Glycobiology vol. 15 no. 4 pp. 409–419, 2005 doi:10.1093/glycob/cwi018 Advance Access publication on December 1, 2004

Glycobiology vol. 15 no. 4 © Oxford University Press 2004; all rights reserved. 409

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

2Department 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; 5Department 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 populationof lipooligosaccharide (LOS) inner cores variously substi-tuted with �1-3-linked glucose and O-3, O-6, and O-7 linkedphosphoethanolamine (PEA), as well as glycine, attached toHepII. Combinations of these attachments to the LOS innercore represent immunodominant epitopes that are beingexploited as future vaccine candidates. Historically, eachLOS immunotype was structurally assessed and prescribed acertain unique inner core epitope. We report that a single iso-late, strain NMB, possesses the capacity to produce all of theknown neisserial LOS inner core immunotype structures.Analysis of the inner cores from parental LOS revealed thepresence or absence of �1,3-linked glucose, O-6 and/or O-7linked PEA, in addition to glycine attached at the 7 positionof the HepII inner core. Identification and inactivation of lpt-6 in strain NMB resulted in the loss of both O-6 and O-7linked PEA groups from the LOS inner core, suggesting thatLpt-6 of strain NMB may have bifunctional transferaseactivities or that the O-6 linked PEA groups once attached tothe inner core undergo nonenzymatic transfer to the O-7 posi-tion of HepII. Although O-3 linked PEA was not detected inparental LOS inner cores devoid of �1-3-linked glucose resi-dues, LOS glycoforms bearing O-3 PEA groups accumu-lated in a truncated mutant, NMBlgtK (Hep2Kdo2-lipid A).Because these structures disappeared upon inactivation of thelpt-3 locus, strain NMB expresses a functional O-3 PEAtransferase. The LOS glycoforms expressed by NMBlgtKwere also devoid of glycine attachments, indicating that gly-cine was added to the inner core after the completion of the�-chain by LgtK. In conclusion, strain NMB has the capabil-ity to express all known inner core structures, but in in vitroculture L2 and L4 immunotype structures are predominantlyexpressed.

Key words: heptose PEA transferase/immunotype/lipooli-gosaccharide/Neisseria meningitidis/phosphoethanolamine

Introduction

Neisseria meningitidis causes rapidly fatal sepsis and epi-demic meningitis. The morbidity and mortality of meningo-coccal disease is closely aligned with the amount ofcirculating endotoxin (variously called lipooligosaccharide,LOS) found in patient sera (Brandtzaeg et al., 2002). Notonly is meningococcal LOS a potent activator of theinflammatory response by engaging the TLR4 receptor(Zughaier et al., 2004) that results in the production ofproinflammatory cytokines, it is also less effectively neu-tralized by human serum lipoproteins (Brandtzaeg et al.,1992; Sprong et al., 2004). These conditions ultimatelyresult in profound stimulation of the inflammatoryresponse and the development of meningococcemia.

Meningococcal disease is caused by strains expressing oneof five capsular serogroups—A, B, C, Y, and W-135 (Tzenget al., 2000). The polysaccharides of these capsules, with theexception of serogroup B polysaccharide, which is a poorimmunogen both in its native (Wyle et al., 1972) and conju-gated forms (Devi et al., 1991), are the basis of current vaccineformulations (Zimmer et al., 2004). Consequently, alternativeconserved surface antigens that elicit protective immuneresponses against serogroup B strains are being examined fortheir suitability for inclusion in future vaccine preparations.

Although meningococcal LOS has endotoxic propertiesand is antigenically variable, antimeningococcal LOS anti-bodies that are both opsonic and bactericidal have beendetected in patient sera (Estabrook et al., 1990) and areprotective in experimental models of meningococcal bacter-emia (Plested et al., 2003; Saukkonen et al., 1988). Conju-gation of the meningococcal LOS through the lipid Aterminus to tetanus toxoid results in the conservation of theinner core epitopes and the induction of bactericidal anti-bodies while removing endotoxic functions (Mieszala et al.,2003). However, anti-LOS antibodies are more stronglycross-protective against strains expressing LOS structuressimilar to the immunogen (Andersen et al., 2002; Braun et al.,2002; Gidney et al., 2004). Therefore the use of meningo-coccal LOS as a component of a broadly active vaccine mayrequire the inclusion of multiple inner core structures,reflecting the diversity of structures naturally expressed byinvasive disease isolates.

