.0 – 3708.0 3708.0 0.30 (Sial, 2Hex, HexNAc), PCho, 4Hex,
3Hep, 2PEtn, Kdo-P, Lipid A
RM118 lic2A 758.0 – 2277.0 2277.4 0.40 2Hex, 3Hep, PEtn, Kdo-P, Lipid A
812.9 – 2441.7 2442.4 0.85 PCho, 2Hex, 3Hep, PEtn, Kdo-P, Lipid A
853.9 – 2564.7 2565.5 0.60 PCho, 2Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
1030.9 – 3095.7 3095.6 0.30 (Sial, 2Hex, HexNAc), 2Hex, 3Hep, PEtn,
Kdo-P, Lipid A
1071.9 – 3218.7 3218.7 0.90 (Sial, 2Hex, HexNAc), 2Hex, 3Hep,
2PEtn, Kdo-P, Lipid A
1085.8 – 3260.4 3260.7 0.35 (Sial, 2Hex, HexNAc), PCho, 2Hex,
3Hep, PEtn, Kdo-P, Lipid A
1126.8 – 3383.4 3383.7 0.75 (Sial, 2Hex, HexNAc), PCho, 2Hex,
3Hep, 2PEtn, Kdo-P, Lipid A
RM118 lpsA 703.6 1055.9 2114.1 2115.2 0.45 Hex, 3Hep, PEtn, Kdo-P, Lipid A
744.9 1117.7 2237.3 2238.2 0.25 Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
758.7 1138.8 2279.3 2280.2 1.00 PCho, Hex, 3Hep, PEtn, Kdo-P, Lipid A
799.7 1200.4 2402.8 2403.2 0.60 PCho, Hex, 3Hep, 2PEtn, Kdo-P, Lipid A
1017.6 1526.5 3056.1 3056.4 0.20 (Sial, 2Hex, HexNAc), Hex, 3Hep, 2PEtn,
Kdo-P, Lipid A
1072.6 1609.3 3221.1 3221.5 0.40 (Sial, 2Hex, HexNAc), PCho, Hex, 3Hep,
2PEtn, Kdo-P, Lipid A

4012 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the O-deacylated material from the RM118 lgtC lic3A
double mutant in order to obtain further information on the
nature of the sialylated glycoforms. Comparison of the total
ion electropherogram in negative ion mode (Fig. 3A) and
selective ion scanning for m/z 290, specific for sialic acid
(Fig. 3B), suggested the presence of two major populations
of sialylated glycoforms consistent with the ES-MS spec-
trum (Fig. 2). MS/MS analysis in positive ion mode of the
doubly charged ion at m/z 1691.6 (Fig. 3C) that corre-
sponds to the 3381.6 atomic mass unit glycoform produced
abundant fragment ions at m/z 657 (Neu5Ac-Hex-Hex-
NAc); m/z 528 (Hex-HexNAc-Hex); m/z 366 (Hex-Hex-
NAc); m/z 292 (Neu5Ac) and; m/z 204 (HexNAc). This
series of ions indicated the presence of a Neu5Ac-Hex-
HexNAc-Hex oligosaccharide unit. In order to determine
the location of this tetrasaccharide unit, the wild-type strain
RM118 and a series of mutants thereof (lgtC, lic2A, lpsA)
that corresponded to sequential truncations of the globoside
trisaccharide extending from Hep III were examined, as
well as a lgtF mutant in which there is no chain extension
from Hep I [21]. In SDS/PAGE analysis of the LPS
obtained from the lgtC, lic2A and lpsA mutants before and
after neuraminidase treatment, the enzyme sensitive sialy-
lated glycoforms showed an increased mobility. Corre-
spondingly, the mobility of the major nonsialylated
glycoforms increased as the length of chain from Hep III
was reduced by consecutive glycose truncations (Fig. 1;
lanes 3–4, 7–10), confirming that the sialylated unit was not
attached via the distal heptose residue (Hep III) of the inner

