Schweda1 1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Huddinge, Sweden; 2 Molecular Infectious Diseases Group and Department of Paediatric
Trang 1Structural diversity in lipopolysaccharide expression in nontypeable
Haemophilus influenzae
Identification of L- glycero -D- manno -heptose in the outer-core region in three clinical isolates
Martin Ma˚nsson1, Derek W Hood2, E Richard Moxon2and Elke K H Schweda1
1
Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Huddinge, Sweden;
2
Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Structural elucidation of the lipopolysaccharide (LPS) from
three nontypeable Haemophilus influenzae clinical isolates,
1209, 1207 and 1233 was achieved using NMR
spectro-scopy and ESI-MS on O-deacylated LPS and core
permethylated dephosphorylated OS It was found that the
organisms expressed a tremendous heterogeneous
glyco-form mixture resulting from the variable length of the OS
chains attached to the common structural element of
H influenzae, L-a-D–Hepp-(1fi2)-[PEtnfi6]-L-a-D
–Hepp-(1fi3)-[b-D-Glcp-(1fi4)]-L-a-D
–Hepp-(1fi5)-[PPEtnfi4]-a-Kdop-(2fi6)-Lipid A Notably, the O-6 position of the
b-D-Glcp residue could either be occupied by PCho or
L-glycero-D-manno-heptose (L,D–Hep), which is a location
for L,D–Hep that has not been seen previously in
H influenzae LPS The outer-core L,D–Hep residue was
further chain elongated at the O-6 position by the
struc-tural element b-D-GalpNAc-(1fi3)-a-D-Galp-(1fi4)-b-D -Galp, or sequentially truncated versions thereof The distal heptose residue in the inner-core was found to be chain elongated at O-2 by the globotetraose unit, b-D -GalpNAc-(1fi3)-a-D-Galp-(1fi4)-b-D-Galp-(1fi4)-b-D-Glcp, or sequentially truncated versions thereof Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant
of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclu-sively expresses LPS glycoforms comprising the outer-core
L,D–Hep residue when PCho is not expressed (the lic1 mutant)
Keywords: Haemophilus; lipopolysaccharide; L
-glycero-D-manno-heptose; phase variation; ESI-MSn
Haemophilus influenzae is an important cause of human
disease worldwide and is found in both encapsulated (types
a–f) and unencapsulated (nontypeable) forms Nontypeable
H influenzae (NTHi) strains routinely colonize the
naso-pharynx of healthy carriers and cause otitis media and
respiratory tract infections [1] The outer membrane
com-ponent lipopolysaccharide (LPS) can influence each stage of the pathogenesis of H influenzae infection H influenzae LPS is composed of a membrane-bound lipid A moiety connected to the core oligosaccharide (OS) via a single phosphorylated 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue The carbohydrate regions provide targets for recognition by host immune responses and expression of certain OS epitopes can alter the virulence of the pathogen [2] Some of these OS epitopes have been found to mimic human antigens, possibly allowing the bacteria to evade the host immune system [3,4] The OS portion of H influenzae LPS is subject to high-frequency phase variation (on/off switching of expression) of terminal epitopes, contributing
to the vast LPS heterogeneity usually found within a single strain [2] This heterogeneity may be an advantage to the bacteria, allowing them to better confront different host compartments and microenvironments and to survive the host immune response [5] The availability of the complete genome sequence of H influenzae strain Rd [6] has facili-tated a comprehensive study of LPS biosynthetic loci in the homologous strain RM118 [7] and in the type b strains Eagan (RM153) and RM7004 [8] Gene functions have been identified that are responsible for most of the steps in the biosynthesis of the OS portion of their LPS molecules
Correspondence to E Schweda, University College of
South Stockholm, Clinical Research Centre, NOVUM,
S-141 86 Huddinge, Sweden.
Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23,
E-mail: elke.schweda@kfc.ki.se
Abbreviations: CE, capillary electrophoresis; Kdo, 3-deoxy- D
-manno-oct-2-ulosonic acid; AnKdo-ol, reduced anhydro Kdo; Hep,
heptose; D , D -Hep, D -glycero- D -manno-heptose; L , D -Hep,
L -glycero- D -manno-heptose; Hex, hexose; HexNAc,
N-acetylhexos-amine; HMBC, heteronuclear multiple-bond correlation; lipid A-OH,
O-deacylated lipid A; LPS, lipopolysaccharide; LPS-OH,
O-deacylated LPS; MSn, multiple step tandem mass spectrometry;
Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable Haemophilus
influenzae; OS, oligosaccharide; PCho, phosphocholine; PEtn,
phosphoethanolamine; PPEtn, pyrophosphoethanolamine.
