Download Inner core assembly and structure

Glycobiology vol. 15 no. 4 pp. 409–419, 2005
doi:10.1093/glycob/cwi018
Advance Access publication on December 1, 2004
Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis:
capacity of strain NMB to express all known immunotype epitopes
Charlene M. Kahler1,2, Anup Datta3, Yih-ling Tzeng4,
Russell W. Carlson3, and David S. Stephens4,5,6
2
Department of Microbiology, Monash University, Clayton 3800,
Australia; 3Complex Carbohydrate Research Center, University of
Georgia, 315 Riverbend Road, Athens, GA 30602; 4Division of Infectious
Diseases, Department of Medicine, Emory University School of Medicine,
Woodruff Memorial Building, 1639 Pierce Drive, Atlanta, GA 30322;
5
Department of Medicine, Emory University Hospital, 1364 Clifton Road
NE, Atlanta, GA 30322; and 6Veterans Affairs Medical Center, Research
Service (151-I), 1670 Clairmont Road, Decatur, GA 30033
Received on October 5, 2004; revised on November 25, 2004; accepted on
November 28, 2004
Neisseria meningitidis expresses a heterogeneous population
of lipooligosaccharide (LOS) inner cores variously substituted with ␣1-3-linked glucose and O-3, O-6, and O-7 linked
phosphoethanolamine (PEA), as well as glycine, attached to
HepII. Combinations of these attachments to the LOS inner
core represent immunodominant epitopes that are being
exploited as future vaccine candidates. Historically, each
LOS immunotype was structurally assessed and prescribed a
certain unique inner core epitope. We report that a single isolate, strain NMB, possesses the capacity to produce all of the
known neisserial LOS inner core immunotype structures.
Analysis of the inner cores from parental LOS revealed the
presence or absence of ␣1,3-linked glucose, O-6 and/or O-7
linked PEA, in addition to glycine attached at the 7 position
of the HepII inner core. Identification and inactivation of lpt6 in strain NMB resulted in the loss of both O-6 and O-7
linked PEA groups from the LOS inner core, suggesting that
Lpt-6 of strain NMB may have bifunctional transferase
activities or that the O-6 linked PEA groups once attached to
the inner core undergo nonenzymatic transfer to the O-7 position of HepII. Although O-3 linked PEA was not detected in
parental LOS inner cores devoid of ␣1-3-linked glucose residues, LOS glycoforms bearing O-3 PEA groups accumulated in a truncated mutant, NMBlgtK (Hep2Kdo2-lipid A).
Because these structures disappeared upon inactivation of the
lpt-3 locus, strain NMB expresses a functional O-3 PEA
transferase. The LOS glycoforms expressed by NMBlgtK
were also devoid of glycine attachments, indicating that glycine was added to the inner core after the completion of the
␥-chain by LgtK. In conclusion, strain NMB has the capability to express all known inner core structures, but in in vitro
culture L2 and L4 immunotype structures are predominantly
expressed.
1
To whom correspondence should be addressed; e-mail: charlene.kahler
@med.monash.edu.au
Glycobiology vol. 15 no. 4 © Oxford University Press 2004; all rights reserved.
Key words: heptose PEA transferase/immunotype/lipooligosaccharide/Neisseria meningitidis/phosphoethanolamine
Introduction
Neisseria meningitidis causes rapidly fatal sepsis and epidemic meningitis. The morbidity and mortality of meningococcal disease is closely aligned with the amount of
circulating endotoxin (variously called lipooligosaccharide,
LOS) found in patient sera (Brandtzaeg et al., 2002). Not
only is meningococcal LOS a potent activator of the
inflammatory response by engaging the TLR4 receptor
(Zughaier et al., 2004) that results in the production of
proinflammatory cytokines, it is also less effectively neutralized by human serum lipoproteins (Brandtzaeg et al.,
1992; Sprong et al., 2004). These conditions ultimately
result in profound stimulation of the inflammatory
response and the development of meningococcemia.
Meningococcal disease is caused by strains expressing one
of five capsular serogroups—A, B, C, Y, and W-135 (Tzeng
et al., 2000). The polysaccharides of these capsules, with the
exception of serogroup B polysaccharide, which is a poor
immunogen both in its native (Wyle et al., 1972) and conjugated forms (Devi et al., 1991), are the basis of current vaccine
formulations (Zimmer et al., 2004). Consequently, alternative
conserved surface antigens that elicit protective immune
responses against serogroup B strains are being examined for
their suitability for inclusion in future vaccine preparations.
Although meningococcal LOS has endotoxic properties
and is antigenically variable, antimeningococcal LOS antibodies that are both opsonic and bactericidal have been
detected in patient sera (Estabrook et al., 1990) and are
protective in experimental models of meningococcal bacteremia (Plested et al., 2003; Saukkonen et al., 1988). Conjugation of the meningococcal LOS through the lipid A
terminus to tetanus toxoid results in the conservation of the
inner core epitopes and the induction of bactericidal antibodies while removing endotoxic functions (Mieszala et al.,
2003). However, anti-LOS antibodies are more strongly
cross-protective against strains expressing LOS structures
similar to the immunogen (Andersen et al., 2002; Braun et al.,
2002; Gidney et al., 2004). Therefore the use of meningococcal LOS as a component of a broadly active vaccine may
require the inclusion of multiple inner core structures,
reflecting the diversity of structures naturally expressed by
invasive disease isolates.
