Characterization of novel structural features in the lipopolysaccharide1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, Huddinge, Swede
Trang 1Characterization of novel structural features in the lipopolysaccharide
1
Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, Huddinge, Sweden;
2
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada;3Molecular Infectious
Diseases Group, University of Oxford, Department of Paediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Nontypeable Haemophilus influenzae (NTHi) is a common
commensal of the human upper respiratory tract and is
associated with otitis media in children The structures of the
oligosaccharide portions of NTHi lipopolysaccharide (LPS)
from several otitis media isolates are now well characterized
but it is not known whether there are structural differences in
LPSfrom colonizing, nondisease associated strains
Struc-tural analysis of LPSfrom nondisease associated NTHi
strains 11 and 16 has been achieved by the application of
electrophoresis coupled to ESI-MS, composition and
link-age analyses on O-deacylated LPSand core oligosaccharide
material This is the first study to report structural details on
LPSfrom strains taken from the nasopharynx from healthy
individuals Both strains express identical structures
and contain the common element of H influenzae LPS,
-Hepp-(1fi5)-[PPEtnfi4]-a-Kdop-(2fi6)-lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is
-Glcp residue Notably, the strains express two
acety-lation sites were identified at O-4 of Gal and O-3 of HepIII Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N-acetylneuraminic acid Keywords: carriage; ESI-MS; Haemophilus influenzae; lipopolysaccharide; NMR
Acapsular or nontypeable Haemophilus influenzae (NTHi) is
a major bacterial cause of otitis media and respiratory tract
infections in the first years of life Otitis media is a childhood
disease, which accounts for the highest frequency of
paediatric visits in Western countries [1] NTHi frequently
colonize the nasopharynx Exposure to H influenzae begins
after birth so that from infancy onward, carriage of one or
more strains for periods of days to months is common
Epidemiological studies on nasopharyngeal carriage of
NTHi have been performed with healthy children in day-care centers [2,3] It was found that younger children acquire and eliminate a number of different strains, some of which are shared with other children, whereas other strains are restricted to a single host High rates of carriage did not appear to correlate with occurrence of disease However, it has been observed that colonization levels increase shortly before the onset of disease and otitis prone children tend to
be more heavily colonized Moreover, the number of times
a child is colonized with H influenzae has been found to
be directly related to the frequency of otitis media [4] NTHi causes otitis media when the bacteria opportunistically translocates from the nasopharynx to the middle ear via the eustachian tube, often in the presence of viral respiratory infections [5] Whether all colonizing strains of H influenzae are equally capable of causing otitis media or alternatively, some strains possess particular virulence factors necessary to cause otitis media, is still not known
Cell wall lipopolysaccharide (LPS) is an essential and characteristic surface component of H influenzae and is implicated as a major virulence factor H influenzae elab-orates short-chain LPSwhich lacks O-specific polysaccha-ride chains and is often referred to as lipooligosacchapolysaccha-ride LPSoligosaccharide epitopes of H influenzae can mimic host glycolipids The molecule is heterogeneous due to a number of mechanisms including high frequency switching
of the expression (phase variation) of a number of epitopes
Stock-holm, Clinical Research Centre, NOVUM, S-141 86 Huddinge,
Sweden Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23,
E-mail: elke.schweda@kfc.ki.se
Abbreviations: Ac, acetate; AnKdo-ol, reduced anhydro Kdo;
gHMQC, gradient selected heteronuclear multiple quantum
N-acetylhexosamine; HPAEC, high-performance anion-exchange
lipopolysaccharide; LPS-OH, O-deacylated LPS; lipid A-OH,