(Received 5 September 2002, revised 19 November 2002,
accepted 26 November 2002)
Trang 2Molecular structural studies of LPS from H influenzae
strains [9–19] have resulted in a structural model consisting
of a conserved phosphoethanolamine (PEtn)-substituted
triheptosyl inner-core moiety (labelled HepI–HepIII) in
which each of the heptose residues can provide a point for
attachment of OS chains or noncarbohydrate substituents
(Scheme 1) HepIII and the b-D-Glcp residue that is linked
to HepI (labelled GlcI) have an especially wide range of
alternatives in the substitution-pattern HepIII has been
found to be substituted by a b-D-Glcp residue either at O-2
[12] or O-3 [15] Alternatively, substitution can occur by a
b-D-Galp residue either at O-2 [10] or O-3 [18] Analysis of
LPS from lpsA mutants established in a number of strain
backgrounds supports a role for LpsA in each of the
alternative glycose substitutions of HepIII HepIII has also
been found to be substituted by the noncarbohydrate
substituents Ac (either at O-2 [15] or O-3 [14]), Gly [15,20],
P(at O-4 [11]) and PEtn [9] For GlcI, the O-4 position has
been found to be substituted by a b-D-Galp residue [9] or a
b-D-Glcp residue [10], while in other strains, O-6 was
substituted by phosphocholine (PCho) [12] orD
-glycero-D-manno-heptose (D,D–Hep) [13] In NTHi strains SB 33
and 176 [16,18], disubstitution by b-D-Glcp (at O-4) and
PCho (at O-6) occurs, while in strain RM118 [19],
disubstitution by b-D-Galp (at O-4) and PCho (at O-6)
was found In NTHi strain 1003 [17], disubstitution by Ac
(at O-4) and PCho (at O-6) was shown In each strain
analysed, PCho addition has been shown to be directed by
the products of the lic1 locus Our recent studies have
focussed on the structural diversity of LPS expression and
the genetic basis for that diversity in a representative set
consisting of 24 NTHi clinical isolates obtained from otitis
media patients In the present study we report on the
structural analysis of LPS from three of these isolates (1209,
1207 and 1233) which introduces a new glycosyl substituent
on GlcI
Experimental procedures
Bacterial strains used in this study
NTHi middle ear isolates 1209, 1207 and 1233 were obtained
from the Finnish Otitis Media Study Group [20a] Isolates
1209 and 1207 are from a single patient on the same day but
from different ears 1233 was isolated from a different patient
on a different date Molecular epidemiological data follow-ing DNA sequence analysis showed that 1233 is identical
to 1209 and 1207 except for one nucleotide change in one of the housekeeping genes investigated (unpublished results) Strains 1233lpsA and 1209lic1 were constructed
by transformation of the designated isolate with plasmid clones containing the relevant gene(s) interrupted by an antibiotic resistance cassette, as described previously [7,8] Bacterial cultivation and preparation of LPS
Bacteria were grown in brain-heart infusion broth supple-mented with haemin (10 lgÆmL)1) and NAD (2 lgÆmL)1) LPS was extracted from lyophilized bacteria by using phenol/chloroform/light petroleum, as described by Galanos
et al [21], but modified with a precipitation step of the LPS with diethyl ether/acetone (1 : 5, v/v; 6 vol.) LPS was purified by ultracentrifugation (82 000 g, 4C, 12 h) Chromatography
Gel filtration chromatography and GLC were carried out as described previously [15]
Preparation of OS material O-Deacylation of LPS O-Deacylation of LPS was achi-eved with anhydrous hydrazine as described previously [15,22]
Mild acid hydrolysis of LPS Reduced core OS material was obtained after mild acid hydrolysis (1% aqueous acetic acid, pH 3.1, 100C, 2 h) and simultaneous reduction (borane-N-methylmorpholine complex) of LPS from 1209 (120 mg), 1207, 1233, 1233lpsA (75 mg) and 1209lic1 The insoluble lipid A was separated by centrifugation and the water-soluble part was purified by gel filtration, giving one major OS-containing fraction from 1209 (OS-1, 22.5 mg),
1207, 1233, 1233lpsA (OS-2, 8.0 mg) and 1209lic1 An NH3 -treated part of OS-1 (OS-1¢) was repeatedly chromato-graphed, giving a major (OS-1¢-A, 4.4 mg) and a minor (OS-1¢-B, 1.0 mg) fraction OS-2 was rechromatographed, resulting in i.a OS-2-A (3.0 mg) and OS-2-B (1.8 mg)
material was performed with 48% aqueous HF as described previously [17]
Mass spectrometry GLC-MS was carried out with a Hewlett-Packard 5890 chromatograph equipped with a NERMAG R10–10H quadrupole mass spectrometer ESI-MS was performed as described previously [15] ESI-MSn on permethylated dephosphorylated OS was performed on a Finnigan-MAT LCQ ion trap mass spectrometer (Finnigan-MAT, San Jose, CA, USA) in the positive ion mode The samples were dissolved in methanol/water (7 : 3) containing 1 mM
NaOAc to a concentration of about 1 mgÆmL)1, and were injected into a running solvent of identical composition at
10 lLÆmin)1 Scheme 1 R 1 ¼ H, PCho or D , D –Hep, R 2 , R 4 , R 5 ¼ H, Glc, Gal or
Ac, R 3 ¼ H or Glc, Y ¼ Gly, P or PEtn.
