Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.     

Natural Phosphoryl and Acyl Variants of Lipid A from Neisseria meningitidis Strain 89I Differentially Induce Tumor Necrosis Factor-�� in Human Monocytes

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-α by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-α induction varied by ∼2–10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.The lipid A (LA)² portion of the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) of Gram-negative bacteria is an inflammatory, pathogenic component of the bacterial outer membrane (1–3). Our interests lie in the Neisserial LA as it is implicated as a significant contributor to the pathogenesis of infections due to Neisseria meningitidis and Neisseria gonorrhoeae, which are of major public health concern around the world. In particular, N. meningitidis is the leading cause of epidemic meningitis and fatal sepsis in otherwise healthy individuals (4). On average more than 500,000 cases of meningococcal infection occur annually leading to ∼50,000 deaths, and large epidemic outbreaks can cause periodic spikes in occurrence. N. gonorrhoeae is a major cause of sexually transmitted infections, which can lead to pelvic inflammatory disease in 10–20% of infected women who can suffer from chronic pain, infertility, and ectopic pregnancy as a result (5). In addition, a growing number of studies have shown that gonococcal infection can facilitate the transmission of HIV (6).Numerous studies of LA signaling through the toll-like receptor 4 (TLR4) have increased our knowledge of the relationship between the LA structure and its inflammatory and immunogenic activity. The affinity of LA for monomeric binding to MD-2, which is a critical determinant in the agonist activity of LA for TLR4, has been found to be most potent in the hexaacylated compared with penta- or tetraacylated forms (7, 8). More recently, we and others have shown that triggering receptor expressed on myeloid cells-2 binds LOS and LPS in both myeloid and non-myeloid cells and initiates an inflammatory cytokine response (9, 10).Neisserial LOS lacks the repeating O-antigens of the LPS of the Gram-negative enteric bacteria, and differs also in the position, number, and chain length of the acyl groups on the LA. There can be differences in the acyl groups on the LA moieties within individual strains as well as between strains and species of Gram-negative bacteria (11, 12). The tetraacylated lipid IVa, which is a precursor of Escherichia coli LA has been found to be a TLR4 antagonist in human cells (13).In both LPS and LOS the number of phosphate (P) and phosphoethanolamine (PEA) groups on LA can vary (14), which impacts on the bioactivity of the molecule for innate immune responses. Recent work has shown that whereas hexaacyl monophosphoryl LA was restricted to the myeloid differentiation factor 88 (MyD88)-independent pathway, which resulted in T cell activation, the diphosphoryl LA also engaged the MyD88-dependent pathway, which activated NF-κB resulting in the production of TNF-α and other inflammatory cytokines (15). Because of its immunogenic properties, monophosphoryl LA has been approved for use as an adjuvant in a hepatitis B vaccine in Europe (16).We have observed major differences in the induction of proinflammatory cytokines through TLR4-mediated signaling elicited by various LOS purified from meningococcal and gonococcal strains (17). Little is known about the extent and biological significance of the natural structural variation in the LA of LOS occurring within a Neisserial strain. Recently, we reported that structural analyses of native LOS from different Neisserial strains indicated that differences in both acylation and phosphorylation of LA correlated significantly with the potency of LA to induce inflammatory cytokines (18). In this study, we used thin-layer chromatography (TLC) and matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) to investigate the heterogeneity and inflammatory activity of the structural variants of the LA from N. meningitidis strain 89I, the LOS of which we found previously to be the most potent inducer of TNF-α among a group of seven Neisserial strains studied (17).     

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.     

Phase-variation of the truncated lipo-oligosaccharide of Neisseria meningitidis NMB phosphoglucomutase isogenic mutant NMB-R6

The detection of antibodies specific to meningococcal lipo-oligosaccharides (LOSs; outer-core-->inner-core-->lipid A) in sera of patients convalescent from meningococcal infection suggests the potential use of LOS as a vaccine to combat pathogenic Neisseria spp. Removal of the outer-core region, which expresses glycans homologous to human blood-group antigens, is a required first-step in order to avoid undesirable immunological reactions following vaccination. To this end, we describe here the structural makeup of the LOS produced by serogroup B N. meningitidis NMB isogenic phosphoglucomutase (Pgm) mutant (NMB-R6). The dominant LOS types produced by NMB-R6 expressed a deep-truncated inner-core region, GlcNAc-(1-->2)-LDHepII-(1-->3)-LDHepI-(1-->5)-[Kdo-2-->4]-Kdo-->lipid A, with one PEA unit attached at either O-6 or O-7 of LDHepII, or with two simultaneously PEA moieties attached at O-3 and O-6 or O-3 and O-7 of the same unit. Unexpectedly, this mutation did not completely deactivate the production of Glc, as some LOS molecules were observed to carry Glc at O-4 of LDHepI and at O-3 of LDHepII. A glycoconjugate vaccine comprised of NMB-R6 LOSs is currently being evaluated in our laboratory.     