O-deacylated lipid A; Neu5Ac, N-acetylneuraminic acid; NTHi,
nontypeable Haemophilus influenzae; OS, oligosaccharide; PCho,
phosphocholine; PEtn, phosphoethanolamine; PPEtn,
(Received 7 October 2003, revised 18 December 2003,
accepted 15 January 2004)
Trang 2The availability of the complete genome sequence of
study of LPSbiosynthetic loci from type b and d strains
[7,8] Gene functions have been identified in those strains
that are responsible for most of the steps in the biosynthesis
of the oligosaccharide portions of the LPSmolecules The
inner core of H influenzae LPShas been found to consist
-Hepp-(1fi5)-a-Kdop, in which each of the three heptose
residues can provide a point for elongation by
oligosaccha-ride chains or for attachment of noncarbohydrate
substit-uents H influenzae strains expressing LPSglycoform
populations having biantennary [9–11] and triantennary
[12–14] structures have been reported
Our previous studies have focused on the extent of
conservation and variability of LPSexpression in a
repre-sentative set of clinical isolates of NTHi obtained from otitis
media patients [10,11,13,15–20] and relating this to the role
of the molecule in commensal and virulence behavior
Recently, we demonstrated that terminal sialic acid
con-taining oligosaccharide epitopes are essential virulence
determinants in experimental otitis media [21] The clinical
isolates investigated by us were human middle ear isolates
that were characterized by ribotyping and that span a
strains of both typeable and NTHi strains [22,23] To gain a
better understanding between acquisition and development
of disease caused by NTHi and the possible role of LPS
epitopes in bacterial translocation, we have now extended
our studies to include a number of nondisease associated
isolates which were collected from the nasopharynx of
healthy children in the same study (on the efficacy of a
pneumococcal vaccine) referred to above Here we report
on structural analyses of two of these strains, referred to as
NTHi carriage strains 11 and 16
Experimental procedures
Bacterial strains, growth and LPS extraction
Nondisease associated NTHi strains 11 and 16 are isolates
from the nasopharynx of infants of 21 and 20 months of
age, respectively, and are part of a series of studies aimed at
evaluating conjugate pneumococcal vaccines conducted by
the Finnish Otitis Media Study Group [24,25] These two
strains were selected from a group of 64 carriage NTHi
isolates following phylogenetic analysis to identify isolates
representative of the genetic diversity of these strains
Bacteria were grown in brain/heart infusion broth (Difco)
and LPSextracted from lyophilized bacteria using the
phenol/chloroform/light petroleum method involving
pre-cipitation of the LPSwith diethyl ether/acetone (1 : 5, v/v;
6 vols) as described previously [9]
Chromatography
Gel filtration chromatography was performed on a Bio-Gel
eluents were monitored by a differential refractometer (Bischoff-chromatography, Leonberg, Germany) and frac-tions were collected by lyophilization Gas liquid chroma-tography (GLC) analyses were carried out using an Agilent
6890 instrument with a DB-5 fused silica capillary column
0.25 mm (0.25 lm internal diameter)] and a temperature
High performance anion-exchange chromatography (HPAEC) was performed on a Dionex Series 4500i chromatography system using a CarboPac PA1 column
USA) and pulsed amperometric detection Samples were
Preparation of oligosaccharides
LPSwas achieved as previously described [26] Briefly, LPS (1 mg) was mixed with anhydrous hydrazine (0.2 mL) and
and cold acetone (1.8 mL) was added drop-wise to destroy excess hydrazine The precipitated O-deacylated LPS(LPS-OH) was centrifuged (48 200 g, 5 min), the pellet was washed twice with cold acetone, and then dissolved in water followed by lyophilization
oligosaccha-ride (OS) fractions were obtained from LPS (50 mg, strain 11; 20 mg, strain 16) after mild acid hydrolysis (1%
reduc-tion with borane-N-methylmorpholine The insoluble lipid A (12 and 5.6 mg, respectively) was separated from the hydrolysis mixtures by centrifugation at 21 600 g for
15 min Following purification by gel filtration on the
