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Haemophilus influenzae incorporates choline obtained from environmental sources onto its lipopolysaccharide as phosphorylcholine (ChoP). The decoration of the bacterial surface with ChoP contributes to pathogenesis by allowing for mimicry of the host. As the main reservoir for choline in the host is phosphatidylcholine, we tested whether other choline-containing molecules associated with eukaryotic membranes could provide an alternative source of choline. H. influenzae was able to use glycerophosphorylcholine (GPC), an abundant degradation product of phospholipids, as efficiently as free choline. Utilization of GPC required glpQ, which expresses an enzyme with glycerophosphodiester phosphodiesterase activity. In the absence of free choline, this gene was required for adherent H. influenzae to obtain choline directly from epithelial cells in culture. GlpQ therefore allows choline to be transferred from the host to the bacterial cell surface.

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca2+ chelator BAPTA-AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galalpha1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galalpha1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occurring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

A number of pathogens of the upper respiratory tract express an unusual prokaryotic structure, phosphorylcholine (ChoP), on their cell surface. We tested the hypothesis that ChoP, also found on host membrane lipids in the form of phosphatidylcholine, acts so as to decrease killing by antimicrobial peptides that target differences between bacterial and host membranes. In Haemophilus influenzae, ChoP is a phase-variable structure on the oligosaccharide portion of the lipopolysaccharide (LPS). There was a bactericidal effect of the peptide LL-37/hCAP18 on a nontypeable H. influenzae strain, with an increasing selection for the ChoP(+) phase as the concentration of the peptide was raised from 0 to 10 microgram/ml. Moreover, constitutive ChoP-expressing mutants of unrelated strains showed up to 1,000-fold-greater survival compared to mutants without ChoP. The effect of ChoP on resistance to killing by LL-37/hCAP18 was dependent on the salt concentration and was observed only when bacteria were grown in the presence of environmental choline, a requirement for the expression of ChoP on the LPS. Further studies established that there is transcription of the LL-37/hCAP18 gene on the epithelial surface of the human nasopharynx in situ and inducible transcription in epithelial cells derived from the upper airway. The presence of highly variable amounts of LL-37/hCAP18 in normal nasal secretions (<1.2 to >80 microgram/ml) was demonstrated with an antibody against this peptide. It was concluded that ChoP alters the bacterial cell surface so as mimic host membrane lipids and decrease killing by LL-37/hCAP18, an antimicrobial peptide that may be expressed on the mucosal surface of the nasopharynx in bactericidal concentrations.

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.

Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non-typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of licl and lic2, which contain translational switches based on intragenic tandem repeats of 5'-CAAT-3'. Genetic analysis showed that opaque organisms have an out-of-frame number of repeats in both licl, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galalpha1-4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C-reactive protein (CRP). The addition of ChoP by licl was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galalpha1-4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host-like antigen blocks antibody-mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galalpha1-4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.

Haemophilus influenzae undergoes phase variation in expression of the phosphorylcholine (ChoP) epitope, a structure present on several invasive pathogens residing in the human respiratory tract. In this study, structural analysis comparing organisms with and without this epitope confirmed that variants differ in the presence of ChoP on the cell surface-exposed outer core of the lipopolysaccharide. During nasopharyngeal carriage in infant rats, there was a gradual selection for H. influenzae variants that express ChoP. In addition, genotypic analysis of the molecular switch that controls phase variation predicted that the ChoP+ phenotype was predominant in H. influenzae in human respiratory tract secretions. However, ChoP+ variants of nontypable H. influenzae were more sensitive to the bactericidal activity of human serum unrelated to the presence of naturally acquired antibody to ChoP. Serum bactericidal activity required the binding of C-reactive protein (CRP) with subsequent activation of complement through the classical pathway. Results of this study suggested that the ability of H. influenzae to vary expression of this unusual bacterial structure may correlate with its ability both to persist on the mucosal surface (ChoP+ phenotype) and to cause invasive infection by evading innate immunity mediated by CRP (ChoP- phenotype).