Trang 3NMR spectroscopy
NMR spectra were obtained at 22C for OS and LPS-OH
samples either on a Varian UNITY 600 MHz spectrometer
as described previously [15,17] or on a JEOL JNM-ECP500
spectrometer using the previously described experiments
[15,17], except that a mixing time of 200 ms was used in all
NOESY experiments
Analytical methods
Sugars were identified as their alditol acetates as previously
described [23] Methylation analysis was performed as
described earlier [15] The relative proportions of the various
alditol acetates and partially methylated alditol acetates
obtained in sugar- and methylation analyses correspond to
the detector response of the GLC-MS Permethylation of
dephosphorylated OS was performed in the same way as in
the methylation analyses [15] but without the prior
acety-lation step The absolute configurations of the hexoses were
determined by the method devised by Gerwig et al [24] The
total content of fatty acids was analysed as described
previously [25]
Results
NTHi isolates 1209, 1207 and 1233 and selected mutant
strains were cultivated in liquid media and the LPS was
extracted using the phenol/chloroform/light petroleum
method
Characterization of LPS from NTHi isolate 1209
Compositional sugar analysis of the LPS sample indicated
D-glucose (Glc), D-galactose (Gal),
2-amino-2-deoxy-D-glucose (GlcN) and L-glycero-D-manno-heptose (L,D–
Hep) in the ratio 31 : 43 : 1 : 25, as identified by
GLC-MS of their corresponding alditol acetate and 2-butyl
glycoside derivatives [24] As described earlier, the LPS
contained ester-linked glycine [20] and a low level of
N-acetylneuraminic acid (Neu5Ac) [26] as shown by
high-performance anion-exchange chromatography, following
treatment of samples with 0.1MNaOH and neuraminidase, respectively
Treatment of the LPS with anhydrous hydrazine under mild conditions afforded water-soluble O-deacylated material (LPS-OH) ESI-MS data (Table 1) indicated a heterogeneous mixture of glycoforms consistent with each molecular species containing a conserved PEtn-substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linker to the putative O-deacylated lipid A (lipid A-OH) Quadruply charged ions were observed at m/z 650.1/680.8 (major) and at m/z 690.4/721.2 corresponding
to glycoforms with the respective compositions PCho• Hex3•Hep3•PEtn1)2•P1•Kdo•Lipid A-OH and PCho• Hex4•Hep3•PEtn1)2•P1•Kdo•Lipid A-OH (Fig 1) Quad-ruply charged ions were also observed at m/z 697.2/728.0 and at m/z 737.7/768.6 (minor) indicating the presence of glycoforms containing four heptose residues with the compositions Hex4•Hep4•PEtn1)2•P1•Kdo•Lipid A-OH and Hex5•Hep4•PEtn1)2•P1•Kdo•Lipid A-OH, respect-ively Quadruply charged ions of very low abundance could also be observed at m/z 609.4/640.4 and at m/z 741.2/772.1 consistent with the respective compositions PCho•Hex2• Hep3•PEtn1)2•P1•Kdo•Lipid A-OH and PCho•HexNAc1• Hex4•Hep3•PEtn1)2•P1•Kdo•Lipid A-OH Thus, ESI-MS data indicated the presence of two subpopulations of glycoforms; a major subpopulation in which the glycoform compositions comprised three heptoses and PCho (Hep3-glycoforms), and a minor subpopulation with compositions comprising four heptoses but lacking PCho (Hep4-glyco-forms) NTHi LPS glycoforms with four heptoses have previously been observed ([13], M Ma˚nsson, E R Moxon and E K H Schweda, unpublished results), and in those cases the fourth heptose has theD-glycero-D -manno-confi-guration and is situated in the outer-core region of the LPS
AsD,D–Hep was completely absent in the sugar analysis,
it was concluded that the fourth heptose here has the
L-glycero-D-manno-configuration
Characterization of OS from NTHi isolate 1209 Partial acid hydrolysis of LPS with dilute aqueous acetic acid afforded an insoluble lipid A and core OS material,
Table 1 Negative ion ESI-MS data and proposed compositions for LPS-OH of NTHi isolate 1209 Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; Hep, 192.17; Kdo, 220.18; P, 79.98; PEtn, 123.05; PCho, 165.13 and Lipid A-OH, 953.02 Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage) Peaks representing less than 5% of the base peak are not included in the table Very minor amounts of glycoforms with the compositions PCho•Hex 2 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH and PCho•HexNAc 1 •Hex 4 •Hep 3 •PEtn 1)2 •P 1 •Kdo•Lipid A-OH were also indicated by quadruply charged ions at m/z 609.4/640.4 and at m/z 741.2/772.1.