core LPS. This was most notable for the lpsA mutant, as the
gene product of lpsA is responsible for initiation of chain
extension from Hep III [21], and in this mutant background
the LPS still elaborates the slower migrating sialylated
glycoform. However, the lgtF mutant LPS (Fig. 1; lanes 11–
12) did not contain slower migrating sialylated glycoforms,
indicating the sialylated unit to be located as an extension
from the Hep I residue of the inner core. LgtF has been
shown to be the glycosyltransferase required for initiation of
chain extension from Hep I [21].
As expected ES-MS analysis of the O-deacylated LPS of
each mutant strain (Table 1) revealed a reduction in mass of
the sialylated species by a value corresponding to the
removed residue. Thus the difference in mass between the
O-deacylated LPS of RM118 lic2A (3383.4 Da) and
RM118 lpsA (3221.1 Da) corresponds to the glucose residue
(162.3 atomic mass units) that is normally attached by the
lpsA gene product. In the spectra of O-deacylated LPS from
the parent strain RM118 and RM118 lgtC, Sial-Lac
glycoforms were observed in addition to the higher mass
sialylated glycoforms. As expected Sial-Lac-containing
glycoforms were not observed in the lic2A and lpsA
mutants, as the required lactose acceptor is not present. It
is noteworthy that di-sialylated species were not observed
even in the lgtC mutant, the most appropriate background
for Sial-Lac production [11]. In each of the mutant strains
the higher mass sialylated glycoforms had a molecular
weight consistent with the addition of a complete tetrasac-
charide unit comprising Neu5Ac-Hex-HexNAc-Hex (819
atomic mass units). Closer examination of the ES-MS

revealed no evidence for any glycoforms corresponding to
partial addition of this sialylated unit, suggesting that this
tetrasaccharide is added as a complete unit or not at all
during LPS biosynthesis.
The structure of the sialylated glycoforms was determined
by NMR spectroscopy. To simplify the analysis the simplest
structure still containing the sialylated tetrasaccharide was
chosen for NMR studies, namely LPS from the lpsA
mutant. The one-dimensional
1
H-NMR spectrum of the
O-deacylated LPS was well resolved (Fig. 4). Characteristic
signals were observed in the anomeric region for the H-1
1
H-resonances of a-configured residues at 5.76 p.p.m.
(Hep II), 5.42 p.p.m. (GlcN of O-deacylated lipid A), and
5.16 p.p.m. (Hep I and Hep III). Additionally, signals were
observed in the anomeric region for the H-1
1
H-resonances
of b-configured residues, including the second GlcN residue
of O-deacylated lipid A (4.63 p.p.m.) and the glucose
residue (Glc I) at Hep I (4.54 p.p.m.) consistent with
assignments previously obtained for O-deacylated LPS
from the lpsA mutant that had been grown on media
not containing Neu5Ac [21]. Closer examination of the
b-anomeric region revealed two minor resolved signals at
4.74 and 4.45 p.p.m. due to the anomeric protons from
Fig. 3. CE-ES mass spectrum of O-deacylated LPS from strain RM118
lgtC lic3A grown on BHI plates supplemented with Neu5Ac. (A) Solid

line indicates the total ion electropherogram (TIE), the dotted line
indicates single ion monitoring (SIM) for m/z 290
–
, obtained in neg-
ative ion mode with a high orifice voltage (120 V), and (B) a MS/MS
experiment in positive ion mode on m/z 1691.6
2+
that corresponds to
the Sial-lNnT-containing glycoform without the PCho moiety.
Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4013
residues in the tetrasaccharide unit of the sialylated glyco-
form. The signal at 4.74 p.p.m. produced a spin-system in
a TOCSY experiment that could be assigned to N-acetyl-
b-
D
-glucosamine (b-
D
-GlcNAc) by comparison to the
published data (Fig. 5) [13,14]. Similarly (data not shown)
the signal at 4.45 p.p.m. could be assigned to a b-
D
-
galactose spin-system by comparison to the published data
[13,14]. In the high-field region of the spectrum character-
istic signals were observed for the axial and equatorial H-3
1
H-resonances of the sialic acid residue at 1.81 and
2.76 p.p.m., respectively (Fig. 4). Integration of the H-3
1
H-resonances of the sialic acid residue indicated that the