Original immunotyping and mobility on sodium dodecylsulfate–polyacrylamide gel electrophoresis gels (Mandrellet al., 1977; Tsai et al., 1987; Zollinger et al., 1977), revealedthat meningococcal isolates expressed 12 immunologically

1To whom correspondence should be addressed; e-mail: charlene.kahler @med.monash.edu.au

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distinct LOS structures. Although the original immunotyp-ing scheme was supported by defined chemical structures ofimmunotypes (Figure 1) novel LOS structures continue tobe found (Cox et al., 2003b). Overall, every structure todate contains a conserved inner core consisting of twoL-glycero-D-manno-heptose (Hep) residues (HepI and HepII)and N-acetylglucosamine (GlcNAc), which is attached toO-2 of HepII (Figure 1). The length and composition of theα- and β-chain extensions from HepI as well as the presenceor absence of a γ-chain extension and phosphoethanola-mine (PEA) residues on HepII appear to determine immu-notype (Figure 1). Although the endotoxic properties ofthe meningococcal LOS are confined to the KDO2 lipid A

structure, the inner core of meningococcal LOS containconserved immunodominant epitopes that may be exploitedfor vaccination purposes.

The neisserial LOS inner core structure is conservedbecause all of the genes encoding the inner core glycosyl-transferases are not subjected to phase-variable expression.However, the LOS inner core structure is masked by addi-tions to HepII which are highly variable and heteroge-neous. The presence of the γ-chain extension of α1-3glucose on HepII, which is characteristic of L2 and L5immunotype LOS (Figure 1), is determined by the phase-variable expression of lgtG (Banerjee et al., 1998). lgtG isnot found in all isolates of meningococci, suggesting that itis located on an exchangeable locus (Jennings et al., 1999).In some meningococcal strains, O-3 linked PEA is attachedto HepII in the absence of LgtG activity, thus producingthe L3 immunotype (Figure 1) (Mackinnon et al., 2002).The O-3 linked PEA transferase was subsequently shown tobe encoded by lpt-3, which is not phase-variable but hasbeen functionally inactivated by internal deletions in someisolates (Mackinnon et al., 2002). The addition of O-6 PEAgroups to LOS inner cores is also not universal (Figure 1),suggesting that this gene may be phase-variable or notpresent in some meningococcal isolates. Very little is knownabout the contribution of glycine attachments to the immu-notype of LOS or the proportion of invasive and carriagemeningococcal isolates displaying this substitution. In men-ingococci, this amino acid is ester linked to the O-7 positionof HepII and has been reported to be present in L3 and L4immunotype inner cores (Cox et al., 2002).

In this article, the neisserial O-6 PEA transferase wasidentified as a novel gene, lpt-6, located on an exchange-able island next to lgtG in strain NMB. Mutational ana-lysis of lpt-6 in meningococcal strain NMB results in theloss of both O-6 and O-7 linked PEA groups from theLOS inner core. In contrast, inactivation of lpt-3 in strainNMB resulted in no discernible effect on the LOS innercores, even though Lpt-3 was required for the addition ofO-3 linked PEA groups to truncated LOS glycoformsexpressed by NMBlgtK. Glycine attachments to the innercore of the L2 immunotype structure of strain NMB werealso detected and were shown to be added to the innercore after the completion of the γ-chain by LgtK. Basedon these data, strain NMB has the capacity to produceall of the known LOS immunotype inner core structures,but regulatory mechanisms such as MisR (Tzeng et al.,2004) influence structure and therefore immunotypeexpression.

Results

Identification of the gene, NMA0408, encoding the O-6 PEA transferase

The discovery of Lpt-3 (Mackinnon et al., 2002) led to thehypothesis that the putative meningococcal LOS inner coreO-6 PEA transferase would use the same substrate andacceptor molecules as Lpt-3 and would be highly conservedwith Lpt-3 at the amino acid sequence level. The neisserialgenome databases (www.tigr.org, www.sanger.ac.uk, andwww.stdgen.lanl.gov) contain two other members of the

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 termi-nal 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).

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Lpt-3 protein family, NMB1638 and NMB0415. Bothpredicted proteins share 27% identity and 42% similarity toLpt-3 at amino acid sequence level. Neither gene was foundto encode the O-6 PEA transferase, as lptA encodes a PEAtransferase specific for lipid A head groups (Cox et al.,2003b; unpublished data) and NMB0415 (dca) encodes thepilin phosphorylcholine transferase (renamed pptA; Warrenet al., 2003). These studies suggested that the putative O-6PEA transferase was either not present in the four neisserialgenomes or that the O-6 PEA transferase was a member ofa distantly related protein family.