Choline, although not a nutritional requirement for Haemophilus influenzae, is taken up from the growth medium and incorporated into its lipopolysaccharide (LPS). Incorporated choline is in the form of phosphorylcholine (ChoP) based on the reactivity with the monoclonal antibody with specificity for this structure, TEPC-15. Incorporation of [3H]choline from the growth medium and expression of the TEPC-15 epitope undergo high-frequency phase variation, characteristic of other LPS structures in this species. The expression and phase variation of ChoP require a previously identified locus involved in LPS biosynthesis, lic1. The first gene in lic1, licA, contains a translational switch based on variation in the number of intragenic tandem repeats of the sequence 5'-CAAT-3'. The full-length LicA polypeptide resembles choline kinases of eucaryotes, suggesting that the pathway for choline incorporation into the H. influenzae glycolipid has similarities to the pathway for choline incorporation in eucaryotic lipid synthesis. The display of ChoP, a host-like structure, renders the organism more rather than less susceptible to the bactericidal activity of human serum. The increased serum sensitivity of variants with ChoP correlates with higher serum immunoglobulin G titers to LPS containing this structure. ChoP appears to be a cell surface feature common to a number of pathogens of the human respiratory tract, including Streptococcus pneumoniae and mycoplasmas. In the case of H. influenzae, its primary contribution to pathogenesis does not appear to be antigenic variation to evade host humoral clearance mechanisms.

Haemophilus influenzae undergoes spontaneous phase variation in colony morphology. Organisms from transparent colonies efficiently colonize the nasopharynx in an infant rat model of H. influenzae carriage, whereas organisms from more opaque colonies are deficient at colonization. A genetic approach relying on the transformability of H. influenzae was used to identify a locus contributing to opacity variation. By screening a library of chomosomal DNA from an opaque variant of strain Rd, it was possible to isolate a single clone capable of transforming a transparent Rd host to a more opaque phenotype. A region containing two genes, designated oapA and oapB, was identified. The deduced amino acid sequence of oapB has similarity to a consensus sequence for bacterial lipoproteins. Genetically defined mutations in oapA were transformed into the transparent Rd to confirm that this gene is required for expression of the transparent colony phenotype. Although oapA lacks a signal sequence, gene fusions to phoA show that OapA is secreted in H. influenzae and undergoes phase variation in expression. Mutagenesis of oapA in strain Rd, and type b strain Eagan, resulted in loss of the ability to colonize the nasopharynx of infant rats. The type b mutant, however, was as virulent as its parent strain when inoculated intraperitoneally. This suggests that the contribution of OapA to pathogenesis is limited to events associated with colonization of the mucosal surface.

Colonies of Haemophilus influenzae are heterogeneous in appearance because of phase variation in opacity. The only cell surface component found to have structural variation correlating with differences in opacity was the lipopolysaccharide (LPS). Changes in LPS structure, seen as migration patterns on tricine-SDS-PAGE, appeared to be independent of a previously described mechanism for generating LPS phase variation. The more transparent variants expressing a higher-molecular-weight LPS were serum sensitive and could efficiently colonize the infant rat nasopharynx after intranasal inoculation. In contrast, the fully opaque variant expressing a smaller-molecular-weight LPS was serum resistant, unable to colonize the nasopharynx, and more virulent when intraperitoneally administered. Organisms disseminating into the blood-stream from the nasopharynx changed phenotype from transparent to opaque. These findings demonstrate the potential importance of LPS structures that determine opacity in pathogenesis and colonization of mucosal surfaces.

The structure of the saccharide part of the lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+) has been investigated. The saccharide was obtained from the lipopolysaccharide by mild acid hydrolysis followed by high-performance anion-exchange chromatography, and isolated fractions were studied by methylation analysis, NMR spectroscopy, and FAB mass spectrometry. The major saccharide is a heptasaccharide with the following structure, [formula: see text] in which Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid and PEA is 2-aminoethyl phosphate. Hep is identified as L-glycero-D-manno-heptose. The absolute configuration of the phosphorylated heptose is tentative only.