Observed ions (m/z) Molecular mass (Da)
Relative abundance (%) Proposed composition (M-4H) 4– (M-3H) 3– Observed Calculated
650.1 866.9 2604.0 2604.3 20 PCho•Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 680.8 908.0 2727.1 2727.3 43 PCho•Hex 3 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 690.4 920.7 2765.4 2766.4 8 PCho•Hex 4 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH 721.2 962.0 2888.9 2889.5 7 PCho•Hex 4 •Hep 3 •PEtn 2 •P 1 •Kdo•Lipid A-OH 697.2 930.0 2792.9 2793.5 6 Hex 4 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH 728.0 970.9 2915.8 2916.5 12 Hex 4 •Hep 4 •PEtn 2 •P 1 •Kdo•Lipid A-OH 737.7 983.7 2954.4 2955.6 2 Hex 5 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH 768.6 1025.0 3078.2 3078.7 2 Hex 5 •Hep 4 •PEtn 2 •P 1 •Kdo•Lipid A-OH
Trang 4which was purified by gel filtration chromatography, giving
OS-1 ESI-MS indicated OS-1 to contain O-acetylated (0–3
Ac) and/or O-glycylated glycoforms (0–2 Gly), where the
most abundant peaks within each subpopulation of
glyco-forms corresponded to compositions comprising two acetyl
groups but lacking glycine (data not shown) On treatment
of OS-1 with 1% aqueous NH3 (giving OS-1¢), ESI-MS
showed major doubly charged ions at m/z 785.3 and 879.9
corresponding to the respective compositions PCho•Hex3•
Hep3•PEtn1•AnKdo-ol and Hex4•Hep4•PEtn1•AnKdo-ol
(Table 2)
In order to obtain sequence and branching information,
OS-1 was dephosphorylated and permethylated and
sub-jected to ESI-MSn [27,28] Due to the increased MS
response obtained by permethylation in combination with
added sodium acetate [27], several glycoforms were
observed in the MS spectra (positive mode) that were not
detected in underivatized samples (Fig 2A) Sodiated adduct ions were identified corresponding to the composi-tions Hex1)6•Hep3•AnKdo-ol, HexNAc1•Hex4)5•Hep3•
Hex5)6•Hep4•AnKdo-ol (Tables 3 and 4) The monosac-charide sequence and branching for the different glycoforms were obtained following collision-induced dissociation (CID) of the glycosidic bonds [27,28] Through the ion mass distinction between reducing, nonreducing and inter-nal fragments resulting from the bond ruptures [27], the topology could be determined for all compositions found in the MS profiling spectrum (Tables 3 and 4) For most compositions, the presence of several (2–3) isomeric com-pounds were revealed by identifying product ions in the
MS2spectra resulting from glycosidic cleavage between the heptose residues MS3 experiments were employed when necessary to confirm the structures
Fig 1 Negative ion ESI-MS spectrum of O-deacylated LPS from NTHi isolate 1209 showing quadruply charged ions The peak at m/z 650.1 corresponds to a glycoform with the composition PCho•Hex 3 •Hep 3 •PEtn 1 •P 1 •Kdo•Lipid A-OH The peak at m/z 697.2 corresponds to a glycoform with the composition Hex 4 •Hep 4 •PEtn 1 •P 1 •Kdo•Lipid A-OH Sodiated adduct ions are indicated by asterisks (*).
Table 2 Negative ion ESI-MS data and proposed compositions for NH 3 -treated OS preparations OS-1¢, OS-1¢-A and OS-1¢-B derived from LPS of NTHi isolate 1209 Average mass units were used for calculation of molecular mass values based on proposed compositions as follows: Hex, 162.14; HexNAc, 203.19; Hep, 192.17; AnKdo-ol, 222.20; PEtn, 123.05 and PCho, 165.13 Relative abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage) ND, not detected.
Observed ions (m/z)
(M-2H)2–
Molecular mass (Da) Relative abundance (%)
Proposed composition Observed Calculated OS-1¢ OS-1¢-A OS-1¢-B
623.4 1248.8 1249.0 Trace a ND 5 PCho•Hex 1 •Hep 3 •PEtn 1 •AnKdo-ol
785.3 1572.6 1573.3 70 80 30 PCho•Hex 3 •Hep 3 •PEtn 1 •AnKdo-ol
968.2 1938.4 1938.6 Trace 2 ND PCho•HexNAc 1 •Hex 4 •Hep 3 •PEtn 1 •AnKdo-ol
798.8 1599.6 1600.4 Trace ND 3 Hex 3 •Hep 4 •PEtn 1 •AnKdo-ol
a Trace amounts, defined as peaks representing less than 1% of the base peak.