The neisserial PEA transferases contain overlappingdomains belonging to the metal dependent hydrolasesCOG2194 and sulfatases PF00884 (data not shown). Inter-estingly, Lpt-3 has low but significant amino acid homol-ogy (E = e-06) to COG1368, which contains members of thephosphoglycerol transferase family such as Escherichia coliMdoB. E. coli MdoB is an O-6 phosphoglycerol transferasethat catalyzes the transfer of phosphoglycerol from phos-phatidylglycerol onto position 6 of the glucose residues ofthe membrane-derived oligosaccharide (MDO) backbone(Kennedy, 1996). Because both MdoB and Lpt-6 catalyzean O-6 linkage of a small hydrophobic molecule to a sugarbackbone, the published neisserial genomes were examinedfor other members of this family of proteins. The genomeof strain Z2491 contained a COG1368 member, NMA0408(E = e-125), which was absent in strain MC58. The pre-dicted protein encoded by NMA0408 had no significantamino acid homology with the Lpt-3 family proteins. Analignment of Lpt-A and Lpt-3 with eukaryotic PEA trans-ferases, PigO and PigN, required for the biosynthesis of gly-cosylphosphatididylinositol anchors for cell surface localizedproteins (Eisenhaber et al., 2003), revealed that these trans-ferases shared a motif (data not shown) that may be involvedin metal ion coordination (Eisenhaber et al., 2003). Theamino acid sequence of NMA0408 also shared many ofthese conserved residues.

To determine whether NMA0408 was related to the pres-ence 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 expressimmunotypes L2 and L5, respectively (Figure 1). Asexpected, strain M981 did not contain NMA0408 (data notshown), thus supporting the association between the pres-ence of the locus and expression of LOS inner cores deco-rated with O-6 PEA residues. The complete lpt-6 gene fromstrain NMB contains no homopolymeric tracts and is notpredicted to be phase-variable (data not shown). In strainNMB, unlike the genome organizations in strains MC58and Z2491, NMA0408 is located between orfC and lgtG(Figure 2). In strain NMB, but not strain MC58, orfC is thefirst gene in an operon of three genes including gmhX (Shihet al., 2001) and nlaB (Shih et al., 1999). Although no func-tion has as yet been ascribed to orfC, gmhX is required forthe third step in heptose biosynthesis (Shih et al., 2001)and has been recently renamed gmhB (Valvano et al.,2002), whereas nlaB encodes a lypsophosphatidic acidacyltransferase required for phospholipid biogenesis (Shihet al., 1999). Coexpression of the genes in this operonpresumably allow for coordinated control of endotoxinand membrane biosynthesis. The genetic organization of

this region has been reshuffled in strain MC58, although atthis point in time it is unclear whether outer membrane bio-genesis in this isolate is affected.

NMA0408 encodes O-6 PEA transferase activity

Note in these sections that we have used a unifying nomen-clature for the many LOS structures described in this arti-cle. We have adopted a three-part naming scheme asfollows: origin (usually strain name) followed by detectionratios (usually major [mj] or minor [mi] amounts) followedby a numerical value representing the number of minorstructures.

Previous structural analysis of the LOS expressed byN. meningitidis strain NMB indicated that this invasiveisolate primarily expresses an L2 immunotype structure(76% of the LOS, NMB-Mj), in which Hep II is substi-tuted at O-3 with Glc and at O-6 with PEA (Figure 3).The remaining 15% of structures lack the Glc substitutionat O-3 while retaining the PEA substituent on HepII(NMB-Mi-1 in Figure 3). Because the location of thePEA was not chemically determined (Rahman et al.,1998), it was considered that the PEA attachments onNMB-Mi could be at the O-3 or O-6 position of HepIIand therefore represented either the L3 or L4 immuno-types (Figure 1). Based on this information, the loss ofLpt-6 activity in strain NMB was hypothesized to resultin the complete absence of PEA groups attached at theO-6 position.

NMA0408 was insertionally inactivated in N. meningiti-dis strain NMB by introduction of an erythromycin cassetteinto this locus by transformation and homologousrecombination of the mutagenic cassette from pJKD2621.The correct transformants (NMBlpt-6) containing theNMA0408::ermC cassette were confirmed by colony PCRusing primers 0408-1 and 0408-2 (data not shown). Simi-larly, the lpt-3 gene was also inactivated in strain NMB tocreate NMBlpt-3.

Mass spectrometry (MS) of the OSs derived from theLOS purified from NMB, NMBlpt-3, and NMBlpt-6 were

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 lysophospha-tidic acid acyltransferase (Shih et al., 1999). orfC encodes a conserved hypothetical protein of unknown function.

2031

MC58

lgtG gmhXnlaB

0409 orfCgmhX

orfC

nlaB

Z2491

2031 orfCgmhX

nlaB

NMB

lgtG

lpt-6

lpt-6

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performed and the spectra are shown in Figure 4 and sum-marized in Table I. The spectra for the OSs from NMB andNMBlpt3 were essentially identical to one another withmajor ions at 1897 (1879, anhydro Kdo) and 1840 (1822).The 1840 (1822) ions were consistent with the structuresreported for strain NMB (Rahman et al., 1998) in which theinner core HepII residue contains a Glc residue at O-3, anO-acetylated GlcNAc residue at O-2, and PEA group at O-6.Interestingly, the 1897 (1879) ions were consistent with anadditional molecular species which contains an added gly-cine residue (+57 mass units). The spectrum for NMBlpt-6showed two pairs of ions at 1774 (1756) and 1717 (1699),which are consistent with the OSs from NMB and NMBlpt-3but lacked a PEA substituent.

