Neisserial Lipooligosaccharide Research Articles

Synthesis of branched inner-core oligosaccharides of neisserial lipooligosaccharides

In this study, branched inner-core oligosaccharides of neisserial lipooligosaccharides was successively synthesized. We found that the glycosylation of a 4-O-substituted heptose derivative with a heptosyl donor proceeded smoothly proceeded in toluene and provided corresponding 3,4-di-O-substituted heptose as a single diastereomer. In addition, we found that the fragment condensation of a 3,4-di-O-substituded heptosyl donor with the axially oriented 5-position of a 4-O-substituted Kdo acceptor provided a consecutive branched hexasaccharide with a vicinal trans-diol of l-glycero-d-manno-heptose (Hep) and a vicinal cis-diol of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) in its structure.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

O19.6 Use of Surfactant Vesicles as a Potential Gonococcal Vaccine Delivery System to Generate Antibody Against Neisserial Lipooligosaccharide

BackgroundTo date, no one has been able to exploit the immunological potential of neisserial lipooligosaccharide (LOS) as a vaccine candidate.MethodsWe have developed a glycoconjugate vaccine (TRIAD) that contains native LOS...

The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.

Western Blot Analysis of Neisserial Lipooligosaccharide at the Lower Femtomole Level with Normal Human Sera

Lipooligosaccharide (LOS) is a major immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae. Recent immunochemical studies have found that normal human sera (NHS) contain bactericidal anti-LOS antibodies that bind to the oligosaccharide (OS) moiety of neisserial LOS. Although affinity-purified anti-LOS antibodies can be characterized using 10-100 ng of LOS samples (up to a few tens of pmoles), a more sensitive immunoblotting assay must be established in order to analyze NHS directly and characterize anti-LOS antibodies without affinity purification. We examined analytical PAGE/blot conditions using a 15-well mini gel. For the first time, Western blot detection of LOS at the lower femtomole level was accomplished by both chromogenic and chemiluminescent detection. A model LOS, 15253 LOS, was detected in a low femtomole range (62.5-500 pg, 16-125 femtomole) even with 10 pM of a monoclonal antibody (MAb) 2C7. Furthermore, detection of similar amounts (50-250 femtomole) of neisserial LOSs and Salmonella truncated lipopolysaccharides (LPSs) was also possible with 1:50 and with 1:100 diluted NHS. The results obtained here indicate that the binding of IgG in NHS to the LOS and LPS samples is probably due to their carbohydrate moieties. The detection level accomplished in this study should help not only to further characterize anti-LOS antibodies in blood and body fluids but also to analyze carbohydrate structures that are recognized by them.

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.

The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies

Recently, we isolated human IgG from normal human sera (NHS) using lipooligosaccharide (LOS) from gonococcal strain JW31R as an affinity ligand. We provided evidence that the oligosaccharide (OS) moiety of LOS was immunogenic in humans and that NHS contains functional antibodies that bind to the branched OS. The present study aimed to identify bactericidal antibodies that bind to partial core OS structures or their adjacent sites expressed in the 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. Using 15253 LOS from serum-resistant gonococcal strain 15253 as an affinity ligand, we isolated IgG2 and found that this preparation contained at least three different species. (i) One IgG2 species recognized a cross-reactive epitope that is expressed on 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. (ii) Another IgG2 species was specific for JW31R LOS from a pyocin-resistant gonococcal strain; this IgG-defined epitope was not shared with the aforementioned branched LOSs. (iii) The third IgG2 species bound to the "Salmonella minnesota" Rb and Re mutant lipopolysaccharides (LPSs); this IgG2 recognizes a KDOalpha2-4KDO residue at the reducing end of the carbohydrate moiety of each LPS. The IgG2 was also found to be functional and facilitated the killing of strain 15253. The current results show that neisserial LOS contains several epitopes within its OS moiety that are recognized by human antibodies.