Trang 5For the major Hep3-glycoform with the composition
Hex3•Hep3•AnKdo-ol ([M + Na]+1671.8 Da), ion
selec-tion and collisional activaselec-tion of the precursor ion at m/z
1671.8 provided the MS2 spectrum shown in Fig 3 The
ions at m/z 1453.6 and 1249.5 indicated losses of a single
nonreducing terminal-Hex (t-Hex) and the nonreducing
fragment t-Hex-Hex, respectively The fragment at m/z
1001.5 (loss of t-Hex-Hex–Hep) and its trisaccharide
counterpart at m/z 693.4 resulting from cleavage between
HepII and HepIII, indicated the dihexose moiety to be
attached to HepIII The ions at m/z 753.4
(t-Hex–Hep-AnKdo-ol) and 941.4 (the counterpart) resulting from
cleavage between HepI and HepII, finally, showed a
terminal Hex residue to be linked to HepI Loss of the
terminal AnKdo-ol residue was also observed (as in almost
every MS2spectrum) from the ion at m/z 1393.5
For the Hep3-glycoform with the composition Hex4•
Hep3•AnKdo-ol ([M + Na]+ 1875.9 Da), three isomers
were shown to be present [Table 3, o¼ 3, q ¼ 1 (I), o ¼ 2,
q¼ 2 (II), o ¼ 1, q ¼ 3 (III)] Ions in the MS2spectrum at
m/z 1657.7, 1453.6 and 1249.5 corresponded to losses of t-Hex (from I, II and III), t-Hex-Hex (I, II and III) and t-Hex-Hex-Hex (I and III), respectively Isomer III could be derived from the ion pairs at m/z 753.3/1145.3 (cleavage HepI–HepII) and 1001.5/897.5 (cleavage HepII–HepIII) Isomer II was derived from the fragment pairs at m/z 957.5/ 941.3 and 1205.5/693.3 and isomer I, finally, from the ion pair at m/z 1161.4/737.4 and from the ion at m/z 1409.9
MS3 experiments on selected product ions confirmed the assigned structures For the structures containing HexNAc residues, it was observed that cleavage of the glycosidic bonds were highly favoured on the reducing side of the HexNAc residues [27] To obtain unambiguous results, it was often necessary to perform MS3 experiments on the product ion after loss of t-HexNAc or t-Hex-HexNAc
Fig 2 ESI-MS spectra of permethylated dephosphorylated OS derived
from LPS of NTHi isolates 1209 (A) and 1233 (B) showing singly
charged ions, [M + Na] + (A) Ions corresponding to selected
Hep3-glycoforms are labelled (B) Ions corresponding to selected
Hep4-glycoforms are labelled.
Table 3 Structures of the Hep3-glycoforms of NTHi isolates 1209 and
1233 as indicated by ESI-MSnon permethylated dephosphorylated OS Subscripts denoted by the letters m, n, o, p and q indicate the number
of glycose residues in the following structure:
ND, not determined.
Relative abun-dance a (%) Structure
Relative abundance b
Glycoform 1209 1233 m n o p q 1209 1233
0 0 0 0 1 Trace Hex2 3.6 2.1 0 0 2 0 0 Low Medium
0 0 1 0 1 High Medium
0 0 0 0 2 Trace Trace
0 0 2 0 1 – Trace
0 0 1 0 2 High Medium
0 0 0 0 3 – Trace Hex4 10.4 30.1 0 0 3 0 1 Trace Trace
0 0 2 0 2 Medium Low
0 0 1 0 3 Medium High Hex5 1.9 3.2 0 0 3 0 2 High Medium
0 0 2 0 3 Trace Medium Hex6 0.7 3.2 0 0 3 0 3 High High
HexNAc1Hex4 1.9 1.5 0 0 1 1 3 High High HexNAc1Hex5 0.7 0.9 0 0 2 1 3 Medium Medium
1 1 2 0 2 Medium Medium HexNAc1Hex6 – 1.0 1 1 2 0 3 High
a Relative abundance for each glycoform Estimated from the area
of molecular ion peak relative to the total area in the MS spectrum (expressed as percentage).bRelative abundance for the isomers of each glycoform Estimated from the intensity of the fragments in
MS2 experiments and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace (below 2%).
Trang 6Elucidation of the Hep4-glycoforms introduced an
addi-tional level of complexity, as ions resulting from cleavages of
outer-core glycosidic linkages in some cases could not be
mass differentiated from fragments resulting from HepII–
HepIII ruptures It was thus necessary to identify product
ions resulting from cleavages between HepI and HepII with
unique masses due to the AnKdo-ol moiety For the major
Hep4-glycoform with the composition Hex4•Hep4
•AnKdo-ol ([M + Na]+ 2124.0 Da), ions in the MS2 spectrum
(precursor ion [M + 2Na]2+1073.5 Da) (Fig 4A) at m/z
1905.9, 1701.7 (counterpart at m/z 445.3) and 1453.6
(counterpart at m/z 693.3) corresponded to losses of
t-Hex, t-Hex-Hex and t-Hex-Hex–Hep The fragments at
m/z 1205.6 (loss of t-Hex-Hex–Hep–Hep) and 941.5 (the
counterpart) indicated a dihexose moiety to be linked to
HepIII An MS3experiment on m/z 1205.6 (Fig 4B) resul-ted in fragments at m/z 987.5 (loss of t-Hex), 739.3 (loss of t-Hex–Hep) and 693.3 (corresponding to t-Hex–Hep-Hex), which showed the trisaccharide element Hex–Hep-Hex to
be attached to HepI The topology of the other glycoforms were determined in a similar manner (data not shown)
Table 4 Structures of the Hep4-glycoforms of NTHi isolates 1209 and
1233 as indicated by ESI-MSnon permethylated dephosphorylated OS.