The location of PEA substituents was determined bymethylation, removal of PEA using aqueous HF, and ethy-lation at the site of PEA removal. The results of gas-liquidchromatography (GLC)-MS analysis of partially methy-lated/ethylated alditol acetates (PMAAs) is shown in Table II.The glycosyl linkages for NMB are, as was the mass spec-trum, consistent with the reported structure for this strain(Rahman et al., 1998). The PEA residue was present on O-6of HepII of OSs derived from NMB LOS as evidenced bythe presence of the PMAA derivative of 2,3-linked heptose,which is ethylated at O-6. The results for the OS ofNMBlpt-3 were identical to those obtained for NMB(Table II; Figure 3). Even though O-7 linked PEA groupswere not detected in the methylation/HF/ethylation proce-dure, 31P-nuclear magnetic resonance (NMR) of the OSfrom NMB and NMBlpt-3 gave signals consistent withPEA substitution at O6 and/or O7 (Figure 5). It is possible

Fig. 3. The structures of the oligosaccharide from NMB, NMBlpt-3, and NMBlpt-6 LOS. The MS results indicated the presence of glycine in per-haps 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).

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 oli-gosaccharides (1699, 1718, 1756, and 1779) in that the latter oligosaccha-rides 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 con-tains a normal Kdo residue.

-10

9 0

1 9 0

2 9 0

3 9 0

4 9 0

1 40 0 1 50 0 1 60 0 1 7 0 0 1 80 0 1 90 0 2 00 0

- 1 0

9 0

1 9 0

2 9 0

3 9 0

4 9 0

5 9 0

6 9 0

7 9 0

8 9 0

1822

1840

1879

1897

1699

1718 17741756

0

9 0

1 9 0

2 9 0

3 9 0

4 9 0

1 40 0 1 50 0 1 60 0 1 80 0 1 90 0 2 00 0

1 40 0 1 50 0 1 60 0 1 7 0 0 1 80 0 1 90 0 2 00 01 40 0 1 50 0 1 60 0 1 80 0 1 90 0 2 00 0

1822

1840

1879

1897

1699

1718 17741756

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)

NMBlpt-3 (1822) 1840 Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo (anhydroKdo)

(1879) 1897 Gly1Glc2Gal2GlcNAc2Ac1Hep2PEA1Kdo (anhydroKdo)

NMBlpt-6 (1699) 1717 Glc2Gal2GlcNAc2Ac1Hep2Kdo (anhydroKdo)

(1756) 1774 Gly1Glc2Gal2GlcNAc2Ac1Hep2Kdo (anhydroKdo)

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that the amount of O-7 linked PEA substitution of HepII isbelow the detection level of the former procedure.

In the case of NMBlpt-6, the results (Table II) also sup-port the glycosyl structure found in NMB OSs but withouta PEA substitution (Lpt-6-Mj in Figure 3); a result thatsupports the mass spectral data (Figure 4). 31P-NMR of theOS from NMBlpt-6 OS lacked any detectable 31P signalconfirming the loss of all PEA groups attached to the innercore of this mutant (Figure 5).

In summary, all PEA attachments to the LOS inner corewere lost on inactivation of NMA0408 (now called lpt-6) instrain NMB. These results indicate that all of the detectablePEA attachments on the LOS inner core of strain NMB are

located at the O-6 position and that this isolate expressedL2 (NMB-Mj) and L4 (NMB-Mi) immunotypes (Figure 1).However, although strain NMB contains an intact lpt-3locus, inactivation of this gene did not result in any detect-able change in the spectrum of PEA attachments to theLOS inner core of the parent strain.

Lpt-3 modifies truncated LOS structures in strain NMB

Although O-3 PEA attachments were not detected on theLOS inner core of the wild-type parent strain NMB, O-3PEA attachments are present on the truncated mutantstructure of NMBlgtK (formerly NMBrfaK). NMBlgtKproduces a truncated LOS inner core in which HepII is sub-stituted with PEA substitutents at both the O-3 and O-6positions (Rahman et al., 2001). To determine whetherLpt3 in strain NMB contributed the O-3 PEA groups tothis structure, or whether Lpt6 performed both attach-ments, the lpt-3 and lpt-6 mutations were introduced intoNMBlgtK, and the resulting LOS glycoforms analysed.

The results of MS analysis of the OSs are shown inFigure 6 and summarized in Table III. The OS fromNMBlgtK contains ions that are consistent with a HepII-HepI-Kdo (or anhydroKdo) trisaccharide containing twoPEA substituents (m/z 867/849) or one PEA substituent(m/z 744/726), whereas the OSs from NMBlgtKlpt-3 andNMBlgtKlpt-6 each consist of a trisaccharide with only asingle PEA substituent. In the latter case, two ions of minorintensity were observed (m/z 603/621), indicating that asmall proportion OS with a structure devoid of all PEAsubstituents was present in these mutants.