Neisserial Lipooligosaccharides (LOS) Differentially Modulate Innate Immune Responses in Human Monocytes throughTLR4 Signaling (135.17)

Abstract Neisseria gonorrhoeae and N. meningitidis engage innate immune receptors TLR4 and TLR2, as well as TREM1 and TREM2. In this study, we report differential innate immune activation by Neisserial LOS in monocytes. LOS differentially induced proinflammatory mediators TNF-alpha, IL-1 beta, IL-6 and IL-8 in THP1 monocytic cells as well as IL-10, IL-12, IFN-alpha, MIP-1 alpha, MIP-1 beta, and RANTES in human monocytes. Interestingly, while gonococcal LOS was not able to induce GM-CSF in monocytes, GM-CSF expression was enhanced by meningococcal LOS in both THP1 cells and monocytes. In THP1 cells, the induction of TNF-alpha by LOS was inhibited by anti-TLR4 and anti-CD14 antibody, but not by anti-TLR2, suggesting that the TLR4, but not TLR2, was responsible for the induction of cytokines. The ability of LOS to directly activate the TLR4 pathway was demonstrated by using TLR4-MD2 transfected HEK reporter cells, the activation of which was blocked by lipid IVa, a TLR4-MD2 antagonist. In monocytes, lipid IVa also inhibited the induction of all cytokines as well as CD80 expression by gonococcal LOS, but it only inhibited the activation of TNF-alpha, IL-10 and GM-CSF by meningococcal LOS. These results suggest that Neisserial LOS differentially activate innate immunity in monocytes through the TLR4 pathway, but that the induction of innate immune responses by meningococcal LOS may rely on the engagement of additional pattern recognition receptors.

Structural Requirements for Monoclonal Antibody 2-1-L8 Recognition of Neisserial Lipooligosaccharides

Monoclonal antibodies (MAbs) that bind neisserial lipooligosaccharides (LOS) have been widely used in structural studies of these glycolipids. MAb 2-1-L8 binds LOS with a lactosyl a chain (Gal beta1-4 Glc beta1-4 [Glc-NAc alpha1-2 Hep2 alpha1-3] Hep1 alpha1-KDO) and at least one phosphoethanolamine (PEA) substitution of Hep2, but the requirement for PEA substitution and/or the exact position of this substitution, cyclic or exocyclic, remains unclear. In order to clarify the exact specificity of this MAb, we engineered an isogenic family of lpt mutants that each make LOS with a lactosyl a chain, but that lacked cyclic (-3Hep2), exocyclic (-6Hep2), or any PEA residues. Mass spectrometry showed that mutants that lack either Lpt3 or Lpt6 make small amounts of LOS with two PEA substitutions. Thus, each enzyme is able to phosphoethanolaminylate the alternate site, albeit with low efficiency. LOS made by the mutant that lacked both Lpt3 and Lpt6 was devoid of PEA. LOS made by the deltalpt3 mutant did not bind MAb 2-1-L8 on Western blot analysis, whereas delta pt6 LOS did. Analysis of intact mutants by fluorescence-activated cell sorting confirmed that PEA substitution at 3Hep2, but not at 6Hep2, is needed for optimal binding of MAb 2-1-L8. These data confirm that the MAb 2-1-L8 epitope requires a -3Hep2 cyclic PEA substitution for optimal conformation and that this MAb specifies the PEA-3Hep2 lactosyl LOS structure.

Neisserial lipooligosaccharide and complement activation: Contrasting effects of heptose II substitutions in gonococci and meningococci

Neisserial lipooligosaccharide and complement activation: Contrasting effects of heptose II substitutions in gonococci and meningococci

Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62DeltaLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62DeltaLgtA, producing strain F62DeltaLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62DeltaLgtA indicated that this strain contained two PEA modifications on its LOS. F62DeltaLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62DeltaLgtAlpt3::Tn5.