Subscripts denoted by the letters m, n, o and p indicate the number of
glycose residues in the following structure:
ND, not determined.
Glycoform
Relative
abundance a
(%) Structure
Relative abundanceb
1209 1233 m n o p 1209 1233
0 0 0 1 Low Medium
0 1 0 1 Medium Medium
0 0 0 2 Medium Medium
0 1 0 2 High Medium
Hex5 2.7 12.6 0 2 0 2 Medium Medium
0 1 0 3 Medium Medium
HexNAc1Hex5 0.4 0.7 1 2 0 2 Medium Medium
0 1 1 3 Medium Medium HexNAc1Hex6 0.3 1.2 1 2 0 3 Medium Medium
0 2 1 3 Medium Medium
a Relative abundance for each glycoform Estimated from the area
of molecular ion peak relative to the total area in the MS spectrum
(expressed as percentage) b Relative abundance for the isomers of
each glycoform Estimated from the intensity of the fragments in
MS2 experiments and indicated as follows: high (over 80%),
medium (30–80%), low (2–30%), trace (below 2%).
Fig 3 ESI-MS2 analysis of permethylated dephosphorylated OS derived from LPS of NTHi isolate 1209 Product ion spectrum of [M + Na]+m/z 1671.8 corresponding to a glycoform with the com-position Hex 3 •Hep 3 •AnKdo-ol The proposed structure is shown in the inset.
Fig 4 ESI-MSn analysis of permethylated dephosphorylated OS derived from LPS of NTHi isolate 1209 (A) MS2 spectrum of [M + 2Na]2+ m/z 1073.5 corresponding to a glycoform with the composition Hex 4 •Hep 4 •AnKdo-ol The proposed structure is shown
in the inset (B) MS3spectrum of the fragment ion at m/z 1205.6.
Trang 7A large number of structural types were observed for the
Hep3-glycoforms (15 found) and Hep4-glycoforms (13
found), all of which can be represented by structures I and
II (Hep3-glycoforms) and structure III (Hep4-glycoforms)
in Scheme 2
Structural characterization of the major glycoforms was
achieved by NMR spectroscopy (see below) In order to
decrease the OS heterogeneity and thereby simplify the
elucidation by NMR, OS-1¢ was repeatedly
chromato-graphed on a P-4 column with intermediate selection for the
Hep3- and Hep4-glycoforms (by ESI-MS) The final result
was a major fraction (OS-1¢-A, 4.4 mg) in which the
Hep3-glycoforms accounted for about 95%, and a minor fraction
(OS-1¢-B, 1.0 mg) in which the Hep4-glycoforms accounted
for about 60% (Table 2) Sugar analysis of OS-1¢-A and
OS-1¢-B indicated Glc, Gal and L,D–Hep in ratios of
26 : 48 : 26 and 29 : 37 : 34, respectively Methylation
analysis of OS-1¢-A (Table 5) indicated terminal-Gal
(t-Gal), 4-substituted-Gal (4-Gal), 4-Glc, 3-Gal, 6-Glc,
4,6-disubstituted-Glc (4,6-Glc), 2–Hep, 3,4–Hep and 2,6–Hep in
the relative proportions 27 : 7 : 37 : 2 : 2 : 2 : 17 : 4 : 2
The methylation analysis of OS-1¢-B showed significantly
higher levels of 6-Glc and 6–Hep, of which the latter sugar
derivative indicated the substitution-pattern for the fourth
heptose residue (shown below)
Characterization of OS fractions and LPS-OH
from NTHi isolate 1209 by NMR
The1H NMR resonances of OS material and LPS-OH were
assigned by 1H-1H chemical shift correlation experiments
(DQF-COSY and TOCSY) Subspectra corresponding to
the individual glycosyl residues were identified on the basis
of spin-connectivity pathways delineated in the1H chemical
shift correlation maps, the chemical shift values, and the
vicinal coupling constants From the glycoform
composi-tions of the different oligosaccharide fraccomposi-tions determined
by ESI-MS (Table 2), the spin-systems could more easily be
identified as originating from either the Hep3- or the
Hep4-glycoform population The 13C NMR resonances of OS
fractions and LPS-OH were assigned by heteronuclear
1H-13C chemical shift correlation in the1H detected mode (HSQC) The chemical shift data obtained for the Hep4-glycoforms are summarized in Table 6 and are consistent
Scheme 2 Structures representing the various Hep3- and Hep4-glycoforms in isolates 1209 and 1233 Truncated structures are indicated by ÆÆÆÆ.
Table 5 Linkage analysis data for OS preparations derived from LPS of NTHi isolates 1209 and 1233 Trace amounts (defined as peaks representing less than 3% of the base peak) of 2,3,4,6-Me 4 -Glc [assigned as D -Glcp-(1fi], 2,3,4,6,7-Me 5 –Hep [ L , D –Hepp-(1fi], 2,3,4,6-Me 4 -GalN [ D -GalpNAc-(1fi] and 2,3,6-Me 3 -GlcN [fi4)- D -GlcpNAc-(1fi] were also indicated.