Detection of the lst gene in different serogroups and LOS immunotypes of Neisseria meningitidis

The sialylation of the lipooligosaccharide (LOS) of Neisseria meningitidis is mediated by the LOS sialyltransferase enzyme encoded by the lst gene. PCR using four sets of primers that targeted to different regions of the lst gene was used to survey the distribution of lst in different serogroups and LOS immunotypes of N. meningitidis as well as other Neisseria species. While the lst gene was found in N. meningitidis strains regardless of their capsular serogroup and LOS structures, the gene is also found in N. gonorrhoeae, N. lactamica, N. polysaccharea, and N. subflava biovar subflava. The presence of the lst gene in these organisms and the sialylation of their LOS antigens were discussed.     

Characterization of the alpha-1,2-N-acetylglucosaminyltransferase of Neisseria gonorrhoeae, a key control point in lipooligosaccharide biosynthesis

The biosynthesis of the lipooligosaccharide (LOS) in Neisseria meningitidis has a control point that regulates the extension of the alpha-chain on heptose (I) of the LOS. The gene that encodes the protein responsible for this control had been identified elsewhere, but the enzyme encoded by the gene was not characterized. We have now shown that this same control mechanism operates in the related species, Neisseria gonorrhoeae, using a gene knockout and subsequent characterization of the LOS species produced. We also cloned and expressed the enzyme from both of these pathogens. Using a synthetic acceptor substrate, we have shown unequivocally that the enzyme is an alpha-1,2-N-acetylglucosaminyltransferase. Experiments with both the core oligosaccharide and the synthetic acceptors suggests that the addition of the alpha-1,2-N-acetylglucosamine moiety on the heptose (II) residue precedes the addition of the ethanolamine phosphate at the O3 position on this heptose (II), and that in the absence of the alpha-1,2-N-acetylglucosamine moiety leads to the addition of an extra ethanolamine phosphate on the heptose (II) residue. Our data do not support the hypothesis that ethanolamine phosphate at O3 of heptose (II) is added and is then required for the addition of the N-acetylglucosamine at O2 by the LgtK enzyme. This enzyme represents a control point in the biosynthesis of the LOS of this pathogen and is a potential target for therapeutic intervention.     

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.     

Campylobacter jejuni Lipooligosaccharide Sialylation,Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.     

Deciphering the complex three-way interaction between the non-integrin laminin receptor,galectin-3 and Neisseria meningitidis

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C¹⁷³) of Gal-3 or lysine (K¹⁶⁶) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.     

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.     

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.     

Genetic analysis of conservation and variation of lipooligosaccharide expression in two L8-immunotype strains of Neisseria meningitidis

Neisseria meningitidis strains A1 and M978 both express the lipooligosaccharide (LOS) L8 immunotype [Gu et al., J. Clin. Microbiol. 30 (1992) 2047-2053]. Under different growth conditions, strain A1 did not change its LOS profile whereas strain M978 produced variable LOS profiles on SDS-PAGE. To understand the genetic basis of LOS conservation and variation, their lgt locus encoding glycosyltransferases responsible for the biosynthesis of the alpha-chain of LOS was analyzed. Strain A1 possessed only two genes, lgtA and lgtH, at the lgt locus. The lgtA gene was inactivated due to a frameshift mutation; thus strain A1 expressed only L8 LOS. In contrast, strain M978 contained five genes lgtZ, lgtC, lgtA, lgtB and lgtE at this locus, thus it had a potential to express L1, L3,7 in addition to the L8 LOS. The data showed that strain A1 is a better reference strain for the L8 immunotype because of the stability of L8 LOS expression resulting from its unique lgt locus. In addition, these two strains had two new genetic organizations, lgtAH and lgtZCABE, compared to the reported gene organization at the lgt locus in N. meningitidis.     

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.     

