Phase variation of Opa proteins of Neisseria meningitidis and the effects of bacterial transformation.

Opa proteins are major proteins involved in meningococcal colonization of the nasopharynx and immune interactions. Opa proteins undergo phase variation (PV) due to the presence of the 5'-CTCTT-3' coding repeat (CR) sequence. The dynamics of PV of meningococcal Opa proteins is unknown. Opa PV, including the effect of transformation on PV, was assessed using a panel of Opa-deficient strains of Neisseria meningitidis. Analysis of Opa expression from UK disease-causing isolates was undertaken. Different opa genes demonstrated variable rates of PV, between 6.4 × 10(-4) and 6.9 × 10(-3) per cell per generation. opa genes with a longer CR tract had a higher rate of PV (r(2) = 0.77, p = 0.1212). Bacterial transformation resulted in a 180-fold increase in PV rate. The majority of opa genes in UK disease isolates (315/463, 68.0%) were in the 'on' phase, suggesting the importance of Opa proteins during invasive disease. These data provide valuable information for the first time regarding meningococcal Opa PV. The presence of Opa PV in meningococcal populations and high expression of Opa among invasive strains likely indicates the importance of this protein in bacterial colonization in the human nasopharynx. These findings have potential implications for development of vaccines derived from meningococcal outer membranes.

Adjuvant effects elicited by novel oligosaccharide variants of detoxified meningococcal lipopolysaccharides on Neisseria meningitidis recombinant PorA protein: a comparison in mice.

Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.

Construction of Opa-positive and Opa-negative strains of Neisseria meningitidis to evaluate a novel meningococcal vaccine.

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5-10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4(+) T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp, and that minor sequence differences are permissible. Anti-Opa bactericidal antibody responses following immunisation of mice with recombinant Opa were specific to the Opa variant used in immunisation. No immunomodulatory effects were observed when Opa was contained within meningococcal outer membrane vesicles (OMVs), compared to Opa-negative OMVs. These observations support the incorporation of Opa in meningococcal vaccines.

Immune inhibitory ligand CD200 induction by TLRs and NLRs limits macrophage activation to protect the host from meningococcal septicemia.

Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation.

Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide.

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either l-glycero-d-manno-heptose (ld-Hep) or d-glycero-d-manno-heptose (dd-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode dd-heptosyl- and ld-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.

Sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in Haemophilus influenzae and its effect on virulence.

BACKGROUND: Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence. RESULTS: Using qRT-PCR, we observed decreased transcriptional activity of genes within a cluster that are required for uptake and catabolism of 5-acetyl neuraminic acid (Neu5Ac), when bacteria were cultured in the presence of the sugar. We show that these uptake and catabolic genes, including a sialic acid regulatory gene (siaR), are highly conserved in the H. influenzae natural population. Mutant strains were constructed for seven of the nine genes and their influence upon LPS sialylation and resistance of the bacteria to the killing effect of normal human serum were assessed. Mutations in the Neu5Ac uptake (TRAP transporter) genes decreased virulence in the chinchilla model of otitis media, but the attenuation was strain dependent. In contrast, mutations in catabolism genes and genes regulating sialic acid metabolism (siaR and crp) did not attenuate virulence. CONCLUSION: The commensal and pathogenic behaviour of H. influenzae involves LPS sialylation that can be influenced by a complex regulatory interplay of sialometabolism genes.

Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal.

Typhoid fever is a major public health problem in developing countries, conservatively estimated to occur in 17 million cases and be responsible for 200,000 deaths annually. We investigated the acquisition of natural immunity to Salmonella enterica serovar Typhi in a region where typhoid is endemic by testing sera from an age-stratified sample of 210 healthy participants in Kathmandu, Nepal, for bactericidal activity toward S. Typhi and for anti-Vi capsular polysaccharide antibodies. Bactericidal titers in children were significantly lower than those in newborns and adults (P < 0.0001). Anti-S. Typhi bactericidal geometric mean titers were age dependent, increasing 10-fold during childhood. Anti-Vi polysaccharide antibody geometric mean concentrations were also lower in children than in adults. Data presented here indicate the possibility of a relationship between low levels of bactericidal activity toward S. Typhi in serum and susceptibility to disease, as observed for other polysaccharide-encapsulated bacteria. Bactericidal antibody may be a marker of protective immunity against S. Typhi.