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

T-Glc 14 13 12

T-Gal 14 14 12

3-Gal 13 13 14

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

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.

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To determine the location of the PEA substituents inNMBlgtKlpt-3 and NMBlgtKlpt-6 mutants, each OS wasanalyzed by the methylation/HF treatment/ethylation pro-

cedure and 31P-NMR. The PMAAs for each OS are shownin Table IV. The NMBlgtK OS contains PMAAs of HepIIwith an ethyl group at O3, at O6, at both O6 and O7, and atboth O3 and O6 (the major derivative). Further support forthe presence of the PEA groups at O-3 and O-7 of HepII ofNMBlgtK OS was gathered using 1D 31P spectrum NMRbecause it is relatively easy to distinguish between the 31Psignal for PEA substitution at O-3 from those for substitu-tion at O-6 and O-7 (Figure 5). Together, these results sup-port the previous report (Rahman et al., 2001) that the lgtKmutant produces an HepI-HepII-Kdo OS that containsPEA substituents at both O3 and O6 (LgtK-Mj); withsmaller amounts of OS with a PEA substituent at either O-6(LgtK-Mi-1) or O3 (LgtK-Mi-2); and also a small amountof OS with PEA at both O6 and O7 (LgtK-Mi-3, Figure 7).

In the case of the NMBlgtKlpt-3 mutant, the majorHepII derivative was ethylated at O-6, whereas ethylationat O-3 was not detectable (Table IV). 31P-NMR analysisconfirmed that this OS contained O-6 or O-7 PEA attach-ments to HepII with no structure with O-3 PEA (Figures 5and 7). These results confirmed that mutation of lpt-3 in theNMBlgtK resulted in the loss of O-3 PEA attachments toHepII, thus providing evidence that lpt-3 was an activelytranscribed and translated gene in strain NMB.

For the NMBlgtKlpt-6 mutant, the major HepII deriva-tive was ethylated at O-3 (Table IV) with no detectableHepII derivatives ethylated at O6, O7, or both O6 and O7.There was a lower percentage (10%) of terminally linkedHep, indicating that a small portion of HepII lacked PEAsubstitutions (LgtKLpt-6-Mi, Figure 7). 31P-NMR of theOS from NMBlgtKlpt-6 gave a signal for a PEA substituentonly at O-3 (Figure 5), indicating that this structure resem-bled LgtK-Mi-2 (Figure 7).

These results are consistent with the conclusion that Lpt3adds the PEA to O3 of HepII to the truncated inner core of anNMBlgtK 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 fromNMB, NMBlpt-3, and NMBlpt-6 revealed an additionalmolecular 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, NMBlgtKand the derivatives of this mutant did not contain glycine

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 oli-gosaccharides 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 oligosac-charides with a composition of PEA(1)Hep(2)Kdo.

0

100

200

300

650 700 750 800 850 900 950

0

50

100

150

200

250

650 700 750 800 850 900 950

0

50

100

150

200

250

650 700 750 800 850 900 950

726

744

849

867

726

744

726

744

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

*Minor ions.

Strain Ion [M-H]- Proposed composition

NMBlgtK (726) 744 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)

Table IV. Glycosyl linkage analysis (relative mol %) and location of PEA substituents of the OSs obtained from NMBlgtK, NMBlgtKlpt-3, and NMBlgtKlpt-6 LOSs

ND, not detected.

Glycosyl linkage NMBlgtK NMBlgtKlpt-3 NMBlgtKlpt-6

3,4-HepI 48.0 48.0 48.0

T-HepII Tr 3.0 10.0

T-Hep (6/7-Et) 4.0 46.0 ND

T-Hep (3-Et) 5.0 ND 42.0

T-Hep (3,6-diEt) 38.0 ND ND

T-Hep (6,7-diEt) 5.0 4.0 ND

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on the inner cores. These data suggest that similar to the α-chain extension form HepI, glycine residues cannot beattached to HepII before the γ-chain is complete. Althoughglycine residues are located on the inner cores of manylipopolysaccharides, the point at which this group is addedto the LOS structure has not been previously demonstratedin any other organism.

Discussion

Historically, each meningococcal LOS immunotype wasshown to have unique structures (Figure 1). In addition,genetic analysis of meningococcal immunotype isolates hasindicated that LOS biosynthesis genes are either not presentin every genome or, when present, are phase-variable inexpression (Jennings et al., 1999). This study demonstratesthat the L2/L4 immunotype strain NMB has the geneticand biochemical capacity to express all known LOS immu-notype inner core structures, a feature that has not previ-ously been demonstrated.

