Richards1 1 Institute for Biological Sciences, National Research Council, Ottawa, Canada;2University of Oxford Department of Paediatrics, Weatherall Institute for Molecular Medicine, Joh
Trang 1Identification and structural characterization of a sialylated
lacto-N-neotetraose structure in the lipopolysaccharide
Andrew D Cox1, Derek W Hood2, Adele Martin1, Katherine M Makepeace2, Mary E Deadman2,
Jianjun Li1, Jean-Robert Brisson1, E Richard Moxon2and James C Richards1
1
Institute for Biological Sciences, National Research Council, Ottawa, Canada;2University 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-enzaegrown 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, comprisingfewer residues than the complete oligosaccharide component, were not detected The findingof sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for addingthe 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
lackinga 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 influenzaeLPS 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 aL-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-containingsubstituents 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 killingby 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 alteraSialyla-tions
in the reactivity of LPS with a mAb 3F11 that is specific for the terminal lactosamine disaccharide of the lNnT group followingtreatment 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 utilizingan exogenous source of sialic acid
in the growth medium we identified and localized another sialylated species, namely sialyl-lactose (Sial-Lac) in
H influenzaeLPS [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: Andrew.Cox@nrc.ca
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)
Trang 2the biosynthesis of the glycose residues in the
globotetraose-containingRoad (RM118) LPS glycoforms have been
recently reported [21] Moreover, it is also known that
H influenzaehas 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
encodingan 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 followinginsertional 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 influenzaeLPS
M A T E R I A L S A N D M E T H O D S
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 37C 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)1to
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 lm o.d.· 50 lm internal
diam-eter; Polymicro Technologies, Phoenix, AZ, USA) using
30 mMmorpholine in deionized water (negative ion mode),
pH 9.0, containing5% methanol and 15 mM ammonium
acetate/ammonium hydroxide in deionized water (positive
ion mode), pH 9.0, containing5% methanol A voltage of
25 kV was typically applied at the injection The outlet of
the capillary was tapered to 15 lm internal diameter usinga 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 using300 mbar for a duration of 0.1 min For CE-ESMS/MS experiments
60 nL sample was introduced using300 mbar for 0.2 min The MS/MS data were acquired with dwell times
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 scanningthe third quadrupole
NMR experiments were performed on Varian INOVA
600 and 500 NMR spectrometers usinga 5-mm triple resonance probe with Z gradient as described previously [25] Measurements were made at 25C at concentrations of
2 mgÆmL)1in D2O, subsequent to several lyophilizations with D2O For the proton chemical shift reference, the HDO resonance was set at 4.78 p.p.m at 25C relative to the methyl resonance of external acetone at 2.225 p.p.m All
of the NMR data was acquired usingVarian 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 duringthe 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 fillingfor 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 followingparameters: water presaturation duringthe relaxation delay of 1.0 s, spectral width of 7 p.p.m (COSY;
10 p.p.m.), acquisition time in t2of 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 F1was achieved by the States method The mixingtime for the NOESY and TOCSY experiments was 0.4 and 0.08 s, respectively The phase-sensitive spectra were processed with forward linear prediction in t1, unshifted Gaussian window functions, and zero fillingto 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 duringthe relaxapresatura-tion delay of 1 s, acquisipresatura-tion time of 1 s,
a mixingtime 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 mixingtime 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 duringthe relaxation delay of
1 s, acquisition time of 1.7 s, mixingtimes of 80 ms, selective pulse with 30 Hz bandwidth, and 5120 transients for a duration of 4 h Usingsimilar acquisition parameters, the one-dimensional TOCSY-NOESY experiment [27] was acquired with a NOE mixingtime of 800 ms, spin lock time
of 80 ms, and 20480 transients for a duration of 21 h Electrophoretic analysis of LPS
LPS was prepared and analysed by tricine-SDS/PAGE as described previously [10]
Trang 3Analytical methods and methylation analysis
Sugars were determined as their alditol acetates and
partially methylated alditol acetate derivatives by
GLC-MS as described previously [8]
R E S U L T S
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,
creatingthe 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 influenzaeLPS typically migrates as a complex series of
bands in SDS/PAGE, each band correspondingto gain or
loss of a glycose moiety due to the phase variable nature of
H influenzaeLPS synthesis [3,21,22] In LPS from RM118
lgtC lic3A it was interestingto note that the band
correspondingto 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, correspondingto 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, RM118 lpsA and RM118 lgtF before and after treatment with neuraminidase (as indicated by – and +, respectively) The sialylated tetrasaccharide-containingglycoforms are indicated by an asterisk All strains were grown on media con-tainingNeu5Ac 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.