Genes necessary for the expression and phase variation of lipopolysaccharide epitopes of a virulent Haemophilus influenzae type b isolate (RM.7004) are contained within two chromosomal loci designated lic-1 and lic-2. Mutations were introduced into both lic-1 and lic-2, and the virulence of the double mutant was compared with that of the wild type in infant rats. These mutations in RM.7004 resulted in a significantly reduced incidence of bacteremia following intranasal inoculation, although nasopharyngeal colonization was similar for the mutant and wild-type strains. In contrast, no differences in bacteremia were observed when the mutant and wild-type strains were inoculated intraperitoneally.

Phase variation of lipopolysaccharide epitopes of an Haemophilus influenzae serotype b strain (strain RM.7004) occurs through a mechanism which depends on multiple tandem repeats of the DNA sequence 5'-CAAT-3' situated within the chromosomal locus lic1. We report here that the same tetranucleotide repeats are also found in two other genomic loci (lic2 and lic3) of RM.7004. Similar to lic1, there are multiple tandem repeats of 5'-CAAT-3' present at the 5' ends of long open reading frames in lic2 and lic3. Variation in the number of repeats of CAAT, by shifting the upstream initiation codons in or out of phase with the remainder of the open reading frame, could switch on or off the translation of downstream genes. Similar to previously reported findings for lic1, site-directed mutations in the open reading frame downstream (3') from the repeats of CAAT in lic2 abolished phase variation and identified DNA sequences required for the expression of additional oligosaccharide epitopes. When we used an oligonucleotide comprising five repeats of CAAT or DNA sequences specific for lic1, lic2, and lic3 as probes, a survey of other encapsulated H. influenzae strains (serotypes a through f) and nontypable H. influenzae strains (including biotype aegyptius) showed that the chromosome of H. influenzae can have from two to five regions which contain multiple tandem repeats of CAAT in addition to other sequences which hybridize to lic1 and lic2.

Multiple carbohydrate structures on the outer-membrane lipopolysaccharide (LPS) of the gram-negative pathogen H. influenzae undergo high frequency, reversible loss, indicative of phase variation. Characterization of a genetic locus, lic-1, responsible for expression of two LPS epitopes displaying phase variation, showed it to comprise four genes. The first gene mediates phase variation. At its 5' end, within the open reading frame, are a variable number of tandem repeats of the tetramer CAAT. By shifting upstream initiation codons in or out of frame, these 4 bp units create a translational switch. The phenotype of organisms corresponds to the number of 4 bp units. Phase variation between three levels of expression ( +, +, and -) of lic-1-derived epitopes is caused by differences in the three phases of translation of the 5' terminus of this gene. Phase variation also allows for selection of organisms displaying certain LPS epitopes in vivo.

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is able to display an extensive repertoire of different LPS structures through the loss and acquisition of multiple oligosaccharide epitopes in various combinations. This marked heterogeneity of LPS molecules has complicated the analysis of the structure of LPS and its role in pathogenesis. A genomic library was screened for the ability to transform H. influenzae to express novel LPS epitopes defined by reactivity with oligosaccharide specific monoclonal antibodies. A chromosomal locus, lic-1, involved in expression of at least three different epitopes (recognized by monoclonal antibodies 4C4, 12D9, and 6A2), was identified on a 5.6-kilobase restriction endonuclease fragment. Transformation of H. influenzae with subclones from within lic-1 was used to generate a series of isogenic and phenotypic variants. All transformants displayed phase variation for their newly acquired epitopes. Altered binding specificities of LPS with monoclonal antibodies correlated with changes in sugar compositional analysis. The expression of two epitopes was eliminated by introduction of site-specific mutations in lic-1, confirming the role of lic-1 in oligosaccharide biosynthesis.