In earlier work, N. meningitidis strain NMB was found toexpress a mixture of two major LOS inner cores: 76% con-tained Hep II substituted at O-3 with Glc and at O-6 withPEA (designated the L2 immunotype), whereas 15% ofstructures lacked the Glc substitution at O-3 and retainedthe PEA substituent on HepII (Rahman et al., 1998). Addi-

tional analysis of the parent LOS structure presented hererevealed that structures that lacked glucose contained anO-6 PEA (L4 immunotype) and that glycine and O-7 linkedPEA can also be attached to the inner cores expressed bythis isolate. The intensity of the ions observed on MS ana-lysis suggested a 50/50 ratio of structures with or withoutglycine.

The presence of glycine on strain NMB OS had not beenpreviously determined because prior analyses had largelybeen done on de-O-acetylated preparations, which wouldhave removed the glycine residue that appears to beattached as an ester linkage presumably to O-7 of HepII asreported for other meningococcal LOS (Cox et al., 2002)and the O-acetyl group attached to O-6 of the terminalGlcNAc attached to O-2 of HepII. Although glycineappears to be a ubiquitous addition to the inner cores ofLPS from many Gram-negative bacteria (Cox et al., 2002;Gamian et al., 1996; Li et al., 2001), the stage at which thisresidue is added during LOS biosynthesis and the geneencoding this transferase have not been identified. Analysisof the inner core mutant, NMBlgtK, has revealed that all ofthe LOS glycoforms expressed by this isolate are devoid ofglycine attachments to HepII. Thus the attachment of gly-cine to HepII likely occurs after the γ-chain is completed,presumably because the conformation of the inner core ofthe truncated structure is inaccessible to the glycine trans-ferase. A similar mechanism for the inability of LgtF toattach the first residue of the α-chain (β1→4) glucose) toHepI in this mutant has been presented (Kahler et al.,1996). Structural modeling of the truncated inner cores ofvarious meningococcal LOS mutants have indicated thatthe conformation of these structures do differ significantlyas the α-chain is added to HepI (Plested et al., 2003).

To further determine whether the minor NMB LOSstructure, NMB-Mi, (Figure 5), corresponded to L3, L4, orL6 immunotype inner cores structures (Figure 1), the lpt-6and lpt-3 loci were insertionally inactivated and the struc-tures of these mutants examined for changes in PEA con-tent and location. Lpt-6 was identified as a member ofCOG1368 that contains E. coli MdoB, the sn-1 phospho-glycerol transferase required for the biosynthesis of MDOsin E. coli (Jackson et al., 1984). Inactivation of lpt-6 instrain NMB resulted in the loss of all PEA groups from theLOS inner core, whereas insertional mutagenesis of lpt-3led to no detectable changes in the amount or location onthe inner core of PEA residues in the wild-type parent. Ifstrain NMB expressed a mixture of L3, L4, and L6 innercores, inactivating each PEA transferase would result in theenrichment of separate subpopulations of LOS inner corestructures. Instead, NMBlpt-6 expressed a uniform popula-tion of structures with no PEA groups (Lpt-6-Mj, Figure 5),supporting the hypothesis that Lpt-6 was necessary for theaddition of both O-6 and O-7 linked PEA groups to HepIIof the inner core.

The analysis of the NMBlgtKlpt-6 mutant structures inwhich structures containing O-6 and O-7 linked PEA were loston inactivation of lpt-6 and structures decorated with O-3PEA groups accumulated also supported this hypothesis.Taken together, these results indicate that Lpt-6 in strainNMB may be a bifunctional transferase adding both O-6and O-7 PEA groups to the LOS inner core. Alternatively,

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.

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O-6 linked PEA may be transferred nonenzymatically tothe O-7 position of HepII, whereas the vacated O-6 posi-tion is filled again by Lpt-6 to produce a structure in whichboth O-6 and O-7 positions are occupied with PEA. Theextent to which nonenzymatic transfer could occur wouldbe partly based on the occupancy of the O-7 position by theglycine residue which is ∼ 50% in strain NMB LOS.

Wright et al. (2004) have also recently identified lpt-6 andhave insertionally inactivated this allele in two meningococ-cal strains 89I (L4 immunotype) and 35E (L2 immunotype).In both instances these strains produced inner cores withonly O-6 PEA groups that were lost on inactivation of lpt-6,suggesting that this allele encodes a monfunctional trans-ferase. Although there was no evidence of nonenzymatictransfer of PEA groups from O-6 to O-7 on the LOS innercores from these strains, it is currently unknown whetherglycine substitution at the O-7 position of HepII was at suf-ficiently high levels in these LOSs to prevent transfer occur-ing at a detectable rate.