Original immunotyping and mobility on sodium dodecyl
sulfate–polyacrylamide gel electrophoresis gels (Mandrell
et al., 1977; Tsai et al., 1987; Zollinger et al., 1977), revealed
that meningococcal isolates expressed 12 immunologically
409
C.M. Kahler et al.
distinct LOS structures. Although the original immunotyping scheme was supported by defined chemical structures of
immunotypes (Figure 1) novel LOS structures continue to
be found (Cox et al., 2003b). Overall, every structure to
date contains a conserved inner core consisting of two
L-glycero-D-manno-heptose (Hep) residues (HepI and HepII)
and N-acetylglucosamine (GlcNAc), which is attached to
O-2 of HepII (Figure 1). The length and composition of the
α- and β-chain extensions from HepI as well as the presence
or absence of a γ-chain extension and phosphoethanolamine (PEA) residues on HepII appear to determine immunotype (Figure 1). Although the endotoxic properties of
the meningococcal LOS are confined to the KDO2 lipid A
structure, the inner core of meningococcal LOS contain
conserved immunodominant epitopes that may be exploited
for vaccination purposes.
The neisserial LOS inner core structure is conserved
because all of the genes encoding the inner core glycosyltransferases are not subjected to phase-variable expression.
However, the LOS inner core structure is masked by additions to HepII which are highly variable and heterogeneous. The presence of the γ-chain extension of α1-3
glucose on HepII, which is characteristic of L2 and L5
immunotype LOS (Figure 1), is determined by the phasevariable expression of lgtG (Banerjee et al., 1998). lgtG is
not found in all isolates of meningococci, suggesting that it
is located on an exchangeable locus (Jennings et al., 1999).
In some meningococcal strains, O-3 linked PEA is attached
to HepII in the absence of LgtG activity, thus producing
the L3 immunotype (Figure 1) (Mackinnon et al., 2002).
The O-3 linked PEA transferase was subsequently shown to
be encoded by lpt-3, which is not phase-variable but has
been functionally inactivated by internal deletions in some
isolates (Mackinnon et al., 2002). The addition of O-6 PEA
groups to LOS inner cores is also not universal (Figure 1),
suggesting that this gene may be phase-variable or not
present in some meningococcal isolates. Very little is known
about the contribution of glycine attachments to the immunotype of LOS or the proportion of invasive and carriage
meningococcal isolates displaying this substitution. In meningococci, this amino acid is ester linked to the O-7 position
of HepII and has been reported to be present in L3 and L4
immunotype inner cores (Cox et al., 2002).
In this article, the neisserial O-6 PEA transferase was
identified as a novel gene, lpt-6, located on an exchangeable island next to lgtG in strain NMB. Mutational analysis of lpt-6 in meningococcal strain NMB results in the
loss of both O-6 and O-7 linked PEA groups from the
LOS inner core. In contrast, inactivation of lpt-3 in strain
NMB resulted in no discernible effect on the LOS inner
cores, even though Lpt-3 was required for the addition of
O-3 linked PEA groups to truncated LOS glycoforms
expressed by NMBlgtK. Glycine attachments to the inner
core of the L2 immunotype structure of strain NMB were
also detected and were shown to be added to the inner
core after the completion of the γ-chain by LgtK. Based
on these data, strain NMB has the capacity to produce
all of the known LOS immunotype inner core structures,
but regulatory mechanisms such as MisR (Tzeng et al.,
2004) influence structure and therefore immunotype
expression.
Results
Fig. 1. The substitution patterns of meningococcal LOS structures as
demonstrated by structures of L1–L7 immunotype (Difabio et al., 1990;
Gamian et al., 1992; Kogan et al., 1997; Michon et al., 1990; Pavliak et al.,
1993). The conserved inner core region is shown with variable attachments
denoted as R1–R5. The composition of the α-chain (R1) is governed by
the phase-variable expression of the lgtA–E transferases (Jennings et al.,
1999) and lst, which encodes the sialyltransferase that attaches the terminal α-Neu5Ac (sialic acid) group. Note that attachment of glycine to the
inner cores of L1 and L5–7 immunotypes have not been investigated using
current techniques. This figure is modified from Kogan et al. (1997).
410
Identification of the gene, NMA0408, encoding the O-6 PEA
transferase
The discovery of Lpt-3 (Mackinnon et al., 2002) led to the
hypothesis that the putative meningococcal LOS inner core
O-6 PEA transferase would use the same substrate and
acceptor molecules as Lpt-3 and would be highly conserved
with Lpt-3 at the amino acid sequence level. The neisserial
genome databases (www.tigr.org, www.sanger.ac.uk, and
www.stdgen.lanl.gov) contain two other members of the
N. meningitidis inner core structure
Lpt-3 protein family, NMB1638 and NMB0415. Both
predicted proteins share 27% identity and 42% similarity to
Lpt-3 at amino acid sequence level. Neither gene was found
to encode the O-6 PEA transferase, as lptA encodes a PEA
transferase specific for lipid A head groups (Cox et al.,
2003b; unpublished data) and NMB0415 (dca) encodes the
pilin phosphorylcholine transferase (renamed pptA; Warren
et al., 2003). These studies suggested that the putative O-6
PEA transferase was either not present in the four neisserial
genomes or that the O-6 PEA transferase was a member of
a distantly related protein family.
The neisserial PEA transferases contain overlapping
domains belonging to the metal dependent hydrolases
COG2194 and sulfatases PF00884 (data not shown). Interestingly, Lpt-3 has low but significant amino acid homology (E = e-06) to COG1368, which contains members of the
phosphoglycerol transferase family such as Escherichia coli
MdoB. E. coli MdoB is an O-6 phosphoglycerol transferase
that catalyzes the transfer of phosphoglycerol from phosphatidylglycerol onto position 6 of the glucose residues of
the membrane-derived oligosaccharide (MDO) backbone
(Kennedy, 1996). Because both MdoB and Lpt-6 catalyze
an O-6 linkage of a small hydrophobic molecule to a sugar
backbone, the published neisserial genomes were examined
for other members of this family of proteins. The genome
of strain Z2491 contained a COG1368 member, NMA0408
(E = e-125), which was absent in strain MC58. The predicted protein encoded by NMA0408 had no significant
amino acid homology with the Lpt-3 family proteins. An
alignment of Lpt-A and Lpt-3 with eukaryotic PEA transferases, PigO and PigN, required for the biosynthesis of glycosylphosphatididylinositol anchors for cell surface localized
proteins (Eisenhaber et al., 2003), revealed that these transferases shared a motif (data not shown) that may be involved
in metal ion coordination (Eisenhaber et al., 2003). The
amino acid sequence of NMA0408 also shared many of
these conserved residues.