11-OS-2 (2.4 mg) and 11-OS-3 (2.3 mg) were obtained from strain 11 From strain 16, two fractions, 16-OS-1 (6.7 mg) and 16-OS-2 (1 mg) were collected
The lipid A portions were purified by partition using chloroform/methanol/water (2 : 1 : 1; v/v/v) After centri-fugation at 48 000 g for 5 min, the chloroform phase was evaporated to dryness using a stream of nitrogen
16-OS-1 (1 mg each) were incubated with 48% aqueous HF
an ice bath and HF was evaporated under a stream of nitrogen gas to give 11-OS-1HF and 16-OS-1HF, respect-ively, which were then dissolved in water and lyophilized 11-OS-2 and 11-OS-3 (< 1 mg each) were dephosphory-lated in the same way
Mass spectrometry Electrospray ionization mass spectrometry (ESI-MS) was recorded on a VG Quattro triple quadrupole mass spectro-meter (Micromass, Manchester, UK) in the negative ion
Trang 3mode LPS-OH and OS samples were dissolved in a mixture
of water/acetonitrile (1 : 1, v/v) Sample solutions were
injected via a syringe pump into a running solvent of water/
performed in the positive ion mode on a Finnigan LCQ
iontrap mass spectrometer (Finnigan-MAT, San Jose, CA,
methanol/water (7 : 3; v/v) The applied flow rate was
negative ion mode on the Finnigan LCQ ion trap mass
spectrometer as described by Kussak et al [27] Briefly, the
lipid A was dissolved in chloroform/methanol (1 : 1; v/v)
and introduced into the mass spectrometer at a flow rate of
out in the positive ion mode with a PrinCE-C 660
instrument (Prince Technologies, Emmen, the Netherlands)
coupled to an API 3000 mass spectrometer (Perkin-Elmer/
Sciex, Concord, Canada) via a MicroIonspray interface as
described previously [16] GLC-MSwas carried out with a
Hewlett Packard 5890 chromatograph (Agilent
Technolo-gies) equipped with a NERMAG R10-10H quadrupole
mass spectrometer using the same conditions for GLC as
described above except for the initial temperature for
NMR spectroscopy
NMR spectra were recorded on solutions in deuterium
Spectra were acquired on a JEOL Esquire 500 spectrometer
(JEOL, Tokyo, Japan) using standard pulse sequences
Chemical shifts are reported in p.p.m referenced to internal
times of 50 and 180 ms, gradient selected heteronuclear
single quantum coherence and gradient selected
hetero-nuclear multiple quantum coherence (gHMQC)
experi-ments were performed using standard pulse sequences For
interresidue correlation, two-dimensional NOESY
experi-ments with a mixing time of 200 ms were used
Analytical methods
Sugars were identified as their alditol acetates as described
previously [28] Methylation analysis of LPS-OH was
accomplished on acetylated material, which was obtained
by treatment of the sample with acetic anhydride (0.2 mL)
Methylation was performed with methyl iodide in
dimethyl-sulfoxide in the presence of lithium methylsulfinylmethanide
[29] The methylated compounds were recovered using a
SepPak C18 cartridge (Waters, Milford, MA, USA) and
subjected to sugar analysis or sequential analysis by multiple
of the various alditol acetates and partially methylated
alditol acetates obtained in sugar- and methylation-analyses
are reported as the detector responses of the GLC-MS
Absolute configurations of glycoses were determined by the
method of Gerwig et al [30] The presence of glycine was
determined by HPAEC following treatment of LPSwith
N-Acetylneu-raminic acid was determined by treating LPS-OH (0.2 mg)
by HPAEC as previously described without further purification [18] The enzyme cleaves terminal N-acetylneu-raminic acid (Neu5Ac) residues linked a-2,3, a-2,6 or a-2,8
to oligosaccharides Fatty acids were identified as their methyl esters, as described previously [31]
Results
Characterization of LPS Nondisease associated NTHi strains 11 and 16 are naso-pharyngeal isolates obtained from the Finnish Otitis Media Study Group The strains were grown in liquid culture and the respective LPSs were isolated by phenol/chloroform/ light petroleum extraction [9] Compositional analysis of
-manno-heptose (Hep) as the constituent sugars in the ratios Glc/Gal/GlcN/Hep 1.0 : 0.3 : 0.8 : 0.8 (w/w/w/w) (strain 11) and 1.0 : 0.2 : 0.6 : 1.4 (w/w/w/w) (strain 16) identified