sialylated glycoform was present at levels of  10% of the
total LPS glycoform population. Higher percentages were
anticipated from the MS analyses, but perhaps the levels of
sialylated species were over-estimated in the MS studies due
to the presence of the sialic acid residue in these glycoforms
that would be more readily ionized in the negative ion mode.
The
1
H NMR spectrum of the inner core LPS structure
was assigned using COSY and TOCSY experiments
(Table 2). The ring sizes and relative stereochemistries of
the component monosaccharides were established from the
1
H chemical shifts and the magnitude of the coupling
constants. The sequence of glycosyl residues of the inner
core was determined from interresidue
1
H–
1
HNOEmeas-
urements between anomeric and aglyconic protons on
adjacent glycosyl residues. The NMR data was almost
identical to that found previously [21] for the O-deacylated
LPS of the RM118 lpsA mutant that had been grown
on media not containing Neu5Ac. Under these growth
conditions, the RM118 lpsA mutant does not elaborate
detectable amounts of LPS containing sialylated glycoforms
[21]. The structure of the oligosaccharide chain of the
sialylated glycoforms was determined from a series of
selective excitation NMR experiments [27]. Initially the

axial resonance of the sialic acid residue at 1.81 p.p.m.
was selectively irradiated in a one-dimensional NOESY
Fig. 4. Anomeric region of the
1
H-NMR spectrum of the O-deacylated LPS obtained from strain RM118 lpsA grown on BHI plates supplemented with
Neu5Ac. Inset, complete
1
H-NMR spectrum. The spectrum was recorded in D
2
O at pH 7.0 and 295 K.
Fig. 5. Ring
1
H region of the TOCSY spectrum of the O-deacylated
LPS from strain RM118 lpsA grown on BHI plates supplemented with
Neu5Ac corresponding to the spin system from the residue at 4.74 p.p.m.
The spectrum was recorded in D
2
O at pH 7.0 and 295 K.
4014 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002
experiment, revealing signals that by comparison to pub-
lished data [13], could be assigned at 2.76 (H-3eq), 3.68
(H-4), 3.86 (H-5) and at 4.05 p.p.m. via an interresidue
NOE. The latter signal was then irradiated in a one-
dimensional TOCSY step following the one-dimensional
NOESY step which revealed a signal at 4.56 p.p.m. that
subsequently was found to correspond to the anomeric
resonance of the b-
D
-galactose (Gal II) residue substituted
by sialic acid (Fig. 6A). Selective irradiation of the anomeric

resonance of the b-
D
-GlcNAc residue at 4.74 p.p.m. in a
one-dimensional TOCSY experiment revealed the ring
1
H-signals at 3.81 (H-2), 3.73 (H-3 and H-4), and
3.59 p.p.m. (H-5). In a subsequent one-dimensional
NOESY step irradiation of the H-3/H-4 signals at
3.73 p.p.m. produced signals at 4.74 and 4.56 p.p.m.
corresponding to the anomeric
1
H-resonances of the
b-
D
-GlcNAc and the b-
D
-galactose (Gal II) residue substi-
tuting the b-
D
-GlcNAc, respectively (Fig. 6B). By compar-
ison to the published data [13,14,28] it was clear that the
chemical shifts of the b-
D
-GlcNAc residue were consistent
with substitution at the 4-position. Thus, these experiments
established the sequence of the terminal trisaccharide as
a-Neu5Ac-(2–3)-b-
D
-Gal-(1–4)-b-
D