To determine whether NMA0408 was related to the presence or absence of O-6 PEA groups on the LOS inner core,
the locus was amplified by polymerase chain reaction
(PCR) from strain NMB and strain M981, which express
immunotypes L2 and L5, respectively (Figure 1). As
expected, strain M981 did not contain NMA0408 (data not
shown), thus supporting the association between the presence of the locus and expression of LOS inner cores decorated with O-6 PEA residues. The complete lpt-6 gene from
strain NMB contains no homopolymeric tracts and is not
predicted to be phase-variable (data not shown). In strain
NMB, unlike the genome organizations in strains MC58
and Z2491, NMA0408 is located between orfC and lgtG
(Figure 2). In strain NMB, but not strain MC58, orfC is the
first gene in an operon of three genes including gmhX (Shih
et al., 2001) and nlaB (Shih et al., 1999). Although no function has as yet been ascribed to orfC, gmhX is required for
the third step in heptose biosynthesis (Shih et al., 2001)
and has been recently renamed gmhB (Valvano et al.,
2002), whereas nlaB encodes a lypsophosphatidic acid
acyltransferase required for phospholipid biogenesis (Shih
et al., 1999). Coexpression of the genes in this operon
presumably allow for coordinated control of endotoxin
and membrane biosynthesis. The genetic organization of
MC58
2031
lgtG
gmhX
nlaB
orfC
Z2491
0409
lpt-6
orfC
gmhX
nlaB
NMB
2031
lgtG
lpt-6
orfC
gmhX
nlaB
Fig. 2. The organization of the lpt-6 locus in meningococcal isolates,
strains MC58, Z2491 and NMB. gmhX encodes an enzyme necessary for
heptose biosynthesis (Shih et al., 2001), and nlaB encodes a lysophosphatidic acid acyltransferase (Shih et al., 1999). orfC encodes a conserved
hypothetical protein of unknown function.
this region has been reshuffled in strain MC58, although at
this point in time it is unclear whether outer membrane biogenesis in this isolate is affected.
NMA0408 encodes O-6 PEA transferase activity
Note in these sections that we have used a unifying nomenclature for the many LOS structures described in this article. We have adopted a three-part naming scheme as
follows: origin (usually strain name) followed by detection
ratios (usually major [mj] or minor [mi] amounts) followed
by a numerical value representing the number of minor
structures.
Previous structural analysis of the LOS expressed by
N. meningitidis strain NMB indicated that this invasive
isolate primarily expresses an L2 immunotype structure
(76% of the LOS, NMB-Mj), in which Hep II is substituted at O-3 with Glc and at O-6 with PEA (Figure 3).
The remaining 15% of structures lack the Glc substitution
at O-3 while retaining the PEA substituent on HepII
(NMB-Mi-1 in Figure 3). Because the location of the
PEA was not chemically determined (Rahman et al.,
1998), it was considered that the PEA attachments on
NMB-Mi could be at the O-3 or O-6 position of HepII
and therefore represented either the L3 or L4 immunotypes (Figure 1). Based on this information, the loss of
Lpt-6 activity in strain NMB was hypothesized to result
in the complete absence of PEA groups attached at the
O-6 position.
NMA0408 was insertionally inactivated in N. meningitidis strain NMB by introduction of an erythromycin cassette
into this locus by transformation and homologous
recombination of the mutagenic cassette from pJKD2621.
The correct transformants (NMBlpt-6) containing the
NMA0408::ermC cassette were confirmed by colony PCR
using primers 0408-1 and 0408-2 (data not shown). Similarly, the lpt-3 gene was also inactivated in strain NMB to
create NMBlpt-3.
Mass spectrometry (MS) of the OSs derived from the
LOS purified from NMB, NMBlpt-3, and NMBlpt-6 were
411
C.M. Kahler et al.
490
390
1897
1840
290
1879
1822
190
90
- 10
0
1 40 0
1 50 0
1 60 0
890
1700
1 80 0
1 90 0
2 00 0
1718
1774
1756
1699
790
690
590
490
390
290
190
90
Fig. 3. The structures of the oligosaccharide from NMB, NMBlpt-3, and
NMBlpt-6 LOS. The MS results indicated the presence of glycine in perhaps 50% of the LOS for each strain. The location of the glycine residue
was not determined by this study but is assumed to be the same as that
previously reported by Cox et al. (2002).
performed and the spectra are shown in Figure 4 and summarized in Table I. The spectra for the OSs from NMB and
NMBlpt3 were essentially identical to one another with
major ions at 1897 (1879, anhydro Kdo) and 1840 (1822).
The 1840 (1822) ions were consistent with the structures
reported for strain NMB (Rahman et al., 1998) in which the
inner core HepII residue contains a Glc residue at O-3, an
O-acetylated GlcNAc residue at O-2, and PEA group at O-6.
Interestingly, the 1897 (1879) ions were consistent with an
additional molecular species which contains an added glycine residue (+57 mass units). The spectrum for NMBlpt-6
showed two pairs of ions at 1774 (1756) and 1717 (1699),
which are consistent with the OSs from NMB and NMBlpt-3
but lacked a PEA substituent.
The location of PEA substituents was determined by
methylation, removal of PEA using aqueous HF, and ethylation at the site of PEA removal. The results of gas-liquid
chromatography (GLC)-MS analysis of partially methylated/ethylated alditol acetates (PMAAs) is shown in Table II.