Methylated sugara T gm
b
Relative detector response (%)
Linkage assignment
a 2,3,4,6-Me 4 -Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl- D -glucitol-1-d 1 , etc b Retention times (T gm ) are reported relative to 2,3,4,6-Me -Glc (T 1.00).
Trang 8with each D-sugar residue being present in the pyranosyl
ring form Further evidence for this conclusion was
obtained from NOE data which also served to confirm
the anomeric configurations of the linkages and, together
with an HMBC experiment on OS-1¢, determined the
monosaccharide sequence
Characterization of the Kdo-lipidA-OH element ESI-MS
data (Table 1), fatty acid compositional analysis (yielding
3-hydroxytetradecanoic acid) and NMR experiments on
LPS-OH (data not shown, giving similar results as for
NTHi strains 486 [15] and 1003 [17]) indicated the presence
of the usual Kdo-lipid A-OH element in isolate 1209 As
observed earlier [15,17], two spin-systems could be traced
for the single a-linked Kdo residue, probably due to the
partial occurrence of PEtn attached to the phosphate group
at O-4 of Kdo [11,12,15]
Structure of the core region of the Hep3-glycoforms In
the 1H NMR spectra of OS-1¢-A (Fig 5A) and OS-1¢
(Fig 5B), anomeric resonances corresponding to the
inner-core region and GlcI could be observed at d 5.70–5.63
(HepII), 5.15–5.04 (HepI), 5.12 (HepIII) and 4.51 (GlcI),
respectively In addition, anomeric signals corresponding to
a globotetraose unit and sequentially truncated versions
thereof linked to HepIII, were identified at d 4.96 (t-a-D
-Galp), 4.93 (weak, 3-a-D-Galp), 4.70 (4-b-D-Glcp), 4.64
(weak, t-b-D-GalpNAc), 4.52 (4-b-D-Galp) and 4.46 (t-b-D
-Galp) as previously described for strain RM118 [12]
Spin-systems for the additional glycose residues shown by MSn
(see Scheme 2) could not be rationalized due to weak and
overlapping signals Signals for methyl protons of PCho were observed at d 3.25 and spin-systems for ethylene protons from this residue and from PEtn were similar to those observed earlier [15,17] 1H-31P NMR correlation studies (data not shown) demonstrated PCho to be located
at O-6 of GlcI and PEtn to substitute HepII at O-6, as previously observed in strain RM118 [12] and NTHi strain
1003 [17] The positions of the O-acetyl groups were determined from NMR experiments on OS-1 Intense signals from methyl protons of the O-acetyl groups were observed at d 2.20/2.18, which correlated to13C signals at d 21.1 in the HSQC spectrum For GlcI, a spin-system was found where characteristic downfield shifts were obtained for the signals from H-4 and C-4, consistent with acetylation
at O-4 as previously described for NTHi strain 1003 [17] HepIII was indicated to be acetylated at O-3 as character-istic downfield shifts were obtained for the signals from H-3 and C-3, as previously described for strain 1003 [17] A crosspeak from the ester-linked glycine substituent was also observed at d 3.99/40.7 (in the HSQC spectrum) due to correlation between the methylene proton and its carbon From the combined data, the structure in Scheme 3 is proposed for the globotetraose-containing Hep3-glycoform (HexNAc1Hex4) of NTHi isolate 1209
Structure of the core region of the Hep4-glycoforms In the 1H NMR spectrum of OS-1¢-B (Fig 5C), anomeric resonances of the three heptose residues (HepI–HepIII) in the inner-core region were observed at d 5.73–5.62 (1H, not resolved), 5.18–5.05 (1H, not resolved) and 4.99 (1H, not resolved) The Hep ring systems were identified on the basis
Table 6.1H and13C NMR chemical shifts for Hep4-glycoforms of OS-1¢-B derived from LPS of NTHi isolate 1209 Data was recorded in D 2 O at
22 C Pairs of deoxyprotons of reduced AnKdo were identified in the DQF-COSY spectrum at 2.19–1.55 p.p.m.
Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6 A /C-6 H-6 B H-7 A /C-7 H-7 B
HepI fi3,4)- L -a- D –Hepp-(1fi 5.05–5.18a 4.00–4.06a 4.01–4.04a 4.25b –c 4.13b – –
97.4–98.8 a 71.1–71.2 a 73.8 74.3 – 68.5 – HepII fi2)- L -a- D –Hepp-(1fi 5.62–5.73a 4.18 3.94 3.96 3.77 4.57 3.71 3.89
6›
GlcI fi6)-b- D -Glcp-(1fi 4.51 3.40 3.44 3.52 3.59 3.87 3.96
GlcII fi4)-b- D -Glcp-(1fi 4.54 3.41 3.72 3.71 3.71 3.84 4.00
GalII* fi4)-b- D -Galp-(1fi 4.52 3.59 3.75 4.05 3.79 d – –
62.7 40.8
a Several signals were observed for HepI and HepII due to heterogeneity in the AnKdo moiety b H-4/H-6 of HepI were identified at d 4.25/ 4.13 by NOE from GlcI c –, not obtained owing to the complexity of the spectrum d Tentative assignment from NOE data.