Separation and identification of neisserial lipooligosaccharide oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

We determined the optimal conditions for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS) released from neisserial lipooligosaccharides (LOS) by mild acid hydrolysis. We efficiently obtained detailed composition, sequence, and linkage information about high M r LOS. We found that HPAE-PAD can discriminate isobaric (same M r) molecules of different structure, for example, nLc 4 and Gb 4, distinguish α from β chain extensions, and determine the number of phosphoethanolamine (PEA) substituents. HPAE-PAD provided quantitative information that could be used to compare the relative abundances of OS. We used HPAE-PAD to identify all of the known LOS α chain antennae. When used with antibody-binding profiles and exoglycosidase digestion results, HPAE-PAD can provide nearly complete structures rapidly.

Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis: capacity of strain NMB to express all known immunotype epitopes

Neisseria meningitidis expresses a heterogeneous population of lipooligosaccharide (LOS) inner cores variously substituted with alpha1-3-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 alpha1,3-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 lpt-6 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 alpha1-3-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 gamma-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.

Synthesis of a 3,4‐Di‐O‐Substituted Heptose Structure: A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide.

We have synthesized a tetrasaccharide containing a 3,4-dibranched L-glycero-D-manno-heptose (Hep), β-lactosyl-(1⇄4)-[L-α-D-Hep-(1⇄3)]-L-α-D-Hep 19, by using a mannose (Man) derivative as an acceptor. Prior to the construction of the branched Hep, we confirmed that the 3,4-dibranched Man structure could be synthesized using a 3-branched Man 6 as an acceptor. Glycosylation of the acceptor 6 using hepta-O-acetyl-α-lactosyl trichloroacetimidate (7) gave the desired 3,4-dibranched structure, β-lactosyl-(1⇄4)-[α-Man-(1⇄3)]-α-Man 8. As expected, β-lactosyl-(1⇄4)-[L-α-D-Hep-(1⇄3)]-α-D-Man 14 was also obtained by glycosylating the 4-OH acceptor 13 with 7 in a similar manner. The Man residue of 14 was converted into the Hep unit by Swern oxidation, Grignard reaction, and oxidative cleavage followed by reduction. Thus, we constructed the 3,4-dibranched Hep structure 19 by using the 3-branched Man 13 as an acceptor. The current results demonstrate that the gauche orientation of the O-3 and O-4 units of the Man configuration does not prevent the formation of the 3,4-di-O-substituted structure. This approach should provide an alternative method to synthesize the 3,4-dibranched Hep structure expressed in LOS produced by pathogenic Gram-negative bacteria such as the Neisserial and Haemophilus species. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Synthesis of a 3,4‐Di‐O‐substituted Heptose Structure: A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide

AbstractWe have synthesized a tetrasaccharide containing a 3,4‐dibranched L‐glycero‐D‐manno‐heptose (Hep), β‐lactosyl‐(1⇄4)‐[L‐α‐D‐Hep‐(1⇄3)]‐L‐α‐D‐Hep 19, by using a mannose (Man) derivative as an acceptor. Prior to the construction of the branched Hep, we confirmed that the 3,4‐dibranched Man structure could be synthesized using a 3‐branched Man 6 as an acceptor. Glycosylation of the acceptor 6 using hepta‐O‐acetyl‐α‐lactosyl trichloroacetimidate (7) gave the desired 3,4‐dibranched structure, β‐lactosyl‐(1⇄4)‐[α‐Man‐(1⇄3)]‐α‐Man 8. As expected, β‐lactosyl‐(1⇄4)‐[L‐α‐D‐Hep‐(1⇄3)]‐α‐D‐Man 14 was also obtained by glycosylating the 4‐OH acceptor 13 with 7 in a similar manner. The Man residue of 14 was converted into the Hep unit by Swern oxidation, Grignard reaction, and oxidative cleavage followed by reduction. Thus, we constructed the 3,4‐dibranched Hep structure 19 by using the 3‐branched Man 13 as an acceptor. The current results demonstrate that the gauche orientation of the O‐3 and O‐4 units of the Man configuration does not prevent the formation of the 3,4‐di‐O‐substituted structure. This approach should provide an alternative method to synthesize the 3,4‐dibranched Hep structure expressed in LOS produced by pathogenic Gram‐negative bacteria such as the Neisserial and Haemophilus species. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Biochemical properties of Neisseria gonorrhoeae LgtE.