The glycosyl linkages for NMB are, as was the mass spectrum, consistent with the reported structure for this strain
(Rahman et al., 1998). The PEA residue was present on O-6
of HepII of OSs derived from NMB LOS as evidenced by
the presence of the PMAA derivative of 2,3-linked heptose,
which is ethylated at O-6. The results for the OS of
NMBlpt-3 were identical to those obtained for NMB
(Table II; Figure 3). Even though O-7 linked PEA groups
were not detected in the methylation/HF/ethylation procedure, 31P-nuclear magnetic resonance (NMR) of the OS
from NMB and NMBlpt-3 gave signals consistent with
PEA substitution at O6 and/or O7 (Figure 5). It is possible
412
-10
1 40 0
1 50 0
1 60 0
1700
1 80 0
1 90 0
2 00 0
Fig. 4. The MALDI-TOF MS spectra of the oligosaccharides released
from the LOS of NMB (top), and of the NMBlpt-6 mutant (bottom). The
spectrum for the oligosaccharides of the LOS from the NMBlpt-3 mutant
is not shown but was identical to that for the NMB oligosaccharides
shown at the top. The ions for the NMB oligosaccharides (1822, 1840,
1879, and 1897) differ from those respective ions for the NMBlpt-6 oligosaccharides (1699, 1718, 1756, and 1779) in that the latter oligosaccharides all lack a PEA group (m/z 123 mass units). The difference between
the NMB ions 1879 and 1897 and the corresponding NMB pair of ions at
1822 and 1840 is m/z 57 is consistent with the presence of a glycine residue
being present on those oligosaccharides with m/z 1879 and 1897. This is
also true for the NMBlpt-6 oligosaccharide ion pair at 1756 and 1779
compared to the ion pair at 1699 and 1718. The mass difference between
the ions in each pair (e.g., 1879 and 1897) is m/z 18 and is due to the fact
that the former ion contains an anhydro Kdo residue and the latter contains a normal Kdo residue.
Table I. The ions observed from the MALDI-TOF MS analysis of the
OSs obtained from NMB, NMBlpt3, and NMBlpt-6 and their proposed
compositions
Strain
Ion [M-H]−
Proposed composition
NMB
(1822) 1840
Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo
(anhydroKdo)
(1879) 1897
Gly1Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo
(anhydroKdo)
(1822) 1840
Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo
(anhydroKdo)
(1879) 1897
Gly1Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo
(anhydroKdo)
(1699) 1717
Glc2Gal2GlcNAc2Ac1Hep2Kdo
(anhydroKdo)
(1756) 1774
Gly1Glc2Gal2GlcNAc2Ac1Hep2Kdo
(anhydroKdo)
NMBlpt-3
NMBlpt-6
N. meningitidis inner core structure
Table II. Glycosyl linkage analysis (relative mol %) and location of PEA
substituents in the OSs from NMB, NMBlpt-3, and NMBlpt-6 LOSs
Glycosyl linkage
NMB
NMBlpt-3
NMBlpt-6
located at the O-6 position and that this isolate expressed
L2 (NMB-Mj) and L4 (NMB-Mi) immunotypes (Figure 1).
However, although strain NMB contains an intact lpt-3
locus, inactivation of this gene did not result in any detectable change in the spectrum of PEA attachments to the
LOS inner core of the parent strain.
T-Glc
14
13
12
T-Gal
14
14
12
3-Gal
13
13
14
Lpt-3 modifies truncated LOS structures in strain NMB
4-Glc
15
15
15
T-GlcNAc
12
12
13
4-GlcNAc
10
10
11
3,4-HepI
12
12
12
2,3-HepII
—
—
11
2,3-Hep (6-Ethyl)
10
11
ND
Although O-3 PEA attachments were not detected on the
LOS inner core of the wild-type parent strain NMB, O-3
PEA attachments are present on the truncated mutant
structure of NMBlgtK (formerly NMBrfaK). NMBlgtK
produces a truncated LOS inner core in which HepII is substituted with PEA substitutents at both the O-3 and O-6
positions (Rahman et al., 2001). To determine whether
Lpt3 in strain NMB contributed the O-3 PEA groups to
this structure, or whether Lpt6 performed both attachments, the lpt-3 and lpt-6 mutations were introduced into
NMBlgtK, and the resulting LOS glycoforms analysed.
The results of MS analysis of the OSs are shown in
Figure 6 and summarized in Table III. The OS from
NMBlgtK contains ions that are consistent with a HepIIHepI-Kdo (or anhydroKdo) trisaccharide containing two
PEA substituents (m/z 867/849) or one PEA substituent
(m/z 744/726), whereas the OSs from NMBlgtKlpt-3 and
NMBlgtKlpt-6 each consist of a trisaccharide with only a
single PEA substituent. In the latter case, two ions of minor
intensity were observed (m/z 603/621), indicating that a
small proportion OS with a structure devoid of all PEA
substituents was present in these mutants.
that the amount of O-7 linked PEA substitution of HepII is
below the detection level of the former procedure.
In the case of NMBlpt-6, the results (Table II) also support the glycosyl structure found in NMB OSs but without
a PEA substitution (Lpt-6-Mj in Figure 3); a result that
supports the mass spectral data (Figure 4). 31P-NMR of the
OS from NMBlpt-6 OS lacked any detectable 31P signal
confirming the loss of all PEA groups attached to the inner
core of this mutant (Figure 5).
In summary, all PEA attachments to the LOS inner core
were lost on inactivation of NMA0408 (now called lpt-6) in
strain NMB. These results indicate that all of the detectable
PEA attachments on the LOS inner core of strain NMB are
Fig. 5. The 31P-NMR spectra for the oligosaccharides release from the LOS of (A) NMB, (B) the NMBlpt-3 mutant, (C) the NMBlpt-6 mutant, (D) the
NMBlgtK mutant, (E) the NMBlgtKlpt-3 double mutant, and (F) the NMBlgtKlpt-6 double mutant. The assignments of the 31P resonances are based on
the previous report by Rahman et al. (2001) describing the structure of the NMBlgtK LOS. The resonances labeled 6/7PEA are due to PEA substitutions
at O-6 and/or O-7 of HepII, while those labeled 3 PEA are due to PEA substitutions at O-3 of HepII.