Gonococcal lipooligosaccharide is a ligand for the asialoglycoprotein receptor on human sperm

In the present study, we show that Neisseria gonorrhoeae lipooligosaccharide (LOS) can bind to the asialoglycoprotein receptor (ASGP-R) on human sperm. This work demonstrates the presence of ASGP-R on human sperm. Binding of purified ASGP-R ligand decreased in the presence of gonococci. Binding of purified iodinated gonococcal LOS identified a protein of molecular weight corresponding to that of human ASGP-R. The presence of excess unlabelled LOS blocked binding of iodinated gonococcal LOS. Binding of wild-type gonococcal LOS to sperm was higher than that of mutant LOS lacking the galactose ligand for ASGP-R. These data suggest that the ASGP-R on human sperm cells recognizes and binds wild-type gonococcal LOS. This interaction may contribute to the transmission of gonorrhea from infected males to their sexual partners.     

Population coverage analysis of T-Cell epitopes of Neisseria meningitidis serogroup B from Iron acquisition proteins for vaccine design

Although the concept of Reverse Vaccinology was first pioneered for sepsis and meningococcal meningitidis causing bacterium, Neisseria meningitides, no broadly effective vaccine against serogroup B meningococcal disease is yet available. In the present investigation, HLA distribution analysis was undertaken to select three most promiscuous T-cell epitopes out of ten computationally validated epitopes of Iron acquisition proteins from Neisseria MC58 by using the population coverage tool of Immune Epitope Database (IEDB). These epitopes have been determined on the basis of their binding ability with maximum number of HLA alleles along with highest population coverage rate values for all the geographical areas studied. The comparative population coverage analysis of moderately immunogenic and high immunogenic peptides suggests that the former may activate T-cell response in a fairly large proportion of people in most geographical areas, thus indicating their potential for development of epitope-based vaccine.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Immunologic and genetic characterization of lipooligosaccharide variants in a Neisseria meningitidis serogroup C strain

Neisseria meningitidis shows great variation in expression of structurally different lipooligosaccharides (LOS) on its cell surface. To better understand the LOS diversity that may occur within an individual strain, a group C wild-type strain, BB305-Tr4, and two stable isogenic LOS variants, Tr5 and Tr7, were selected for this study. SDS-PAGE analysis showed a size reduction of Tr5 and Tr7 LOS compared to that of Tr4. Immunoblotting showed that parental Tr4 LOS reacted with L1, L2 and L3,7 antibodies, variant Tr5 LOS with L1 and L6 antibodies, while Tr7 LOS was non-typeable. Genetic analysis showed that the gene organization at the lgt-1 locus in the three strains was lgtZ,C,A,B,H4 in Tr4, lgtZ,C,A,H4 in Tr5 and lgtZ,C,A,H9 in Tr7. The genetic differences in the three strains were consistent with their phenotypic changes. Sequence comparison revealed two independent recombination events. The first was the recombination of repeated DNA fragments in the flanking regions to delete lgtB in Tr5. The second was the recombination of a fragment of two genes, lgtB and lgtH4, to create an inactive lgtH9 allele with a mosaic structure in Tr7. These findings suggest that besides phase variation, homologous recombination can contribute to the genetic diversity of the lgt locus and to the generation of LOS variation in N. meningitidis.     

Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae

Neisserial lipooligosaccharide (LOS) contains three oligosaccharide chains, termed the alpha, beta, and gamma chains. We used Southern hybridization experiments on DNA isolated from various Neisseria spp. to determine if strains considered to be nonpathogenic possessed DNA sequences homologous with genes involved in the biosynthesis of these oligosaccharide chains. The presence or absence of specific genes was compared to the LOS profiles expressed by each strain, as characterized by their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and their reactivities with various LOS-specific monoclonal antibodies. A great deal of heterogeneity was seen with respect to the presence of genes encoding glycosyltransferases in Neisseria. All pathogenic species were found to possess DNA sequences homologous with the lgt gene cluster, a group of genes needed for the synthesis of the alpha chain. Some of these genes were also found to be present in strains considered to be nonpathogenic, such as Neisseria lactamica, N. subflava, and N. sicca. Some nonpathogenic Neisseria spp. were able to express high-molecular-mass LOS structures, even though they lacked the DNA sequences homologous with rfaF, a gene whose product must act before gonococcal and meningococcal LOS can be elongated. Using a PCR amplification strategy, in combination with DNA sequencing, we demonstrated that N. subflava 44 possessed lgtA, lgtB, and lgtE genes. The predicted amino acid sequence encoded by each of these genes suggested that they encoded functional proteins; however, structural analysis of LOS isolated from this strain indicated that the bulk of its LOS was not modified by these gene products. This suggests the existence of an additional regulatory mechanism that is responsible for the limited expression of these genes in this strain.