Strain NMB Research Articles

The Use of High-Throughput DNA Sequencing in the Investigation of Antigenic Variation: Application to Neisseria Species

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3′ end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.

MisR/MisS Two-Component Regulon in Neisseria meningitidis

Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and beta-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His(6) protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.

Physicochemical characterization and biological activity of lipooligosaccharides and lipid A fromNeisseria meningitidis

Meningococcal endotoxin is the major contributor to the pathogenesis of fulminant sepsis and meningitis of meningococcal disease and is a potent activator of the MyD88-dependent and MyD88-independent pathways via the MD-2/TLR4 receptor. To understand better the biological properties of meningococcal endotoxin that initiates these events, the physicochemical structure of Neisseria meningitidis lipopoly(oligo)saccharide (LOS) of the serogroup B wild-type strain NMB (NeuNAc-Gal beta-GlcNAc-Gal beta-Glc beta-Hep2(GlcNAc,Glc alpha)PEA-Kdo2-lipid A, 1,4'-bisphosphorylated +/- PEA, PEtN) and the genetically-defined mutants (gmhB, Kdo2 -lipid A; kdtA, meningococcal lipid A; gmhB-lpxL1, Kdo2penta-acylated lipid A and NMB-lpx1, penta-acylated meningococcal LOS) were assessed in relation to bioactivity. Confirming previous work, Kdo2lipid A was the minimal structure required for optimal activation of the MD-2/TLR4 pathway of human macrophages. Meningococcal lipid A alone was a very weak agonist in stimulating human macrophages, even at high doses. Penta-acylated LOS structures demonstrated a moderate reduction in TLR4/MyD88-dependent signaling and a dramatic decrease in TLR4-TRIF-dependent signaling. For a better understanding of these results, we have performed an analysis of physicochemical parameters of the LOS structures such as the gel-to-liquid crystalline phase transition of the acyl chains, the inclination angle of the diglucosamine backbone with respect to the membrane surface, and the aggregate structure, and have found a very significant correlation of these parameters with biological activities extending our concept of endotoxicity.

O-Acetylation of the Terminal N-Acetylglucosamine of the Lipooligosaccharide Inner Core in Neisseria meningitidis: INFLUENCE ON INNER CORE STRUCTURE AND ASSEMBLY

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the gamma-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.

Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence ofNeisseria meningitidis

Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galbeta-GlcNAc-Galbeta-Glcbeta-Hep2 (GlcNAc, Glcalpha) 3-deoxy-D-manno-2-octulosonic acid (KDO2)-lipid A; 1,4' bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in Neisseria meningitidis

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.

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.

The MisR/MisS Two-component Regulatory System Influences Inner Core Structure and Immunotype of Lipooligosaccharide in Neisseria meningitidis

Lipooligosaccharide (LOS) of Neisseria meningitidis is the major inflammatory mediator that contributes to meningococcal pathogenesis. Variable attachments to the HepII residue of the LOS inner core together with the alpha-chain heterogeneity result in immunologically distinct LOS structures, which may be selected for during human infection. Lpt-3, a phosphoethanolamine (PEA) transferase, and LgtG, a glucosyltransferase, mediate the substitution of PEA or glucose at the O-3 position of HepII in L3 or L2 LOS immunotypes, respectively. Inactivation of a two-component response regulator, encoded by NMB0595, in N. meningitidis strain NMB resulted in the loss of all PEA decorations on the LOS inner core expressed by the NMB0595 mutant. When compared with the parental strain NMB that predominantly expresses L2 immunotype LOS and other minor LOS structures, the NMB0595 mutant expresses a pure population of a novel LOS structure completely substituted at the HepII O-3 position with glucose, but lacking other PEA decorations on the inner core. Quantitative real time PCR experiments showed increased transcription of lgtG in the NMB0595 mutant, and no significant change in lpt-3 transcription. Inactivation of lgtG resulted in LOS inner cores without glucose, but these structures, even though the lpt-3 transcription was unaffected, also lacked the O-3-linked PEA. Consistently, a double mutation of lgtG and misR in strain NMB yielded a LOS structure without PEA or Glc substitution of HepII. These data indicated a new pathway for the regulation of LOS inner core structure in N. meningitidis through an environmental sensing two-component regulatory system, named misR(NMB0595)/misS(NMB0594) for regulator and sensor of the meningococcal inner core structure.

The msbB mutant of Neisseria meningitidis strain NMB has a defect in lipooligosaccharide assembly and transport to the outer membrane.

A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galbeta1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.

Polymorphisms in pilin glycosylation Locus of Neisseria meningitidis expressing class II pili.

We have located a locus, pgl, in Neisseria meningitidis strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (pglF, pglB, pglC, pglB2, orf2, orf3, orf8, and avtA) were present in the pgl clusters of different meningococcal isolates. The Class I pilus-expressing strains Neisseria gonorrhoeae MS11 and N. meningitidis MC58 each contain a pgl cluster in which orf2 and orf3 have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained pgl clusters in which pglB had been replaced by pglB2 and an additional novel ORF, orf8, had been inserted between pglB2 and pglC. Insertional inactivation of the eight ORFs of the pgl cluster of strain NMB showed that pglF, pglB2, pglC, and pglD, but not orf2, orf3, orf8, and avtA, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as pglF and pglD mutants retained wild-type levels of serum resistance. Although pglB2 and pglC mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that pglB2 and pglC mutants expressed almost 50% less capsule than wild-type NMB. A mutation in orf3, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.