Trang 9of the observed small J1,2-values and their a-configurations
were confirmed by the occurrence of single intraresidue
NOE between the respective H-1 and H-2 resonances as
observed earlier [15,17] Several signals for methylene
protons of AnKdo-ol were observed in the DQF-COSY
and TOCSY spectra of OS-1¢-B in the region d 2.19–1.55
As previously observed [10,15,17], several anhydro-forms of
Kdo are formed during the hydrolysis by elimination of
phosphate or pyrophosphoethanolamine from the C-4
position, which causes both the signal splitting of the
methylene protons and the appearance of several anomeric
signals for HepI and HepII (Table 6) The occurrence of intense transglycosidic NOE connectivities between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3 (LPS-OH and OS-1¢-B) and HepI H-1/Kdo H-5 and H-7 (LPS-OH) confirmed the sequence of the heptose-contain-ing trisaccharide unit and the point of attachment to Kdo as
L-a-D–Hepp-(1fi2)-L-a-D–Hepp-(1fi3)-L-a-D –Hepp-(1fi5)-a-Kdop 1H-31P NMR correlation studies demonstrated PEtn to be linked to O-6 of HepII as a31P resonance at d 0.03 correlated to the signals from H-6 of HepII (d 4.57) and the methylene proton pair of PEtn (d 4.13)
Fig 5 600 MHz 1 H NMR spectra of OS-1¢-A (A), OS-1¢ (B) and OS-1¢-B (C) derived from LPS of NTHi isolate 1209 showing the anomeric regions (A) Anomeric resonances that are characteristic for the Hep3-glycoforms are labelled Also indicated is an ethylene proton signal from PCho at d 4.38 (C) Anomeric resonances that are characteristic for the Hep4-glycoforms are labelled.
Trang 10In OS-1¢-B, relatively large J1,2-values (about 7.7 Hz) of
the anomeric resonances observed at d 4.54, 4.52 (weak),
4.51, 4.51 and 4.46 indicated each of the corresponding
residues to have the b-anomeric configuration The residues
with anomeric signals at d 4.96 (not resolved) and 4.96
(weak, J 4.0 Hz) were identified as having the a-anomeric
configuration Further evidence for the anomeric
configu-rations was obtained from the occurrence of intraresidue
NOE between the respective H-1, H-3 and H-5 resonances
(b-configuration) or between H-1 and H-2
(a-configur-ation) On the basis of the chemical shift data and the large
J2,3, J3,4and J4,5-values (9 Hz), the residues with anomeric
shifts of d 4.51 and 4.54 could be attributed to the 6-Glc
(GlcI) and 4-Glc (GlcII) identified by methylation analysis
(Table 5) On the basis of low J3,4and J4,5-values (< 4 Hz)
and chemical shift data, the residues with anomeric
resonances at d 4.51, 4.46, 4.96 (weak, J 4.0 Hz) and
4.52 were attributed to the t-Gal (GalI, GalII and GalIII)
and 4-Gal (GalII*) identified by linkage analysis The
residue with anomeric signal at d 4.96 (not resolved) was
attributed to the 6–Hep (HepIV) identified in the methyla-tion analysis, on the basis of the small J1,2-value and chemical shift data
Interresidue NOE were observed between the proton pairs GalI H-1/HepIV H-6, HepIV H-1/GlcI H-6A, H-6B and GlcI H-1/HepI H-4 and H-6 (Fig 6) which established the presence of the tetrasaccharide unit b-D-Galp-(1fi6)-L -a-D–Hepp-(1fi6)-b-D-Glcp-(1fi4)-L-a-D–Hepp-(1fi This monosaccharide sequence was also confirmed by transgly-cosidic correlations in an HMBC experiment, where corre-lations were seen between GalI C-1/HepIV H-6, GalI H-1/ HepIV C-6 and HepIV H-1/GlcI C-6 The occurrence of interresidue NOE connectivities between the proton pairs GalII H-1/GlcII H-4 and GlcII H-1/HepIII H-1 and H-2 (Fig 6) established the sequence of a disaccharide unit and its attachment point to HepIII as b-D-Galp-(1fi4)-b-D -Glcp-(1fi2)-L-a-D–Hepp-(1fi The lactose element was shown to be further chain extended by an a-D-Galp residue
as transglycosidic NOE between GalIII H-1/GalII* H-4 and GalII* H-1/GlcII H-4 were observed Spin-systems for Scheme 3 Structure proposed for a Hep3-glycoform (HexNAc1Hex4) of NTHi isolate 1209.
Fig 6 Selected regions from the 600 MHz 2D NOESY spectrum (mixing time 200 ms) of OS-1¢-B derived from LPS of NTHi isolate 1209 Both regions were plotted at the same contour levels Cross-peaks that are characteristic for the Hep4-glycoforms are labelled.