413
C.M. Kahler et al.
Table IV. Glycosyl linkage analysis (relative mol %) and location of PEA
substituents of the OSs obtained from NMBlgtK, NMBlgtKlpt-3, and
NMBlgtKlpt-6 LOSs
300
867
200
Glycosyl linkage
744
100
726
849
NMBlgtK
NMBlgtKlpt-3
3,4-HepI
48.0
48.0
48.0
T-HepII
Tr
3.0
10.0
46.0
ND
T-Hep (6/7-Et)
T-Hep (3-Et)
0
650
700
750
800
850
900
950
4.0
NMBlgtKlpt-6
5.0
ND
42.0
T-Hep (3,6-diEt)
38.0
ND
ND
T-Hep (6,7-diEt)
5.0
4.0
ND
250
ND, not detected.
744
200
150
100
726
50
0
650
700
750
800
850
900
950
800
850
900
950
250
744
200
150
100
726
50
0
650
700
750
Fig. 6. The MALDI-TOF MS spectra of the oligosaccharides released
from the LOS of (top) NMBlgtK, (middle) NMBlgtKlpt-6, and (bottom)
NMBlgtKlpt-3. The pairs of ions, for example, 849 and 867, represent oligosaccharides with anhydro Kdo and normal Kdo, respectively. The ions
at m/z 849 and m/z 867 are due to oligosaccharides with a composition of
PEA(2)Hep(2)Kdo and those ions at m/z 726 and 744 are due to oligosaccharides with a composition of PEA(1)Hep(2)Kdo.
Table III. The ions observed from the MALDI-TOF MS analysis of the
OSs obtained from NMBlgtK, NMBlgtKlpt-3, and NMBlgtKlpt-6 and
their proposed compositions
Strain
Ion [M-H]-
NMBlgtK
(726) 744
Proposed composition
Hep2PEA1Kdo (anhydro Kdo)
(849) 867
Hep2PEA2Kdo (anhydro Kdo)
NMBlgtKlpt-3
(726) 744
Hep2PEA1Kdo (anhydro Kdo)
NMBlgtKlpt-6
(726) 744
Hep2PEA1Kdo (anhydro Kdo)
(603) 621*
Hep2Kdo (anhydro Kdo)
*Minor ions.
To determine the location of the PEA substituents in
NMBlgtKlpt-3 and NMBlgtKlpt-6 mutants, each OS was
analyzed by the methylation/HF treatment/ethylation pro414
cedure and 31P-NMR. The PMAAs for each OS are shown
in Table IV. The NMBlgtK OS contains PMAAs of HepII
with an ethyl group at O3, at O6, at both O6 and O7, and at
both O3 and O6 (the major derivative). Further support for
the presence of the PEA groups at O-3 and O-7 of HepII of
NMBlgtK OS was gathered using 1D 31P spectrum NMR
because it is relatively easy to distinguish between the 31P
signal for PEA substitution at O-3 from those for substitution at O-6 and O-7 (Figure 5). Together, these results support the previous report (Rahman et al., 2001) that the lgtK
mutant produces an HepI-HepII-Kdo OS that contains
PEA substituents at both O3 and O6 (LgtK-Mj); with
smaller amounts of OS with a PEA substituent at either O-6
(LgtK-Mi-1) or O3 (LgtK-Mi-2); and also a small amount
of OS with PEA at both O6 and O7 (LgtK-Mi-3, Figure 7).
In the case of the NMBlgtKlpt-3 mutant, the major
HepII derivative was ethylated at O-6, whereas ethylation
at O-3 was not detectable (Table IV). 31P-NMR analysis
confirmed that this OS contained O-6 or O-7 PEA attachments to HepII with no structure with O-3 PEA (Figures 5
and 7). These results confirmed that mutation of lpt-3 in the
NMBlgtK resulted in the loss of O-3 PEA attachments to
HepII, thus providing evidence that lpt-3 was an actively
transcribed and translated gene in strain NMB.
For the NMBlgtKlpt-6 mutant, the major HepII derivative was ethylated at O-3 (Table IV) with no detectable
HepII derivatives ethylated at O6, O7, or both O6 and O7.
There was a lower percentage (10%) of terminally linked
Hep, indicating that a small portion of HepII lacked PEA
substitutions (LgtKLpt-6-Mi, Figure 7). 31P-NMR of the
OS from NMBlgtKlpt-6 gave a signal for a PEA substituent
only at O-3 (Figure 5), indicating that this structure resembled LgtK-Mi-2 (Figure 7).
These results are consistent with the conclusion that Lpt3
adds the PEA to O3 of HepII to the truncated inner core of an
NMBlgtK mutant, which lacks both GlcNAc and glycine.
Glycine attachment to the inner core of meningococcal LOS
occurs after γ-chain extension
MS analysis of the OSs derived from the LOS purified from
NMB, NMBlpt-3, and NMBlpt-6 revealed an additional
molecular species that contained an added glycine residue:
1894 (1897) for both NMB and NMBlpt-3 and 1774(1756)
for NMBlpt-6 (Figure 4; Table I). In contrast, NMBlgtK
and the derivatives of this mutant did not contain glycine
N. meningitidis inner core structure
Fig. 7. The four structures of the oligosaccharide from NMBlgtK,
NMBlgtKlpt-3, and NMBlgtK-lpt-6 are shown. NMBlgtK expresses four
structures including the predominant structure, LgtK-Mj, and the three
structures, LgtK-Mi-1, LgtK-Mi-2, LgtKMi-3, detected in trace amounts.
