Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae.

The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b.

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.     

Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DL42, which constitutively expressed LOS reactive with MAb 5G8. This library was used to transform a second Hib strain (DL180) that normally does not express this LOS epitope, and a recombinant clone was identified that bound MAb 5G8. Subcloning of different regions of the Hib DL42 DNA insert in this recombinant plasmid determined that a 1.9-kb EcoRI fragment, designated lex-2, was responsible for transforming Hib strain DL180 to reactivity with MAb 5G8. Nucleotide sequence analysis revealed the presence of two contiguous open reading frames (ORFs) in lex-2, the first of which contained 18 tandem repeats of the nucleotide tetramer GCAA near its 5' end. Sequence analysis of PCR-derived products from MAb 5G8-reactive and -nonreactive Hib DL180 colonies established that 18 GCAA repeats were associated with expression of the LOS epitope that bound MAb 5G8. Mutational analysis determined that a functional ORF 2 was essential for expression of the MAb 5G8-reactive LOS epitope. The nucleotide tetramer GCAA repeat present in ORF 1 was also detected in at least two different chromosomal regions in all Hib strains tested. The availability of the cloned lex-2 locus should facilitate future analysis of the complex regulatory mechanisms involved in expression of LOS epitopes by this pathogen.     

Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703-3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the beta-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, and hgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.     

Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b.

The major outer membrane protein (P2) of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 37,000 to 40,000 has been previously shown to function as a porin and also as a target for antibodies protective against experimental Hib disease. The gene encoding the Hib P2 protein was cloned by using a shuttle vector capable of replication in both Escherichia coli and H. influenzae. The amino acid sequence of the amino terminus of the Hib P2 protein was determined and used to design an oligonucleotide probe corresponding to the first 20 amino acids of this protein. This oligonucleotide probe was used to identify Hib chromosomal DNA fragments containing the Hib P2 gene. These DNA fragments were ligated into the plasmid vector pGJB103 and then used to transform a rec-1 mutant of H. influenzae Rd. Recombinant clones expressing the Hib P2 protein were identified in a colony blot-radioimmunoassay by using a monoclonal antibody specific for a surface epitope of the Hib P2 protein. The gene encoding this Hib protein was present on a 10-kilobase Hib DNA insert in the recombinant plasmid. Transformation experiments involving the recombinant plasmid suggested that unregulated synthesis of Hib P2 is a lethal event in E. coli. The recombinant Hib P2 protein was exposed on the surface of the recombinant H. influenzae strain. This recombinant strain was used to develop a system for detecting polyclonal serum antibodies directed against surface determinants of the Hib P2 protein. The availability of the gene encoding the Hib P2 protein should facilitate investigation of both the immunogenicity and the structure-function relationship(s) of this major outer membrane protein.     

Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b.

Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.     

Characterization of a mutant of Haemophilus influenzae type b lacking the P2 major outer membrane protein.

An isogenic mutant of Haemophilus influenzae type b (Hib) lacking the ability to express the P2 major outer membrane (porin) protein was constructed and characterized in various model systems. Linker insertion mutagenesis of a cloned Hib DNA insert containing the P2 structural gene was used in conjunction with a genetic transformation system to obtain a transformant unreactive with a P2-specific monoclonal antibody. This transformant was shown to lack detectable P2 protein by both protein staining and immunoblot methods. The P2 mutant exhibited a generation time in complex broth medium that was significantly longer than that of the wild-type parent strain. The P2 mutant was also unable to produce detectable bacteremia in infant rats after intraperitoneal challenge, while the wild-type parent strain produced bacteremia in all animals challenged with this strain. Reintroduction of a wild-type copy of the P2 gene into this mutant yielded a transformant strain that had a generation time in vitro identical to that of the wild-type parent strain and that was also fully virulent in the infant rat model. These findings suggest that the ability to synthesize the P2 protein may be necessary but not sufficient for full expression of virulence by this pathogen.     

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.     

Expression of the heat-modifiable major outer membrane protein of Haemophilus influenzae type b is unrelated to virulence.

The heat-modifiable major outer membrane protein (P1) of Haemophilus influenzae type b (Hib) has been shown to be both exposed on the cell surface and capable of inducing the synthesis of antibodies protective against experimental Hib disease. Chemical mutagenesis of a recombinant plasmid containing the Hib gene encoding P1 resulted in inactivation of P1 expression by this plasmid. The mutated P1 gene was transformed into Hib to obtain an isogenic mutant lacking only the ability to synthesize this surface protein. In addition, the P1 gene was inserted into a plasmid shuttle vector and used to construct a recombinant Hib strain that overexpressed the P1 protein. Lack of P1 expression did not affect the ability of Hib to grow in vitro. Neither the absence nor the overproduction of P1 affected expression of capsular polysaccharide and lipooligosaccharide by Hib. The P1-negative mutant and the P1-overexpressing strain were both as susceptible to the bactericidal activity of pooled normal human serum as was the wild-type parent strain, while the P1-negative mutant was as resistant to the bactericidal activity of normal infant rat serum as was the wild-type parent strain. The P1-negative mutant was no less virulent than was the wild-type parent strain in an animal model system, such that both the numbers of animals infected by this mutant and the mean magnitudes of the resultant bacteremias were essentially identical to those obtained with challenge by the wild-type parent strain. Similarly, overexpression of P1 did not detectably affect the virulence of Hib. These data indicate that this protective protein antigen plays no detectable role in the expression of virulence by Hib, as assessed in an animal model system.     

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.     

Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection

To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The tonB inactivation was shown to make P. aeruginosa unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the tonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, the tonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant. These results indicate clearly that the intact tonB gene-and, therefore, the TonB protein encoded by it-is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P. aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P. aeruginosa to infect the animal host.     

Reactivity of Haemophilus influenzae type b anti-pili antibodies

The reactivity of anti-pilus antibodies to native and denatured Haemophilus influenzae b (Hib) pili was studied using rabbit serum prepared against piliated H. influenzae b strain M43 (p+) and adsorbed with its non-piliated variant, strain M42 (p-). The specificity of the adsorbed serum for Hib pili was documented by immunogold electron microscopy and by immunoprecipitation, which revealed the 24 kDa pilin band from strain M43 (p+) that was not seen on strain M42 (p-). In immunodot assays, the anti-pilus antibodies reacted with the native pili present on the outer membrane of strain M43 (p+), but on Western blot assay using denatured outer membranes, the anti-pilus antibodies did not react with the 24 kDa pilin subunit. These data demonstrate that the anti-pilus antibodies in the adsorbed serum recognize conformational epitopes that depend on the tertiary or quaternary structure of Hib pili.     

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.     

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

Utilization of transferrin-bound iron by Haemophilus influenzae requires an intact tonB gene.

Haemophilus influenzae can utilize iron-loaded human transferrin as an iron source for growth in vitro. H. influenzae tonB mutants, containing a chloramphenicol acetyltransferase gene within their tonB genes, could bind iron-charged human transferrin to their cell surfaces, but they were unable to utilize this serum glycoprotein as the sole source of iron for growth in vitro. In contrast, these tonB mutants were able to utilize an iron chelate (ferric ammonium citrate) for growth. Transformation of a tonB mutant with a plasmid encoding a wild-type H. influenzae tonB gene restored the ability of a tonB mutant to utilize iron-charged human transferrin. These results indicate that the uptake of iron from human transferrin by H. influenzae is a TonB-dependent process.     

Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates

In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.