The macrophage scavenger receptor A is host-protective in experimental meningococcal septicaemia.

Macrophage Scavenger Receptor A (SR-A) is a major non-opsonic receptor for Neisseria meningitidis on mononuclear phagocytes in vitro, and the surface proteins NMB0278, NMB0667, and NMB1220 have been identified as ligands for SR-A. In this study we ascertain the in vivo role of SR-A in the recognition of N. meningitidis MC58 (serogroup B) in a murine model of meningococcal septicaemia. We infected wild-type and SR-A(-/-) animals intraperitoneally with N. meningitidis MC58 and monitored their health over a period of 50 hours. We also determined the levels of bacteraemia in the blood and spleen, and measured levels of the pro-inflammatory cytokine interleukin-6 (IL-6). The health of SR-A(-/-) animals deteriorated more rapidly, and they showed a 33% reduction in survival compared to wild-type animals. SR-A(-/-) animals consistently exhibited higher levels of bacteraemia and increased levels of IL-6, compared to wild-type animals. Subsequently, we constructed a bacterial mutant (MC58-278-1220) lacking two of the SR-A ligands, NMB0278 and NMB1220. Mutation of NMB0667 proved to be lethal. When mice were infected with the mutant bacteria MC58-278-1220, no significant differences could be observed in the health, survival, bacteraemia, and cytokine production between wild-type and SR-A(-/-) animals. Overall, mutant bacteria appeared to cause less severe symptoms of septicaemia, and a competitive index assay showed that higher levels of wild-type bacteria were recovered when animals were infected with a 1ratio1 ratio of wild-type MC58 and mutant MC58-278-1220 bacteria. These data represent the first report of the protective role of SR-A, a macrophage-restricted, non-opsonic receptor, in meningococcal septicaemia in vivo, and the importance of the recognition of bacterial protein ligands, rather than lipopolysaccharide.

Naturally-occurring human serum antibodies to inner core lipopolysaccharide epitopes of Neisseria meningitidis protect against invasive meningococcal disease caused by isolates displaying homologous inner core structures.

Sera from healthy infants (under 1 year old), toddlers (3-4 years) and adults (18-65 years) were assayed for their ability to bind to inner core (ic) lipopolysaccharide (LPS) epitopes of Neisseria meningitidis. Antibodies (Abs) reacting to inner core structures, including different substitutions of the first heptose (HepI) and second heptose (HepII) residues of the LPS backbone, truncated and fully extended LPS glycoforms, were detected and for each structure, these inner core antibodies showed an age-related pattern of acquisition. A novel column-based methodology was used to affinity purify IgG antibodies in which purified inner core LPS (derived from a mutant MC58) was covalently linked to Sepharose 4B. Comparison of reactivity before and after affinity purification of the pooled sera showed that the purified Abs bound to the surface of N. meningitidis organisms displaying truncated and extended LPS with a homologous inner core region, promoted the deposition of C3b, were opsonophagocytic in vitro and decreased bacteraemia when used to passively protect infants rats. In addition, the purified Abs were bactericidal in vitro against the mutant strain displaying truncated LPS with a homologous inner core region. These results demonstrate that naturally occurring serum human antibodies to N. meningitidis LPS can access inner core epitopes of encapsulated organisms with a fully extended LPS.

Neisseria meningitidis escape from the bactericidal activity of a monoclonal antibody is mediated by phase variation of lgtG and enhanced by a mutator phenotype.