Although inactivation of lpt-3 in the parental strain NMBbackground produced no discernible differences in the spec-trum of LOS structures produced, Lpt-3 activity was presentin this isolate. First, LOS glycoforms with O-3 linked PEAgroups attached to HepII accumulate in mutants, such asNMBlgtK (this study), NMBpgm (formerly NMBR6; Mon-teiro et al., 2003), NMBlgtF and NMBgalE (formerly SS3;Lee et al., 1995; data not shown). Furthermore, inactivationof lpt-3 in NMBlgtK results in the loss of those glycoformswith O-3 PEA groups. The lpt-3 locus from strain NMB hasbeen sequenced and similar to this locus from strain MC58,appears to have no polymeric tracts either within the openreading frame or in the putative promoter region that wouldindicate phase-variable expression. Quantitative real-timePCR of these loci from both strain NMB and MC58 alsoreveal that lpt-3 is transcribed at similar levels in both isolates(data not shown). Thus although strain NMB possesses Lpt-3 activity, the O-3 PEA groups are not present on completedLOS structures expressed by this strain.

In conclusion, parental strain NMB expresses a mixtureof L2 and L4 immunotype structures. We have previouslyshown that conversion of L4 cores to L2 cores is under theregulatory 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 com-mon 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 histori-cal observation that immunotyping antibodies generatedagainst L2 immunotype structures cross-react with all ofthese structures (Tsai et al., 1987). Pavliak et al. (2004)recently demonstrated that a tetravalent LOS conjugate vac-cine was capable of elicting a protective immune response ininfant rats against meningococcal challenge, thus providingthe first direct evidence that such a vaccine could poten-tially elicit a cross-protective immune response againstpathogenic Neisseria. Importantly, the LOS structuresexpressed by strain NMB contain all four of the epitopesused in the teravalent LOS conjugate vaccine.

Additionally, this study shows that strain NMB has thecomplete repertoire of genes necessary to build each knownmeningococcal LOS inner core (Figure 1), including the L3

immunotype. However, under laboratory growth condi-tions, the wild-type parent expresses a mixture of L2 and L4inner cores. Thus other regulatory processing pathwaysappear to be present in strain NMB that determine thespectrum of LOS inner core structures that are expressedunder different growth conditions. Isolates expressing L2/L4 immunotypes contain an archetypal LOS biosynthesispathway while meningococcal strains expressing the otherLOS immunotype structures may represent naturally occur-ing mutants in this pathway.

Materials and methods

Bacterial strains, media, and growth conditions

Meningococcal strains were grown with 3.5% CO2 at 37°Cunless 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% NaHCO3was used. Brain heart infusion medium (37 g/L brain heartinfusion) with 1.25% fetal bovine serum was used when kan-amycin selection was required. Antibiotic concentrationsused for E. coli strains were 100 µg/ml ampicillin, 50 µg/mlkanamycin, 100 µg/ml spectinomycin, and 300 µg/ml eryth-romycin; and for N. meningitidis were 80 µg/ml kanamycin,3 µg/ml erythromycin, 60 µg/ml spectinomycin, and 5 µg/mltetracycline. E. coli strain JM109 cultured on Luria Bertanimedium was used for cloning and propagation of plasmids.Meningococci were transformed as previously described(Janik et al., 1976). E. coli strains were transformed by themethod of Chung et al. (1989).

Construction of NMBlpt-3 and NMBlpt-6

NMBlpt-3. An internal section of the lpt-3 locus in strainNMB was amplified using lpt3-1 (5′-GCTGACATTTGT-GATTGCTGCG-3′) and lpt3-2 (5′-CTCATTGCGGAT-AAACATATTCCG-3′) primers and cloned into the HincIIsite of pHSG576 (Takeshita et al., 1987) to formpJKD2618. The aphA-3 cassette was liberated frompUC18K (Menard et al., 1993) using SmaI sites and clonedinto the unique EcoRV site of the pJKD2618 to createpJKD2619. The orientation of aphA-3 and in-frame fusionwith lpt-3 in pJKD2619 were confirmed by directional PCRand sequencing (data not shown). The plasmid was used totransform N. meningitidis strain NMB and the transfor-mants (NMBlpt-3) were identified by resistance to kanamy-cin. Inactivation of lpt-3 was confirmed by colony PCR(data not shown). NMBlgtKlpt-3 mutants were constructedby transforming chromosomal DNA from NMBlpt-3 intoCMK1 (NMBlgtK) (Kahler et al., 1996; Rahman et al., 2001).

NMBlpt-6. A unique HincII site was incorporatedinto NMA0408 by splice-overlap extension PCR. Twointernal fragments of NMA0408 were amplified fromN. 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 molaramounts of these products were mixed together and used