LgtK-Mi-1 and LgtK-Mi-3 are the predominant structures expressed by
NMBlgtKlpt-3 double mutants. The predominant structure expressed by
the NMBlgtKlpt-6 double mutant is LgtK-Mi-2 with trace amounts of
LgtKLpt-6-Mi.
on the inner cores. These data suggest that similar to the αchain extension form HepI, glycine residues cannot be
attached to HepII before the γ-chain is complete. Although
glycine residues are located on the inner cores of many
lipopolysaccharides, the point at which this group is added
to the LOS structure has not been previously demonstrated
in any other organism.
Discussion
Historically, each meningococcal LOS immunotype was
shown to have unique structures (Figure 1). In addition,
genetic analysis of meningococcal immunotype isolates has
indicated that LOS biosynthesis genes are either not present
in every genome or, when present, are phase-variable in
expression (Jennings et al., 1999). This study demonstrates
that the L2/L4 immunotype strain NMB has the genetic
and biochemical capacity to express all known LOS immunotype inner core structures, a feature that has not previously been demonstrated.
In earlier work, N. meningitidis strain NMB was found to
express a mixture of two major LOS inner cores: 76% contained Hep II substituted at O-3 with Glc and at O-6 with
PEA (designated the L2 immunotype), whereas 15% of
structures lacked the Glc substitution at O-3 and retained
the PEA substituent on HepII (Rahman et al., 1998). Addi-
tional analysis of the parent LOS structure presented here
revealed that structures that lacked glucose contained an
O-6 PEA (L4 immunotype) and that glycine and O-7 linked
PEA can also be attached to the inner cores expressed by
this isolate. The intensity of the ions observed on MS analysis suggested a 50/50 ratio of structures with or without
glycine.
The presence of glycine on strain NMB OS had not been
previously determined because prior analyses had largely
been done on de-O-acetylated preparations, which would
have removed the glycine residue that appears to be
attached as an ester linkage presumably to O-7 of HepII as
reported for other meningococcal LOS (Cox et al., 2002)
and the O-acetyl group attached to O-6 of the terminal
GlcNAc attached to O-2 of HepII. Although glycine
appears to be a ubiquitous addition to the inner cores of
LPS from many Gram-negative bacteria (Cox et al., 2002;
Gamian et al., 1996; Li et al., 2001), the stage at which this
residue is added during LOS biosynthesis and the gene
encoding this transferase have not been identified. Analysis
of the inner core mutant, NMBlgtK, has revealed that all of
the LOS glycoforms expressed by this isolate are devoid of
glycine attachments to HepII. Thus the attachment of glycine to HepII likely occurs after the γ-chain is completed,
presumably because the conformation of the inner core of
the truncated structure is inaccessible to the glycine transferase. A similar mechanism for the inability of LgtF to
attach the first residue of the α-chain (β1→4) glucose) to
HepI in this mutant has been presented (Kahler et al.,
1996). Structural modeling of the truncated inner cores of
various meningococcal LOS mutants have indicated that
the conformation of these structures do differ significantly
as the α-chain is added to HepI (Plested et al., 2003).
To further determine whether the minor NMB LOS
structure, NMB-Mi, (Figure 5), corresponded to L3, L4, or
L6 immunotype inner cores structures (Figure 1), the lpt-6
and lpt-3 loci were insertionally inactivated and the structures of these mutants examined for changes in PEA content and location. Lpt-6 was identified as a member of
COG1368 that contains E. coli MdoB, the sn-1 phosphoglycerol transferase required for the biosynthesis of MDOs
in E. coli (Jackson et al., 1984). Inactivation of lpt-6 in
strain NMB resulted in the loss of all PEA groups from the
LOS inner core, whereas insertional mutagenesis of lpt-3
led to no detectable changes in the amount or location on
the inner core of PEA residues in the wild-type parent. If
strain NMB expressed a mixture of L3, L4, and L6 inner
cores, inactivating each PEA transferase would result in the
enrichment of separate subpopulations of LOS inner core
structures. Instead, NMBlpt-6 expressed a uniform population of structures with no PEA groups (Lpt-6-Mj, Figure 5),
supporting the hypothesis that Lpt-6 was necessary for the
addition of both O-6 and O-7 linked PEA groups to HepII
of the inner core.
The analysis of the NMBlgtKlpt-6 mutant structures in
which structures containing O-6 and O-7 linked PEA were lost
on inactivation of lpt-6 and structures decorated with O-3
PEA groups accumulated also supported this hypothesis.
Taken together, these results indicate that Lpt-6 in strain
NMB may be a bifunctional transferase adding both O-6
and O-7 PEA groups to the LOS inner core. Alternatively,
415
C.M. Kahler et al.
O-6 linked PEA may be transferred nonenzymatically to
the O-7 position of HepII, whereas the vacated O-6 position is filled again by Lpt-6 to produce a structure in which
both O-6 and O-7 positions are occupied with PEA. The
extent to which nonenzymatic transfer could occur would
be partly based on the occupancy of the O-7 position by the
glycine residue which is ∼ 50% in strain NMB LOS.
Wright et al. (2004) have also recently identified lpt-6 and
have insertionally inactivated this allele in two meningococcal strains 89I (L4 immunotype) and 35E (L2 immunotype).
In both instances these strains produced inner cores with
only O-6 PEA groups that were lost on inactivation of lpt-6,
suggesting that this allele encodes a monfunctional transferase. Although there was no evidence of nonenzymatic
transfer of PEA groups from O-6 to O-7 on the LOS inner
cores from these strains, it is currently unknown whether
glycine substitution at the O-7 position of HepII was at sufficiently high levels in these LOSs to prevent transfer occuring at a detectable rate.