Trang 4lgtCsingle 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 influenzae 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
Trang 5the 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 scanningfor 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 extendingfrom 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), confirmingthat 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, indicatingthe 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 correspondingto 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 lpsAgene 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 comprisingNeu5Ac-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 duringLPS biosynthesis
The structure of the sialylated glycoforms was determined
by NMR spectroscopy To simplify the analysis the simplest structure still containingthe sialylated tetrasaccharide was chosen for NMR studies, namely LPS from the lpsA mutant The one-dimensional 1H-NMR spectrum of the O-deacylated LPS was well resolved (Fig 4) Characteristic signals were observed in the anomeric region for the H-1
1H-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-11H-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 containingNeu5Ac [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-containingglycoform without the PCho moiety.
Trang 6residues 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
1H-resonances of the sialic acid residue at 1.81 and
2.76 p.p.m., respectively (Fig 4) Integration of the H-3
1H-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
The1H NMR spectrum of the inner core LPS structure
was assigned using COSY and TOCSY experiments
(Table 2) The ringsizes and relative stereochemistries of
the component monosaccharides were established from the
1H chemical shifts and the magnitude of the coupling
constants The sequence of glycosyl residues of the inner
core was determined from interresidue1H–1H NOE
meas-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 containingNeu5Ac Under these growth
conditions, the RM118 lpsA mutant does not elaborate
detectable amounts of LPS containingsialylated 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 the1H-NMR spectrum of the O-deacylated LPS obtained from strain RM118 lpsA grown on BHI plates supplemented with Neu5Ac Inset, complete1H-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.
Trang 7experiment, revealingsignals 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 followingthe one-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
1H-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
correspondingto the anomeric 1H-resonances of the
b-D-GlcNAc and the b-D-galactose (Gal II) residue
substi-tutingthe 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 arisingfrom the anomeric
resonance at 4.45 p.p.m [14] This inference was confirmed
by a TOCSY/NOESY selective excitation experiment,
where followingselective 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 confirmingthis linkage (data not shown)
and establishingthe 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 establishingthe 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 causingsequential truncations from Hep III are illustrated in Fig 8
D I S C U S S I O N
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-containingglycoforms were also characterized in LPS from RM118 mutant strains with sequential truncations in the globoside unit (lgtC, lic2A, and lpsA) 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.
Lipid A-OH
Inner core
5.76 Hep II H-1
4.05 Hep I H-6 Sial-lNnT unit
1.81
3.68 3.86 4.05 Gal-II H-3
Trang 8responsible 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-containingglycoforms 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-containingstruc-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-containingglycoforms, 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.
Trang 9residual 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-containingglycoforms
pointingto 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-Gthat 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 involvingblock 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 The SDS/PAGE profiles and MS data indicated the 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 interestingbiosynthetic scheme for the attachment of this structure in H influenzae One explanation is that biosyn-thesis is more akin to the biosynbiosyn-thesis of an O-antigen, where each repeatingunit 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 influenzae strain RM118 Mutant strains with truncated oligosaccharide extensions from Hep III residue are as indicated.
Trang 10A C K N O W L E D G E M E N T S
We thank D Krajcarski for ES-MS and S Larocque for assistance with
NMR experiments.
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