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as a template for PCR amplification using 0408-3 (5′-CTGT-TCCACAGGCTGAAAATCC-3′) and 0408-4 (5′-GCTTTGTCCAAATCGGCAATGC-3′) primers. The inter-nal fragment of lpt-6 containing the unique HincII site wascloned 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 (NewEngland Biolabs, Beverly, MA) and ligated into the HincII siteof pJKD2620 to form pJKD2621. The plasmid was purifiedand used to transform N. meningitidis strain NMB using theplate transformation method (Janik et al., 1976). Transformants(NMBlpt-6) were selected for resistance to erythromycin andthe inactivation of lpt-6 confirmed by colony PCR (data notshown). NMBlgtKlpt-6 mutants were constructed by trans-forming 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 har-vested from an overnight aerobic culture using a modifiedversion of the phenol-chloroform–petroleum ether extrac-tion 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% aceticacid (10 ml) for 2 h at 100°C. The hydrolysate was centri-fuged at 10,000 × g for 20 min, and the supernatant was col-lected. The pellet was washed once with 5 ml water andcentrifuged again. The water wash was added to the super-natant, and any remaining lipid A was extracted withdiethyl ether (three times, 5 ml volumes each time). Theaqueous phase, containing the OSs, was lyophilized. Thelyophilized OSs were dissolved in 0.5 ml water, filtered withMicrofilterfuge tubes containing 0.45-µm pore size Nylon-66 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.

Glycosyl composition analyses

The glycosyl compositions of the LOS and OS (0.5 mgeach) samples were performed by the preparation and anal-ysis of alditol acetates as previously described (York et al.,1985). Briefly, the samples were hydrolyzed in 2 M trifluo-roacetic acid (0.5 ml) in a closed vial at 120°C for 3 h. Theresulting 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 modifiedNaOH method. The sample (1 mg) was dissolved in dime-thyl sulfoxide (100 µl), powdered NaOH (100 mg) wasadded, and the reaction mixture was stirred rapidly at roomtemperature for 30 min. Methylation was performed by thesequential additions of methyl iodide (10, 10, and 20 µl) at10-min intervals. After an additional 20 min stirring, 1 mlof 1 M sodium thiosulfate was added, and the methylatedglycans were recovered in the organic phase by extractionwith chloroform (0.5 ml × 3). The permethylated productwas further purified by reverse-phase chromatography

using a Sep-Pak C18 cartridge (Waeghe et al., 1983). Themethylated glycan was hydrolyzed with 2 M trifluoroaceticacid (120°C, 3 h), reduced with NaB2H4, acetylated, andanalyzed by GLC and GLC-MS (York et al., 1985). GLCand GLC-MS analyses were performed using capillary col-umns (length, 30 m; inner diameter, 0.32 mm) with heliumas the carrier. A DB-5 column (J&W Scientific, now Agi-lent Technologies, Wilmington, DE) was used for ami-noglycosyl derivatives, and an SP2330 column (Supelco,Bellefonte, PA) was used for the neutral glycosyl derivatives.GLC equipment consisted of HP5890 gas chromatographequipped with a flame ionization detector (Hewlett-Packard).GLC-MS (electron ionization) was performed using aHewlett-Packard 5970 MSD.

The location of phosphate substituents (e.g., PEA) wasdetermined by methylating the OS as just described. Thiswas followed by removal of the phosphate substituents bytreating the samples with cold aqueous 48% HF (100 µl) for24 h at 4°C as described (Kenne et al., 1993). The samplewas then dissolved in dimethyl sulfoxide and NaOH asdescribed, followed by ethylation with ethyl iodide. ThePMAAs were then prepared and analyzed by GLC-MS asdescribed.

Mass spectroscopy analyses

Matrix-assisted laser desorption ionization time of flight(MALDI-TOF) MS was performed with either an AppliedBiosystem Voyager or an HP MALDI-TOF spectrometersystem in the negative-ion mode. Approximately 2 µl of a 1mg/ml lipid A solution in chloroform-methanol (3:1, v/v)was mixed with 1 µl trihydroxyacetophenone matrix solu-tion (∼ 93.5 mg trihydroxyacetophenone per ml of metha-nol) and applied to the probe for mass analysis. Spectrawere calibrated externally with E. coli lipid A (Sigma,St. Louis, MO). The OSs were dissolved in distilled water ata final concentration of 2 µg/µl, and 1 µl was mixed withthe dihydroxybenzoic acid in methanol matrix for analysis.

31P-NMR spectroscopy

Each sample, 1–2 mg, was prepared for NMR analysis by atwofold lyophilization from D2O, dissolved in D2O, andanalyzed. NMR spectra were recorded on Varian 300 MHzspectrometers at 27°C. The data was referenced to external85% phosphoric acid (0.0 ppm).

Acknowledgments

C. Kahler thanks John Davies and David Stephens for con-tinued support and J.C. Wright and R. Moxon foracknowledging this work in their recent publication. Thiswork was funded by NIH grant R01 AI033517-10 to D.S.S.

Abbreviations

GLC, gas-liquid chromatography; LOS, lipooligosaccha-ride; MDO, membrane-derived oligosaccharide; MALDI-TOF, matrix-assisted laser desorption ionization time offlight; MS, mass spectrometry; NMR, nuclear magneticresonance; OS, oligosaccharide; PCR, polymerase chain

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reaction; PEA, phosphoethanolamine; PMAA, partiallymethylated/ethylated aldtitol acetate.

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