Although inactivation of lpt-3 in the parental strain NMB
background produced no discernible differences in the spectrum of LOS structures produced, Lpt-3 activity was present
in this isolate. First, LOS glycoforms with O-3 linked PEA
groups attached to HepII accumulate in mutants, such as
NMBlgtK (this study), NMBpgm (formerly NMBR6; Monteiro et al., 2003), NMBlgtF and NMBgalE (formerly SS3;
Lee et al., 1995; data not shown). Furthermore, inactivation
of lpt-3 in NMBlgtK results in the loss of those glycoforms
with O-3 PEA groups. The lpt-3 locus from strain NMB has
been sequenced and similar to this locus from strain MC58,
appears to have no polymeric tracts either within the open
reading frame or in the putative promoter region that would
indicate phase-variable expression. Quantitative real-time
PCR of these loci from both strain NMB and MC58 also
reveal that lpt-3 is transcribed at similar levels in both isolates
(data not shown). Thus although strain NMB possesses Lpt3 activity, the O-3 PEA groups are not present on completed
LOS structures expressed by this strain.
In conclusion, parental strain NMB expresses a mixture
of L2 and L4 immunotype structures. We have previously
shown that conversion of L4 cores to L2 cores is under the
regulatory control of a two-component system, MisR/S,
which controls the expression of lgtG (Tzeng et al., 2004).
The L2 structure expressed by strain NMB also has common inner core features with L3 (O-7 linked glycine), L5
(O-3 linked glucose and acetylation of terminal GlcNAc),
and L6 (O-7 linked PEA) cores, which explains the historical observation that immunotyping antibodies generated
against L2 immunotype structures cross-react with all of
these structures (Tsai et al., 1987). Pavliak et al. (2004)
recently demonstrated that a tetravalent LOS conjugate vaccine was capable of elicting a protective immune response in
infant rats against meningococcal challenge, thus providing
the first direct evidence that such a vaccine could potentially elicit a cross-protective immune response against
pathogenic Neisseria. Importantly, the LOS structures
expressed by strain NMB contain all four of the epitopes
used in the teravalent LOS conjugate vaccine.
Additionally, this study shows that strain NMB has the
complete repertoire of genes necessary to build each known
meningococcal LOS inner core (Figure 1), including the L3
416
immunotype. However, under laboratory growth conditions, the wild-type parent expresses a mixture of L2 and L4
inner cores. Thus other regulatory processing pathways
appear to be present in strain NMB that determine the
spectrum of LOS inner core structures that are expressed
under different growth conditions. Isolates expressing L2/
L4 immunotypes contain an archetypal LOS biosynthesis
pathway while meningococcal strains expressing the other
LOS immunotype structures may represent naturally occuring mutants in this pathway.
Materials and methods
Bacterial strains, media, and growth conditions
Meningococcal strains were grown with 3.5% CO2 at 37°C
unless specified otherwise. GC base agar (Difco, Detroit,
MI), supplemented with 0.4% glucose and 0.68 mM Fe(NO3)3,
or GC broth with same supplements and 0.043% NaHCO3
was used. Brain heart infusion medium (37 g/L brain heart
infusion) with 1.25% fetal bovine serum was used when kanamycin selection was required. Antibiotic concentrations
used for E. coli strains were 100 µg/ml ampicillin, 50 µg/ml
kanamycin, 100 µg/ml spectinomycin, and 300 µg/ml erythromycin; and for N. meningitidis were 80 µg/ml kanamycin,
3 µg/ml erythromycin, 60 µg/ml spectinomycin, and 5 µg/ml
tetracycline. E. coli strain JM109 cultured on Luria Bertani
medium was used for cloning and propagation of plasmids.
Meningococci were transformed as previously described
(Janik et al., 1976). E. coli strains were transformed by the
method of Chung et al. (1989).
Construction of NMBlpt-3 and NMBlpt-6
NMBlpt-3. An internal section of the lpt-3 locus in strain
NMB was amplified using lpt3-1 (5′-GCTGACATTTGTGATTGCTGCG-3′) and lpt3-2 (5′-CTCATTGCGGATAAACATATTCCG-3′) primers and cloned into the HincII
site of pHSG576 (Takeshita et al., 1987) to form
pJKD2618. The aphA-3 cassette was liberated from
pUC18K (Menard et al., 1993) using SmaI sites and cloned
into the unique EcoRV site of the pJKD2618 to create
pJKD2619. The orientation of aphA-3 and in-frame fusion
with lpt-3 in pJKD2619 were confirmed by directional PCR
and sequencing (data not shown). The plasmid was used to
transform N. meningitidis strain NMB and the transformants (NMBlpt-3) were identified by resistance to kanamycin. Inactivation of lpt-3 was confirmed by colony PCR
(data not shown). NMBlgtKlpt-3 mutants were constructed
by transforming chromosomal DNA from NMBlpt-3 into
CMK1 (NMBlgtK) (Kahler et al., 1996; Rahman et al., 2001).
NMBlpt-6. A unique HincII site was incorporated
into NMA0408 by splice-overlap extension PCR. Two
internal fragments of NMA0408 were amplified from
N. meningitidis strain Z2491 using the primer pairs 0408-1
(5′-CTTCGGTCTGGTTTGTGGTGC-3′)/0408-6 (5′CAAATGTCGACTTCCACGTTGCG-3′) and 0408-5
(5′-CAACGTGGAAGTCGACATTTGCG-3′)/0408-2 (5′GCAGATAACGGTCGAAACTTTCC-3′). Equal molar
amounts of these products were mixed together and used
N. meningitidis inner core structure
as a template for PCR amplification using 0408-3 (5′-CTGTTCCACAGGCTGAAAATCC-3′) and 0408-4 (5′GCTTTGTCCAAATCGGCAATGC-3′) primers. The internal fragment of lpt-6 containing the unique HincII site was
cloned into the HincII site of pHSG576 to form pJKD2620.