-GlcNAc. Examination
of the two-dimensional TOCSY and NOESY spectra
revealed characteristic signals for a 3-linked b-
D
-galactose
(Gal I) residue at 3.60 (H-2), 3.73 (H-3), and 4.17 p.p.m.
(H-4) in the spin-system arising from the anomeric
resonance at 4.45 p.p.m. [14]. This inference was confirmed
by a TOCSY/NOESY selective excitation experiment,
where following selective irradiation of the anomeric proton
at 4.45 p.p.m. in the TOCSY step, subsequent irradiation of
the H-3 proton at 3.73 p.p.m. in the NOESY step revealed
the anomeric proton at 4.74 p.p.m. of the b-
D
-GlcNAc
residue, thus confirming this linkage (data not shown)
and establishing the structure of the tetrasaccharide to
be, a-Neu5Ac-(2–3)-b-
D
-Gal-(1–4)-b-
D
-GlcNAc-(1–3)-b-
D
-
Gal. Due to the low intensity of the sialylated glycoforms
and a high degree of overlap in the b-anomeric region of the
spectrum, NMR methods were not successful in confirming
the linkage position of the glucose residue at Hep I, to
which the terminal tetrasaccharide unit was expected to be
the attached. This is the only glucose residue present in the

LPS of the RM118 lpsA mutant. Thus a methylation
analysis was performed in order to determine its linkage
position. Methylation analysis was carried out on the
dephosphorylated O-deacylated LPS as it is known that
phosphorylated residues are not readily identified following
methylation analysis and a PCho residue substitutes the
glucose residue in the majority of glycoforms. GLC-MS
analysis of the partially methylated alditol acetates identified
the expected substitution patterns for the inner core LPS,
with t-Glc, t-Hep, 2-Hep and 3,4-Hep residues identified in
approximately equimolar amounts. Less intense signals
were also identified for 3-Gal and 4-Glc establishing the
glucose residue to be 4-substituted and confirming the
3-linkages for the galactose residues of the Sial-lNnT unit
(Fig. 7). This confirms that a terminal Sial-lNnT unit is
linked to the Hep I residue of the LPS inner core in the
sialylated glycoform. The structure of the sialylated glyco-
forms of the O-deacylated LPS of H. influenzae strain
RM118 and mutants causing sequential truncations from
Hep III are illustrated in Fig. 8.
DISCUSSION
Structural analysis of H. influenzae strain RM118 lpsA LPS
has revealed glycoforms containing a Sial-lNnT unit when
the organism is grown on solid medium containing Neu5Ac.
This structure was found to extend from the proximal
heptose residue (Hep I) and is expressed in LPS glycoforms
from the wild-type RM118 strain which also expresses a
globoside unit (a-
D
-Galp-(1–4)-b-

D
-Galp-(1–4)-b-
D
-Glcp-
(1–) from the distal heptose residue (Hep III). Sial-lNnT-
containing glycoforms were also characterized in LPS from
RM118 mutant strains with sequential truncations in the
globoside unit (lgtC, lic2A,andlpsA). Sial-lNnT-containing
glycoforms were not observed however, in LPS from the
RM118 lgtF mutant strain. LgtF is the glucosyltransferase
Table 2.
1
H NMR assignments of O-deacylated LPS from H. influenzae strain RM118 lpsA grown on media containing sialic acid. Assignments at
295 K, relative to HOD 4.78 p.p.m. ND, Not determined.
H-1 H-2 H-3 H-4 H-5 NOEs
Lipid A-OH
a-GlcN 5.42 3.94 ND ND ND –
b-GlcN 4.63 3.82 3.70 ND ND ND
Inner core
Hep I 5.16 4.14 4.07 ND ND 4.31 Kdo H-5
Hep II 5.76 4.27 ND ND ND 4.07 Hep I H-3
Hep III 5.16 4.06 ND ND ND 4.27 Hep II H-2
5.76 Hep II H-1
t-b-Glc (Glc I) 4.54 3.47 3.55 ND ND 4.25 Hep I H-4
4.05 Hep I H-6
Sial-lNnT unit
-4)-b-Glc-(1-(Glc I) ND ND ND ND ND ND
-3)-b-Gal-(1-(Gal I) 4.45 3.60 3.73 4.17 ND ND
-4)-b-GlcNAc-(1- 4.74 3.81 3.73 3.74 3.59 3.73 Gal-I H-3
-3)-b-Gal-(1-(Gal II) 4.56 ND 4.05 ND ND 3.74 GlcNAc H-4