by GLC-MSof the derived alditol acetates and 2-butyl glycoside derivatives In addition, small amounts of glycine and traces of N-acetyl neuraminic acid (Neu5Ac) were detected as substituents of LPSby HPAEC following
with the exception that no Neu5Ac was found in strain 16 [16,18] It was estimated from ESI-MS data (see below) that less than 10% of all glycoforms were substituted with glycine, their compositions are given in tables below The presence of Neu5Ac was confirmed in a precursor ion monitoring MS/MS experiment (negative ion mode) by scanning for loss of m/z 290 (Neu5Ac) following CE-ESI-MS/MS (see below)
The lipid A portions obtained from LPSof both strains after mild acid hydrolysis (see below) were investigated by ESI-MS in the negative ion mode after first partitioning
in chloroform/methanol/water (2 : 1 : 1; v/v/v) [27] The spectra were virtually identical revealing, inter alia, mole-cular ions for both a diphosphorylated (m/z 1825, minor) and a monophosphorylated (m/z 1744, major) lipid A moiety substituted with four 3-hydroxytetradecanoic and two tetradecanoic acids
O-Deacylation of LPSfrom both strains by treatment with anhydrous hydrazine under mild conditions afforded water soluble LPS-OH material, which was subjected to methylation analyses and analyses by mass spectrometric techniques Methylation analysis revealed the presence of terminal Glc, 2-substituted-Hep, 3,4-disubstituted Hep and 2,3,6-trisubstituted-Hep as the major components in both strains (Table 1) The data is consistent with triantennary structures, containing the common inner core
LPSlinked to the conserved lipid A consisting of a
samples (negative ion mode) revealed abundant molecular signals corresponding to triply and quadruply
Trang 4nated ions The MSdata presented in Table 2 pointed to
the presence of glycoforms in which each molecular
species contains the conserved phosphoethanolamine
(PEtn)-substituted triheptosyl inner core moiety attached
via a phosphorylated Kdo linked to the O-deacylated
lipid A For strain 11, as observed earlier, populations of
glycoforms were observed which differed by 123 Da (i.e a
PEtn group) which was consistent with either phosphate
or pyrophosphoethanolamine (PPEtn) substitution at the
O-4 position of the Kdo residue [12] In the spectrum of
LPS-OH derived from strain 11, abundant quadruply
charged ions at m/z 691.4 and 722.0, together with their
corresponding triply charged ions at m/z 922.1 and 963.0,
indicated the presence of glycoforms with the respective
A-OH In addition, minor ions corresponding to Hex3
glycoforms with one PCho were detected at m/z 650.1 and
681.1 and with no PCho at m/z 609.0 and 639.4 In the
spectrum of LPS -OH from strain 16, one major peak was
observed at m/z 691.4 corresponding to a composition
addi-tion, a minor triply charged ion signal at m/z 907.5
trace amounts of PPEtn containing glycoforms in
strain 16 (Table 2) could be confirmed in a precursor
ion monitoring tandem mass spectrometry experiment
(negative ion mode) by scanning for loss of m/z 220
(PPEtn) following on-line separation by capillary
electro-phoresis (CE-ESI-MS/MS) Being of low abundance, ions
corresponding to sialylated species indicated by HPAEC
were not observed in the full MSspectra However, their
presence in strain 11 could be confirmed in a precursor
ion monitoring tandem mass spectrometry experiment
(negative ion mode) by scanning for loss of m/z 290
(Neu5Ac) following CE-ESI-MS/MS The resulting
spec-trum revealed a minor quadruply charged ion at m/z 754
at m/z 786, 796 and 827 were observed corresponding to disialylated Hex3 glycoforms having the respective
Lipid A-OH (data not shown) In agreement with compositional analysis (see above) no signals correspond-ing to sialylated compounds were detected for strain 16 in this experiment