Bacteria adapt to environmental changes through high-frequency switches in expression of specific phenotypes. Localized hypermutation mediated by simple sequence repeats is an important mechanism of such phase variation (PV) in Neisseria meningitidis. Loss or gain of nucleotides in a poly(C) tract located in the reading frame results in switches in expression of lgtG and determines whether a glucose or a phosphoethanolamine (PEtn) is added at a specific position in the inner core lipopolysaccharide (LPS). Monoclonal antibody (MAb) B5 is bactericidal for N. meningitidis strain 8047 when PEtn is present in the inner core LPS and lgtG is switched "off." Escape from the bactericidal activity of this antibody was examined by subjecting strain 8047 to multiple cycles of growth in the presence of MAb B5 and human serum. Escape variants with alterations in the lgtG repeat tract rapidly accumulated in bacterial populations during selection with this antibody. Strain 8047 was outcompeted in this assay by the 8047 Delta mutS strain due to the elevated PV rate of this mismatch repair mutant and hence the greater proportion of preexisting phase variants of lgtG in the inoculum. This mutS mutant was also more virulent than strain 8047 during escape from passive protection by MAb B5 in an in vivo infant rat model of bacteremia. These results provide an example of how PV rates can modulate the occurrence and severity of infection and have important implications for understanding the evolution of bacterial fitness in species subject to environmental variations that occur during persistence within and transmission between hosts.

Duplicate copies of lic1 direct the addition of multiple phosphocholine residues in the lipopolysaccharide of Haemophilus influenzae.

The genes of the lic1 operon (lic1A to lic1D) are responsible for incorporation of phosphocholine (PCho) into the lipopolysaccharide (LPS) of Haemophilus influenzae. PCho plays a multifaceted role in the commensal and pathogenic lifestyles of a range of mucosal pathogens, including H. influenzae. Structural studies of the LPS of nontypeable H. influenzae (NTHI) have revealed that PCho can be linked to a hexose on any one of the oligosaccharide chain extensions from the conserved inner core triheptosyl backbone. In a collection of NTHI strains we found several strains in which there were two distinct but variant lic1D DNA sequences, genes predicted to encode the transferase responsible for directing the addition of PCho to LPS. The same isolates were also found to express concomitantly two PCho residues at distinct positions in their LPS. In one such NTHI isolate, isolate 1158, structural analysis of LPS from lic1 mutants confirmed that each of the two copies of lic1D directs the addition of PCho to a distinct location on the LPS. One position for PCho addition is a novel heptose, which is part of the oligosaccharide extension from the proximal heptose of the LPS inner core. Modification of the LPS by addition of two PCho residues resulted in increased binding of C-reactive protein and had consequential effects on the resistance of the organism to the killing effects of normal human serum compared to the effects of glycoforms containing one or no PCho. When bound, C-reactive protein leads to complement-mediated killing, indicating the potential biological significance of multiple PCho residues.

Identification of the functional initiation codons of a phase-variable gene of Haemophilus influenzae, lic2A, with the potential for differential expression.

Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5' CAAT repeat tract is preceded by four 5' ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.

Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: incorporation of disialic acid.

The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an alpha-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., Månsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both alpha-2,3- and alpha-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.

Identification of Neisseria meningitidis nonlipopolysaccharide ligands for class A macrophage scavenger receptor by using a novel assay.

Macrophages (Mphi) may play an important role in the pathogenesis of invasive meningococcal infection. Previously, we have shown that the class A Mphi scavenger receptor (SR-A) is a major nonopsonic receptor for Neisseria meningitidis on Mphi. SR-A contributes to host defense by binding proinflammatory polyanionic ligands such as lipopolysaccharide (LPS) and by the uptake and killing of live organisms. SR-A-deficient mouse Mphi display a substantial reduction in the number of meningococci ingested compared to wild-type Mphi, and SR-A is required for meningococcal phagocytosis but not for the release of tumor necrosis factor alpha. Although soluble lipid A and lipid(IV)A are reported as ligands for SR-A, we demonstrated that LPS and LPS expression were not essential for the uptake of whole meningococci. In the present study, we set out to discover protein ligand(s) for SR-A in N. meningitidis lysates and outer membrane vesicles. Using various microbial mutant strains, we determined that molecules comprising the membrane capsule and pili, as well as the abundant surface Opa proteins were not essential for SR-A recognition. We developed a binding assay to detect SR-A ligands and identified three candidate proteins expressed on intact organisms, namely, NMB1220, NMB0278, and NMB0667. Soluble forms of these ligands were shown to block the binding of meningococci to CHO cells stably transfected with SR-A. Furthermore, NMB1220 was endocytosed by SR-A on Mphi and prevented internalization of soluble acetylated low-density lipoprotein. Thus, we have identified novel, unmodified protein ligands for SR-A that are able to inhibit meningococcal interactions with macrophages in vitro.