A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the alpha and beta chains of neisserial LOS.

Neisseria gonorrhoeae strain PID2 simultaneously expresses six chemically related lipooligosaccharide structures.

Neisseria gonorrhoeae strain PID2 was isolated from a woman suffering from pelvic inflammatory disease. When LOS expressed by this strain is analyzed on SDS-PAGE gels, at least six different lipooligosaccharide (LOS) components are visualized. We characterized the LOSs made by this strain by exoglycosidase digestion, sugar composition analysis, mass spectrometry, and analysis of the genes needed for its synthesis. DNA sequence analysis showed that the lgt gene cluster in this strain has undergone a rearrangement and that it possesses two copies of lgtA, one copy of lgtB and lgtC, and a hybrid gene containing sequences from lgtB and lgtE. We determined that the hybrid lgtB/E gene retained the lgtE gene function. DNA sequence analysis of the gene organization suggested that an intramolecular recombination between lgtA and lgtD and lgtB and lgtE had occurred via homologous recombination between similar sequences. Our studies demonstrated that fluorophore-assisted carbohydrate electrophoresis can be utilized to rapidly determine the composition of LOS. By combining exoglycosidase digestion, in combination with mass spectrometry analysis and compositional analysis, the data indicate that all of the LOS components produced by PID2 extend off of the alpha chain. The longest alpha chain oligosaccharide structure is Gal-GlcNAc-Gal-GlcNAc-Gal-Glc-Heptose I, and the six LOS components are built up by sequentially adding sugars onto the first heptose. PID2 LOS is the first Neisserial LOS to be shown to be devoid of phosphoethanolamine modifications. Because PID2 can surface express its LOS, it indicates that the addition of phosphoethanolamine is not required for LOS surface expression.

Interaction of Neisseria meningitidis with human dendritic cells.

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-alpha, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1beta, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.

Functional characterization of an isogenic meningococcal alpha-2,3-sialyltransferase mutant: the role of lipooligosaccharide sialylation for serum resistance in serogroup B meningococci.

The neisserial alpha-2,3-sialyltransferase, which is encoded by the lst gene, terminally links sialic acid to the lacto-N-neotetraose residue of neisserial lipooligosaccharide (LOS). We used the recently published nucleotide sequence of the neisserial lst gene to construct an isogenic serogroup B meningococcal lst mutant by insertion of a kanamycin resistance gene. The resulting lst mutant expressed the unsialylated lacto-N-neotetraose structure. Using bactericidal assays and an infant rat model of meningococcal infection, we were able to demonstrate that lst mutation, in contrast to galE mutation, which results in a truncated LOS, or to siaD mutation, which results in loss of the capsule, neither had an effect on resistance to normal human serum, nor did it impair the ability of meningococci to spread systemically in the non-immune host. The lst mutant was serum resistant despite of the fact that the central factor of complement activation, C3b, was deposited on the lst mutant as efficiently as it was on the galE mutant. Thus, the terminal sialic acid residue linked to the wild-type LOS inhibited C3b deposition on the meningocuccus. However, in contrast to the galE mutant, where C3b deposition is promoted by IgM binding, the lst mutant's surface is not a target for IgM molecules. Thus, the lacto-N-neotetraose residue of neisserial LOS alone, without the presence of terminal sialic acid, is sufficient to block IgM epitopes either on the LOS itself, or on other surface molecules. Our data provide further insight into the complex interplay of capsular and LOS sialic acids in serogroup B meningococci with host effector mechanisms, and suggest that LOS sialylation in meningococci is of a less central importance as it is in gonococci.

Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O-deacylated lipid A. Because the phosphorylated diheptose core region of the LOS is essential for the formation of a conserved LOS epitope (D6A) that is immunogenic in humans, care should be taken to maintain stereochemical requirements for the expression of this conserved epitope in the design of effective, nontoxic LOS vaccines.