Characterization of core oligosaccharides Partial acid hydrolysis of LPSfrom both strains with dilute acetic acid afforded insoluble lipid A and core oligosaccha-ride fractions which were separated by gel filtration to give one major (leading) and two minor fractions, 11-OS-1, 11-OS-2 and 11-OS-3 for strain 11 and one major and one minor fraction for strain 16, 16-OS-1 and 16-OS-2 The major oligosaccharides 11-OS-1/16-OS-1 were dephospho-rylated to give 11-OS-1HF/16-OS-1HF Methylation ana-lysis of 11-OS-1 showed terminal Glc, 2-substituted-Hep, 3,4-disubstituted Hep and 2,3-substituted-Hep as the major components (Table 1) Methylation analysis of dephos-phorylated oligosaccharide 11-OS-1HF showed signifi-cantly increased amounts of terminal Glc, terminal Gal and 2,3-disubstituted Hep indicating phosphorylation of the corresponding sugar residues (Table 1) Methylation ana-lysis of dephosphorylated oligosaccharide 16-OS-1HF showed the same derivatives as observed for 11-OS-1HF Methylation analyses of the minor fractions 11-OS-2, 11-OS-3 and 16-OS-2 showed the same major sugar derivatives
to obtain sequence and branching information, oligosac-charides were dephosphorylated and permethylated and
ESI-MSspectrum is shown in Fig 1A and the data is
Relative detector response
Linkage assignment
LPS-OH (strain 11)
LPS-OH (strain 16) 11-OS-1 11-OS-1HF 11-OS-2 11-OS-3 16-OS-1HF 16-OS-2
-GlcpNAc(1-a
Trang 5• 3
b Ions
Trang 6summerized in Table 3 Sodiated molecular ions at m/z
experiments were performed on all molecular ions and in
These data gave evidence for the glycoforms presented in Table 4 The isomeric glycoforms for strain 11 and strain 16 were found to be identical except for strain 16 having only one of the three Hex1 glycoforms expressed
by strain 11 The shared isomeric Hex1 glycoform was
by ions m/z 797.3 and 737.4 due to losses of terminal Hex-Hep and Hep-AnKdo-ol units, respectively No ion due to the loss of a terminal Hep was observed in the
isomeric Hex1 glycoform with a disubstituted HepI residue was defined by ions m/z 1001.5 and 753.1 corresponding to loss of a terminal Hep and terminal Hep-Hep unit from the parent ion Analyses on 11-OS-2 and -3 revealed a third Hex1 isomeric glycoform being substituted at HepII This Hex1 glycoform was identified
ion at m/z 475 due to the loss of a terminal Hep unit
three isomeric Hex2 structures The ion corresponding to the loss of t-Hex-Hep at m/z 1001.4 was further fragmented to give the ion at m/z 753.4 corresponding
to the loss of a monosubstituted HepII This confirmed the structure of the major glycoform in which a hexose moiety substitutes both HepI and HepIII In the same
corresponding to the loss of a Hep-AnKdo-ol unit This confirmed the structure of a glycoform in which a hexose moiety substitutes both HepII and HepIII Furthermore,
t-Hex-Hep-AnKdo-ol at m/z 737.0 was further fragmen-ted to give the ion at m/z 475.4 corresponding to the loss
of terminal HepIII This confirmed the structure of a glycoform in which a hexose moiety substitutes both HepI and HepII No ions indicating a Hex-Hex unit were observed
on the parent ion m/z 1671.8 to give ions m/z 1205.5 and 941.5.4 due to losses of terminal Hex-Hep and Hex-Hep-AnKdo-ol (Fig 1B and Table 4) When the ion at m/z 941.4
Fig 1 ESI-MS spectra (positive mode) of permethylated 11-OS-1HF.
(A) Parent ion mass spectra of permethylated 11-OS-1HF Ions
ion m/z 941.5 and proposed fragmentation pattern.
Table 3 Positive ion ESI-MS data and proposed compositions for glycoforms of dephosphorylated and permethylated oligosaccharide fractions Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; Hep, 192.17; AnKdo-ol, 222.20; Me, 14.03; Na, 22.99.
Trang 7gave, inter alia, a product ion at m/z 475.1 due to the loss of a
t-Hex-Hep unit This confirmed the structure of a glycoform
with one hexose moiety substituting each Hep There was no
indication of any Hex-Hex units in this glycoform either
Electrospray-ionization mass spectrometry on OS
mate-rials demonstrated the heterogeneity in the oligosaccharide
part due to various substituents, in particular acetates and
glycine The ESI-MS spectrum of 11-OS-1 (negative ion
mode, Table 2) showed two major doubly charged ions at
868.0 and 931.1 corresponding to Hex2 and Hex3
ions at m/z 917.4 and 938.2 corresponding to
glycine-substituted species The ESI-MS spectra of 11-OS-2 and
11-OS-3 revealed ions corresponding to acylated Hex2 and
Hex3 glycoforms with the difference that in 11-OS-2
glycoforms with one PCho predominated, while in
11-OS-3 glycoforms with no PCho were most abundant (Table 2)
The ESI-MS spectrum of 16-OS-1 was dominated by two
doubly charged ions at m/z 889.0 and 910.0, corresponding