The ermC‘ marker was liberated from pAErmC’G (Zhou et al.,
1996) by EcoRI, polished using T4 DNA polymerase (New
England Biolabs, Beverly, MA) and ligated into the HincII site
of pJKD2620 to form pJKD2621. The plasmid was purified
and used to transform N. meningitidis strain NMB using the
plate transformation method (Janik et al., 1976). Transformants
(NMBlpt-6) were selected for resistance to erythromycin and
the inactivation of lpt-6 confirmed by colony PCR (data not
shown). NMBlgtKlpt-6 mutants were constructed by transforming chromosomal DNA from NMBlpt-6 into CMK1
(NMBlgtK) (Kahler et al., 1996; Rahman et al., 2001).
LOS and oligosaccharide (OS) isolation
The LOS was prepared from 5 g (dry weight) of cells harvested from an overnight aerobic culture using a modified
version of the phenol-chloroform–petroleum ether extraction procedure as previously described (Kahler et al., 1996).
The LOS preparation was then extracted with a 9:1 ethanol/
water solution to remove any contaminating phospholipids.
The LOS samples were hydrolyzed in aqueous 1% acetic
acid (10 ml) for 2 h at 100°C. The hydrolysate was centrifuged at 10,000 × g for 20 min, and the supernatant was collected. The pellet was washed once with 5 ml water and
centrifuged again. The water wash was added to the supernatant, and any remaining lipid A was extracted with
diethyl ether (three times, 5 ml volumes each time). The
aqueous phase, containing the OSs, was lyophilized. The
lyophilized OSs were dissolved in 0.5 ml water, filtered with
Microfilterfuge tubes containing 0.45-µm pore size Nylon66 membrane filters, applied to a Bio-Gel P-4 column (70 ×
1.6 cm), and eluted with water containing 1% 1-butanol.
Fractions were assayed for carbohydrate using the phenol–
sulfuric acid assay.
using a Sep-Pak C18 cartridge (Waeghe et al., 1983). The
methylated glycan was hydrolyzed with 2 M trifluoroacetic
acid (120°C, 3 h), reduced with NaB2H4, acetylated, and
analyzed by GLC and GLC-MS (York et al., 1985). GLC
and GLC-MS analyses were performed using capillary columns (length, 30 m; inner diameter, 0.32 mm) with helium
as the carrier. A DB-5 column (J&W Scientific, now Agilent Technologies, Wilmington, DE) was used for aminoglycosyl derivatives, and an SP2330 column (Supelco,
Bellefonte, PA) was used for the neutral glycosyl derivatives.
GLC equipment consisted of HP5890 gas chromatograph
equipped with a flame ionization detector (Hewlett-Packard).
GLC-MS (electron ionization) was performed using a
Hewlett-Packard 5970 MSD.
The location of phosphate substituents (e.g., PEA) was
determined by methylating the OS as just described. This
was followed by removal of the phosphate substituents by
treating the samples with cold aqueous 48% HF (100 µl) for
24 h at 4°C as described (Kenne et al., 1993). The sample
was then dissolved in dimethyl sulfoxide and NaOH as
described, followed by ethylation with ethyl iodide. The
PMAAs were then prepared and analyzed by GLC-MS as
described.
Mass spectroscopy analyses
Matrix-assisted laser desorption ionization time of flight
(MALDI-TOF) MS was performed with either an Applied
Biosystem Voyager or an HP MALDI-TOF spectrometer
system in the negative-ion mode. Approximately 2 µl of a 1
mg/ml lipid A solution in chloroform-methanol (3:1, v/v)
was mixed with 1 µl trihydroxyacetophenone matrix solution (∼ 93.5 mg trihydroxyacetophenone per ml of methanol) and applied to the probe for mass analysis. Spectra
were calibrated externally with E. coli lipid A (Sigma,
St. Louis, MO). The OSs were dissolved in distilled water at
a final concentration of 2 µg/µl, and 1 µl was mixed with
the dihydroxybenzoic acid in methanol matrix for analysis.
31
P-NMR spectroscopy
Glycosyl composition analyses
The glycosyl compositions of the LOS and OS (0.5 mg
each) samples were performed by the preparation and analysis of alditol acetates as previously described (York et al.,
1985). Briefly, the samples were hydrolyzed in 2 M trifluoroacetic acid (0.5 ml) in a closed vial at 120°C for 3 h. The
resulting glycoses were reduced with NaBH4, acetylated,
and analyzed by GLC and combined GLC-MS.
Glycosyl linkage analyses
Glycosyl linkage analysis was carried out using a modified
NaOH method. The sample (1 mg) was dissolved in dimethyl sulfoxide (100 µl), powdered NaOH (100 mg) was
added, and the reaction mixture was stirred rapidly at room
temperature for 30 min. Methylation was performed by the
sequential additions of methyl iodide (10, 10, and 20 µl) at
10-min intervals. After an additional 20 min stirring, 1 ml
of 1 M sodium thiosulfate was added, and the methylated
glycans were recovered in the organic phase by extraction
with chloroform (0.5 ml × 3). The permethylated product
was further purified by reverse-phase chromatography
Each sample, 1–2 mg, was prepared for NMR analysis by a
twofold lyophilization from D2O, dissolved in D2O, and
analyzed. NMR spectra were recorded on Varian 300 MHz
spectrometers at 27°C. The data was referenced to external
85% phosphoric acid (0.0 ppm).
Acknowledgments
C. Kahler thanks John Davies and David Stephens for continued support and J.C. Wright and R. Moxon for
acknowledging this work in their recent publication. This
work was funded by NIH grant R01 AI033517-10 to D.S.S.
Abbreviations
GLC, gas-liquid chromatography; LOS, lipooligosaccharide; MDO, membrane-derived oligosaccharide; MALDITOF, matrix-assisted laser desorption ionization time of
flight; MS, mass spectrometry; NMR, nuclear magnetic
resonance; OS, oligosaccharide; PCR, polymerase chain
417
C.M. Kahler et al.
reaction; PEA, phosphoethanolamine; PMAA, partially
methylated/ethylated aldtitol acetate.
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