Transcriptional regulation of divergent capsule biosynthesis and transport operon promoters in serogroup B Neisseria meningitidis.

The clinically important serogroups B, C, Y, and W-135 of Neisseria meningitidis produce sialic acid capsules that are critical in pathogenesis. In each of these serogroups, the capsule transport (ctrABCD) and capsule biosynthesis (synABCD) operons are divergently transcribed from putative promoters located in a 134-bp intergenic region (J. S. Swartley, J. H. Ahn, L. J. Liu, C. M. Kahler, and D. S. Stephens, J. Bacteriol. 178:4052-4059, 1996). In this study we further assessed the role of the intergenic sequence in the transcriptional regulation of the sialic acid capsules of N. meningitidis. Insertional mutagenesis or deletions of the 134-bp sequence in the serogroup B meningococcal strain NMB resulted in a marked reduction or elimination of ctrABCD and synABCD transcription, with a concomitant loss of encapsulation. Chromosomal transcriptional lacZ-ermC reporter fusions of syn and ctr promoters were constructed through allelic exchange. Using these constructs, both operons were found to be constitutively transcribed in meningococci, the biosynthesis operon about fourfold higher than the transport operon. Both promoters showed increased activity during stationary-phase growth. In addition to the promoters, a 70-bp 5' untranslated region (UTR) upstream of synA was found to have a direct repeat and an inverted repeat that overlapped three putative integration host factor binding sites. Mutation of this 70-bp UTR and of the direct repeat upregulated both syn and ctr transcription. Regulation through the synA UTR was absent in a K1 Escherichia coli strain that produces identical capsular polysaccharide, implicating species-specific regulation. Meningococcal sialic acid capsule expression is initiated by divergent promoters in a 134-bp intergenic region, is repressed at the transcriptional level by the 5' UTR of synA, is increased during stationary-phase growth, and shows species-specific regulation. Transcriptional regulation is another important control point for sialic capsule expression in N. meningitidis.

Construction of Acetate Auxotrophs of Neisseria meningitidis to Study Host-Meningococcal Endotoxin Interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.

Relationship between UDP-glucose 4-epimerase activity and oligoglucose glycoforms in two strains of Neisseria meningitidis.

Sodium dodecyl sulfate-polyacrylamide gel analysis of lipooligosaccharide (LOS) from Neisseria meningitidis has demonstrated considerable microheterogeneity in the variable region of LOS due to the presence of novel glycoforms. As a step toward understanding the basis for the expression of these novel glycoforms, we have examined the LOS structures and UDP-glucose 4-epimerase (epimerase) activity levels in two strains (NMB and MA-1) and their respective galE mutants. Strain NMB was found to have low epimerase activity and to contain multiple glycoforms, some of which appear to contain only glucose sugars. The galE mutant had only the oligoglucose glycoforms. Strain MA-1 had higher epimerase activity at both log and stationary phases (2- and 12.5-fold, respectively) and one glycoform with a putative lactosyl structure. Strain MA-1 galE had two glycoforms that contained one or two glucose residues. To understand the molecular basis for the different epimerase activities, we examined the predicted amino acid sequences of the respective galE open reading frames and determined the relative amounts of GalE protein. We found no significant differences between the predicted amino acid sequence of the GalE protein in NMB and that in MA-1. We observed no significant differences in the level of GalE protein between MA-1 and NMB at exponential or stationary phase. We also observed an 8.2-fold drop in epimerase activity in NMB between the log and stationary phases that was not due to the GalE protein level or low glucose levels.

The lipooligosaccharide (LOS) of Neisseria meningitidis Serogroup B Strain NMB contains L2, L3, and novel oligosaccharides, and lacks the lipid-A 4′-phosphate substituent