to the same Hex3 glycoforms as in 11-OS-1 Additional
minor ions corresponding to various acylated Hex1–3
glycoforms containing one or two PCho were observed
The ESI-MS spectrum of 16-OS-2 showed basically
iden-tical ions as 16-OS-1 with increased abundance of ions
corresponding to glycoforms with one PCho (Table 2)
Information on the location of the acetyl groups and glycine in 11-OS-1 was provided by ESI tandem mass spectrometry (MS/MS) following online separation by capillary electrophoresis (CE) The product ion spectrum (positive mode) obtained from the doubly charged ion at
AnKdo-ol) and the proposed fragmentation pattern is shown in Fig 2A The spectrum contained an intense ion at
of Hep (m/z 562) could be seen Loss of the PChoAcHex-Hep fragment resulted in the ion at m/z 1219 giving evidence for the acetyl group to be located at the hexose linked to HepIII The minor ion at m/z 643 showed consecutive losses due to PEtn (m/z 520) and Hep (m/z 328; composition PChoHex) confirming the PChoHexHepIIPEtn segment Information on the location of a second acetyl group was obtained from the CE-ESI-MS/MS spectrum on m/z 912
Fig 2B) Additions of HepAc to the intense ion at m/z
370 corresponding to PChoAcHex could be seen at m/z 604 indicating that HepIII was acetylated The presence of another acetylation site, however, was indicated by the ion
at m/z 1219 to which a minor addition of 42 Da (m/z 1261) was observed This and other minor acetylation sites were deduced from the CE-ESI-MS/MS spectrum on m/z 933
data not shown) in which an addition of 42 Da to the major ion at m/z 370 giving m/z 412 indicated further acetylation of the hexose linked to HepIII To the ions at
additions of 42 Da resulted in m/z 604, 646 and 685, respectively, indicating also diacetylation of HepIII and/or
product ions of significant importance and the corresponding fragments.
a
only observed in 11-OS-2,3.
Trang 8acetylation of the hexose linked to HepII The location of
the major acetyl groups on the hexose linked to HepIII and
HepIII itself could be confirmed and fully established by
NMR (see below) Information on the location of glycine
was obtained from the CE-ESI-MS/MS spectra on m/z 919
shown), respectively These data indicated that glycine
substituted HepIII By analogy, the acylation sites in
16-OS-1 were found to be the same as in 16-OS-116-OS-1-OS-16-OS-1 (data not shown)
NMR spectroscopy on OS samples
using chemical shift correlation techniques (DQF-COSY
and TOCSY experiments) and the chemical shift data are
corresponding to the individual glycosyl residues were
identified on the basis of spin-connectivity pathways
chemical shift values, and the vicinal coupling constants Spin systems for HepIII could not be delineated beyond H-2 The chemical shift data are consistent with each
Further evidence for this conclusion was obtained from NOE data (Table 6), which also served to confirm the anomeric configurations of the linkages and the monosac-charide sequence The Hep ring systems were identified on
were confirmed by the occurrence of single intraresidue NOE between the respective H-1 and H-2 resonances only Several signals for methylene protons of AnKdo-ol were observed in the COSY and TOCSY spectra in the region d 2.24–1.84 This is due to the fact that several anhydro-forms
of Kdo are formed during the hydrolysis as observed previously [9–14,17,19,20] The methyl protons of the O-acetyl groups in 11-OS-1 and 16-OS-1 were observed at
gHMQC spectrum A gHMQC cross peak was observed at
d 4.02/41.3 which was assigned to the ester-linked glycine
Fig 2 CE-ESI-MS/MS (positive ion mode) analysis of 11-OS-1 derived from LPS of NTHi
corresponding to the composition
•PEtn•AnKdo-ol The proposed structures are shown in the insets.
Trang 9Table 5.1H and13C chemical shifts for oligosaccharide preparations 11-OS-1 and 16-OS-1 Data was recorded in D 2 O at 22 C 3
in the DQF-COSY at 2.24–1.84 p.p.m The signal corresponding to PCho methyl protons occurred at 3.24 p.p.m OS*, O-deacylated samples: chemical shifts were identical for both strains OS**, Selected chemical shifts for acetylated residues observed in samples prior to treatment with aqueous ammonia –, result not obtained.