t-a-Neu5Ac 2.76
1.81
3.68 3.86 4.05 Gal-II H-3
Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4015
responsible for the initiation of oligosaccharide extension
from Hep I. In the absence of the glucose acceptor the
sialylated tetrasaccharide unit cannot be attached. In strain
RM118, and the RM118 lgtC mutant strain, we have now
identified two different sialylated species, namely Sial-Lac
and sialyl-lacto-N-neotetraose (Sial-lNnT). Interestingly,
glycoforms containing both sialylated oligosaccharides were
not observed. It is possible that the expression of one
sialylated structure sterically, or in some other way, hinders
the attachment of the second sialylated group. Similarly, the
high molecular weight sialylated glycoform was not
observed in the wild-type RM118 strain when there was
full extension of the globotetraose oligosaccharide (b-
D
-
GalpNAc-(1–4)-a-
D
-Galp-(1–4)-b-
D
-Galp-(1–4)-b-
D
-Glcp-
(1–) from Hep III, again perhaps indicating that steric
interference of the two chains precludes coincident expres-
sion of both. It is noteworthy that the majority of Sial-
lNnT-containing glycoforms contain two PEtn residues. In

the inner core of H. influenzae LPS, one PEtn residue is
stoichiometrically present at the 6-position of the Hep II
residue, and a second PEtn residue is sometimes attached to
the Kdo-P moiety. The percentage of Sial-lNnT-containing
glycoforms that elaborate two PEtn residues is considerably
higher than the percentage of other glycoforms that elabo-
rate two PEtn residues (Fig. 2), although the significance of
this is unclear. Apart from the presumed steric incompat-
ability of the globotetraose-containing RM118 glycoform
with elaboration of the sialylated tetrasaccharide, all Sial-
lNnT units are found in LPS molecules that have only the
fully extended oligosaccharide at Hep III. Due to the phase-
variable expression of the glycosyltransferases LgtC and
Lic2A, a variety of extensions from Hep III are typically
observed in the nonsialylated glycoforms [5,21]. Taken
together, the correlation of fully extended structures from
Hep III and the proportion of two-PEtn-containing struc-
tures with the expression of the Sial-lNnT unit, would
suggest that the addition of this tetrasaccharide unit utilizes
a preferred LPS core structure or is perhaps a late event in
the LPS biosynthesis pathway. Glycoforms in which the
PCho group on Glc I is either absent or present were also
detected in each of the strains investigated. The Sial-lNnT
side-chain is identical to that previously observed in
meningococcal and gonococcal LPS where it is found in
an analogous molecular environment. It is of considerable
interest that this sialylated structure has been found to
increase the serum resistance of those organisms bearing this
group [15]. This phenomenon is of particular importance in
strains that lack a capsular structure, which in itself provides

serum resistance. Recent data from our laboratory indicate
that the ability of acapsular strains of H. influenzae to
elaborate sialylated glycoforms can confer serum
resistance in model systems. In the study of Hood et al.
[11], a RM118 lgtC lic3A mutant strain that cannot
synthesize Sial-Lac, but as detailed in the present study
can express Sial-lNnT-containing glycoforms, was resistant
to the bactericidal effect of human sera compared with the
isogenic RM118 lgtC siaB strain, a strain that is unable to
incorporate any sialic acid residues. It is likely that this
Fig. 6. (A) One-dimensional NOESY-TOCSY spectrum using selective excitation of H-3
ax
1
H-resonance of the Neu5Ac residue in the NOESY step
and of H-3
1
H-resonance of the Gal II residue in the TOCSY step and (B) one-dimensional TOCSY-NOESY spectrum using selective excitation of H-1
1
H-resonance of the GlcNAc residue in the TOCSY step and of H-3/H-4
1
H-resonance of the GlcNAc residue in the NOESY step. (A) The assignments
of the
1
H-resonances of the Gal II residue are indicated. (B) The assignments of the
1
H-resonances of the GlcNAc and Gal II residues are indicated.
The spectrum was recorded in D
2
O at pH 7.0 and 295 K.
4016 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002