The complete structure of the lipooligosaccharide (LOS) from Neisseria meningitidis strain NMB (serotype 2b:P1.2,5), a serogroup B cerebrospinal fluid isolate, was determined. Two oligosaccharide (OS) fractions and lipid-A were obtained following mild acid hydrolysis of the LOS. The structures in these fractions were determined using glycosyl composition and linkage analyses, N spectroscopy and mass spectrometry. One oligosaccharide fraction (OS1) consists of a molecule having a glycosyl sequence identical to that previously reported for the LOS from immunotype L2 N. meningitidis [A. Gamain, M. Beurret, F. Michon, J.-R. Brisson, and H.J. Jennings, J. Biol. Chem., 267, (112) 922–925] i.e., a lacto- N-neotetraose is attached to heptose I (Hep I), with terminally linked N-acetylglucosaminosyl and glucosyl residues attached to Hep II of the inner core. Approximately 70% of this structure is acetylated at O-6 of the terminally linked α- N-acetylglucosaminosyl residue. As with the L2 structure, the NMB LOS contained phosphoethanolamine (PEA) at O-6 or O-7 of the Hep II residue. The second oligosaccharide fraction (OS2) contains a mixture of three different molecules, all of which vary from one another in their glycosyl substitution patterns of the Hep II residue. The most abundant molecule in OS2 has a structure identical to that of OS1, i.e., it has the L2 glycosyl sequence. A second molecule (OS2a) lacks the terminal glucosyl residue at O–3 of Hep II; i.e., it has a glycosyl sequence identical to that of the mild acid released oligosaccharide of N. meningitidis immunotype L3, L4, or L7 LOSs. The third molecule (OS2b) is a novel structure that lacks the terminal N-acetylglucosaminosyl residue linked to O-2 of Hep II. Overall, 76% of OS released from NMB LOS has the L2 structure, 15% is OS2a (L3), and 9% is OS2b. A portion (20%) of the molecules in the NMB LOS preparation also contained terminally linked sialic acid attached to O-3 of the lacto- N-neotetraose galactosyl residue, which is also consistent with the L3, or L4 LOS structures. In contrast to the previously reported structure of N. meningitidis lipid-A [V. A. Kulshin, U. Zähringer, B. Linder, C.E. Frasch, C.-M. Tsai, B.A. Dmitriev, and E.T. Rietschel, J. Bacteriol., 174, (1992) 1793–1800], only 30% of the lipid-A from NMB LOS possesses 4′-phosphate. Comparison with the lipid-A of LOS purified from an isogenic acapsulate mutant, M7, revealed that the 4′-position was almost completely occupied with phosphate. These data emphasize the structural heterogeneity of the OS and phosphate substituents of Hep II, and 4′-phosphorylation of lipid-A of meningococcal LOS.

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK).

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.

Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn916 mutagenesis.

Fourteen Tn916 mutants of Neisseria meningitidis strain NMB were identified as auxotrophs. Among these were eight amino acid auxotrophs, with five different phenotypes, and three isolates restricted in carbon source utilization, growing in the presence of glucose but not on L-lactate, D-lactate, pyruvate, or casamino acids as principal carbon sources.

Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn 916 mutagenesis

Fourteen Tn 916 mutants of Neisseria meningitidis strain NMB were identified as auxotrophs. Among these were eight amino acid auxotrophs, with five different phenotypes, and three isolates restricted in carbon source utilization, growing in the presence of glucose but not on L-lactate, D-lactate, pyruvate, or casamino acids as principal carbon sources.

Lipooligosaccharide biosynthesis in pathogenic Neisseria. Cloning, identification, and characterization of the phosphoglucomutase gene

The lipooligosaccharide (LOS) of pathogenic Neisseria is an important factor in disease pathogenesis. Little is known about the genes involved in neisserial LOS biosynthesis. To elucidate specific LOS biosynthetic genes, we screened a Tn916 library that was constructed in Neisseria meningitidis strain NMB. This strain expresses a single LOS that has an molecular mass of 4.5 kDa and binds monoclonal antibody (mAb) 3F11. This library was screened using a mAb panel that recognizes structural differences in neisserial LOS oligosaccharides. A stable LOS mutant of strain NMB was identified which we designated NMB-R6. This mutant expressed an LOS with an molecular mass of approximately 3.1-3.2 kDa and did not bind mAb 3F11. Genomic DNA from this mutant transformed N. meningitidis strain NMB to the tetracycline resistant NMB-R6 phenotype greater than 10(-4)/recipient/micrograms of DNA. In addition, we transformed Neisseria gonorrhoeae strain 1291 (LOS phenotype molecular mass 4.5 kDa, mAb 3F11+) to the NMB-R6 LOS phenotype with N. meningitidis NMB-R6 genomic DNA. Analysis of N. gonorrhoeae strain 1291-R6 LOS by mass spectroscopy showed that the LOS oligosaccharide structure is GlcNAc-->Hep2phosphoethanolamine-->2-keto-3-deoxymannooctuloson ic acid (where Hep is heptose). Sequence analysis showed that the transposon is inserted into the 3' end of a gene that has homology to the human phosphoglucomutase (PGM) gene. Sequence comparison indicated that the putative PGM gene in N. gonorrhoeae 1291 and N. meningitidis NMB had 92% identity at the DNA level. PGM and glucokinase activity was present in cell free extracts of N. meningitidis NMB and N. gonorrhoeae strain 1291. N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 had no detectable PGM activity, whereas glucokinase activity was similar to the wild type strains. PGM activity can be reconstituted in N. meningitidis strain NMB-R6 by transformation with the cloned PGM gene. SDS-polyacrylamide gel electrophoresis demonstrated that NMB-R6 transformed with the PGM gene expressed the 3F11+, 4.5-kDa LOS of the parent NMB strain. The inability of N. meningitidis NMB-R6 and N. gonorrhoeae strain 1291-R6 to convert glucose 6-phosphate to glucose 1-phosphate results in the truncated LOS phenotype expressed by these mutants.