97.3–99.8 (n.r)
99.5 (n.r)
100.6 (n.r)
104.2 (7.3)
102.0 (3.2)
104.0 (7.8)
100.1 (n.r)
Gal 4-OAc 4.32 3.59 3.93 5.37 – – –
104.5 (n.r)
(2.8)
Gal 3-OAc 4.52 3.80 4.91 4.18 4.04 – –
104.0 (4.1)
(n.r)
Gal2-OAc 4.39 4.89 3.80 4.18 – – –
104.5 (n.r)
75.8 (n.r)
from H-1 of GlcI.
Trang 10substituents The structure of the OSbackbone in 11-OS-1
and 16-OS-1 was determined by detailed NMR analyses
strains the characteristic signal for the methyl groups of
PCho was observed at d 3.24 Anomeric resonances from
HepI–HepIII were identified at d 5.17–5.04, 5.80–5.67 and
5.12–5.10, respectively Subspectra corresponding to GlcI,
GlcII and Gal were identified in the 2D COSY and TOCSY
spectra where the anomeric proton signals were found at d
4.52, 5.31 and 4.39, respectively From the downfield shifted
d 4.08 and 4.28 it was concluded that these residues were
substituted with PCho at those positions which was in
agreement with CE-ESI-MS/MS analysis (see above)
Inter-residue NOE connectivities (Table 6) between proton pairs
GlcI H-1/HepI H-4,6, HepIII H-1/HepII H-2, HepII H-1/
HepI H-3 confirmed the presence of the common inner core
triheptosyl moiety substituted with a glucose residue at
HepI Interresidue NOE between GlcII H-1/HepII H-2,3
H-1 and HepIII H-1 and H-2 indicated substitution at O-2
CE-ESI-MS/MS experiments indicated the major acetylation sites in
the oligosaccharides to be at HepIII and Gal (see above)
The points of substitution on these residues were identified
by NMR on 11-OS-1 and 16-OS-1 prior to de-O-acylation
The chemical shifts of H-2, H-3, C-2 and C-3 of HepIII were
observed at d 4.20, 5.09, 76.6 and 74.3, respectively The
significant downfield shifted H-3 and upfield shifted C-2
-Galp residue, resonances were observed that corresponded
Gal3-OAc, Gal2-OAc, respectively Thus, three spin-systems
and 4.89 (0.25H) corresponding to H-4, H-3 and H-2 of
Gal4-OAc, Gal3-OAc, Gal2-OAc, respectively For Gal2-OAc,
when compared to unsubstituted Gal, downfield shifts were
obtained for the signals from H-2 (+1.31 p.p.m.), H-3
(+0.05 p.p.m.) and C-2 (+4.5 p.p.m.), indicating
acetyla-tion at O-2 For Gal3-OAc, the signals of H-3
(+1.16 p.p.m.), H-2 (+0.22 p.p.m.), H-4 (+0.19 p.p.m.)
and C-3 (+2.4 p.p.m.) were shifted downfield, indicating
acetylation at O-3 Acetylation at O-4 was indicated from
the downfield shifted signals of H-4 (+1.38 p.p.m.), H-3
(+0.18 p.p.m) and C-4 (+1.8 p.p.m.) Substitution sites for the other, minor acyl groups indicated by CE-ESI-MS could not be identified
From the combined data the structure in Fig 3 is proposed for the major di-O-acetylated Hex3 LPSglyco-form of NTHi strains 11 and 16
Discussion
As part of our ongoing studies on the role of H influenzae LPSin disease pathogenesis, we have undertaken a system-atic analysis of LPSfrom a genetically diverse set of human isolates of NTHi The majority of strains collected were from patients with otitis media, but a number of isolates were also obtained from healthy, asymptomatic individuals Until now, no structural data has been available on LPS glycoform patterns from H influenzae strains that were not associated with disease Structural studies have revealed that
17,19,20,33–35] produces LPScontaining the conserved triheptosyl inner core moiety (Fig 4) In addition, gene
Table 6 Proton NOE data for oligosaccharide preparation 11-OS-1
derived from LPS of NT H influenzae carriage strain 11
Measure-ments were made from NOES Y experiMeasure-ments.
Anomeric proton
Observed proton
of HepIII
Fig 3 Structure proposed for the core oligosaccharide of the major Hex3 LPS glycoform of NTHi carriage strains 11 and 16.
phos-phomonoester groups at C-1 and C-4¢ and 3-hydroxytetradecanoic and tetradecanoic as described previously [31].