Digalactoside expression in the lipopolysaccharide of Haemophilus influenzae and its role in intravascular survival.

Digalactoside (galalpha-1-4 galbeta) structures of the lipopolysaccharide (LPS) of Haemophilus influenzae are implicated in virulence. A confounding factor is that tetranucleotide repeats within the lic2A, lgtC, and lex2 genes mediate phase-variable expression of the digalactosides. By deleting these repeats, we constructed recombinant strains of RM153 constitutively expressing either one or two LPS digalactosides. Expression of two digalactosides, rather than one, was associated with increased virulence of H. influenzae in vivo.

Elucidation of the monoclonal antibody 5G8-reactive, virulence-associated lipopolysaccharide epitope of Haemophilus influenzae and its role in bacterial resistance to complement-mediated killing.

The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-alpha1,4-Gal-beta1,4-Glc-beta1,4-Glc-beta attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

Microsatellite instability regulates transcription factor binding and gene expression.

Microsatellites are tandemly repeated simple sequence DNA motifs widely prevalent in eukaryotic and prokaryotic genomes. In pathogenic bacteria, instability of these hypermutable loci through slipped-strand mispairing mediates the high-frequency reversible switching of phenotype expression, i.e., phase variation. Phase-variable expression of NadA, an outer membrane protein and adhesin of the pathogen Neisseria meningitidis, is mediated by changes in the number of TAAA repeats located upstream of the core promoter of nadA. Here we report that loss or gain of TAAA repeats affects the binding of the transcriptional regulatory protein IHF to the nadA promoter. Thus, phase-variable transcription of nadA potentially incorporates interplay between stochastic (mutational) and prescriptive (classical) mechanisms of gene regulation.

Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis.

It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.

lpt6, a gene required for addition of phosphoethanolamine to inner-core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae.

We previously described a gene, lpt3, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the beta-chain heptose (HepII) of the inner-core Neisseria meningitidis lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene, lpt6 (NMA0408), that is required for the addition of PEtn-6 on HepII. The lpt6 gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the lpt3 and lpt6 genes and showed that 36% contained both genes, while 50% possessed lpt3 only and 12% possessed lpt6 only. The translated amino acid sequence of lpt6 has a homologue (72.5% similarity) in a product of the Haemophilus influenzae Rd genome sequence. Previous structural studies have shown that all H. influenzae strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated H. influenzae strains, the lpt6 homologue was invariably present. Structural analysis of LPS from H. influenzae and N. meningitidis strains where lpt6 had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the N. meningitidis and H. influenzae lpt6 genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.

Three genes, lgtF, lic2C and lpsA, have a primary role in determining the pattern of oligosaccharide extension from the inner core of Haemophilus influenzae LPS.

Lipopolysaccharide (LPS) is a virulence determinant of Haemophilus influenzae and exhibits substantial heterogeneity in structure within and between strains. Key factors contributing to this heterogeneity are the genes required to add the first glycose to each of the three heptose residues of the LPS inner core. In each case this addition can facilitate further oligosaccharide extension. lgtF is invariably present in strains and the product has a function in adding the glucose to the first heptose. lic2C is present in half the strains and was found to add a glucose to the second heptose. Insertion of lic2C into a strain that does not naturally contain it resulted in hexose incorporation from the second heptose of the LPS. The product of the lpsA gene can add a glucose or galactose to the third heptose. By allelic replacement of lpsA between strains it is shown that the sequence of the gene can be the sole determinant of this specificity. Thus, lgtF, lic2C and lpsA make significant but very distinct contributions to the conservation and variable patterns of oligosaccharide extensions seen in H. influenzae LPS.