residual serum resistance of the RM118 lgtC lic3A
strain was due to the Sial-lNnT structure. H. influenzae
requires an exogenous source of sialic acid and thus
the ability to sequester sialic acid from the host and to
then utilize this moiety to modify LPS molecules is likely to
play an important role in the virulence of H. influenzae
strains.
Previous studies from our laboratory had identified the
sialyltransferase gene (lic3A) responsible for sialylation of
the lactose moiety [11]. As shown in this study the lic3A
mutant can elaborate Sial-lNnT-containing glycoforms
pointing to the presence of another sialyltransferase gene
in the RM118 genome that specifically adds sialic acid to the
b-
D
-Galp-(1–4)-b-
D
-GlcNAcp-(1–3)-b-
D
-Galp-(1-trisaccha-
ride. Several candidates are currently under investigation for
this role and include HI0871 that is a homologue to the
sialyltransferase gene lst of H. ducreyi [29] and HI1699 that
is a homologue to the sialyltransferase gene lst of N. men-
ingitidis [30]. This latter gene is part of a seven-gene locus,
lsgA-G that when expressed in Escherichia coli results in the
production of chimeric LPS that reacts with MAb 3F11
(specific for the terminal lactosamine disaccharide of lNnT)
[31].
The results of the present study would suggest that the

Sial-lNnT group is added to the LPS as a complete unit via
a mechanism involving block addition of the terminal
tetrasaccharide unit to the Glc I acceptor. This behaviour
differs from the expression of the Sial-lNnT group of
neisserial LPS, which involves sequential addition of
monomeric units, and invariably a series of truncated
Sial-lNnT structures is observed which indeed accounts for
some of the different meningococcal LPS immunotypes.
The presence of truncated Sial-lNnT structures is com-
monly encountered due to the phase variable glyco-
syltransferases of the lgt operon that synthesize the lNnT
structure in meningococcal LPS [32]. Truncated structures
due to incomplete biosynthesis of the Sial-lNnT unit were
not observed in RM118 H. influenzae strains in this study.
TheSDS/PAGEprofilesandMSdataindicatedthe
presence of the Sial-lNnT structure as a complete unit
and no evidence for loss of any residues in this structure
was observed. This profile has also been observed in other
H. influenzae strains (unpublished data). This suggests an
interesting biosynthetic scheme for the attachment of this
structure in H. influenzae. One explanation is that biosyn-
thesis is more akin to the biosynthesis of an O-antigen,
where each repeating unit of the O-antigen is often
transferred as a complete unit to the growing LPS molecule.
An alternative explanation could be that cooperative
glycosylation reactions of a series of glycosyltransferases
are required for synthesis, although such cooperativity has
not been described before in H. influenzae LPS synthesis.
Studies are underway in our laboratory to elucidate the
genetic mechanisms involved.

This study is the first to provide definitive structural
evidence for the presence of a sialylated lacto-N-neotetra-
ose moiety in H. influenzae LPS, a structure which is
potentially of great importance to the virulence of this
organism.
Fig. 7. (A) GC-MS trace of partially methylated alditol acetates derived
from O-deacylated, dephosphorylated LPS from strain RM118 lpsA
grown on BHI plates supplemented with Neu5Ac and (B) fragmentation
pattern of a 4-linked hexose residue.
Fig. 8. Structural model of the sialylated tetrasaccharide-containing glycoforms of O-deacylated LPS of H. in fluenza e strain RM118. Mutant strains
with truncated oligosaccharide extensions from Hep III residue are as indicated.
Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4017
ACKNOWLEDGEMENTS
We thank D. Krajcarski for ES-MS and S. Larocque for assistance with
NMR experiments.
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