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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. 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