Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media

Since Haemophilus influenzae lacks enzymes necessary for synthesis of the porphyrin ring, it has an absolute growth requirement for a porphyrin source. This requirement can be satisfied in vitro by hemoglobin and hemoglobin complexed to haptoglobin. The products of the hgp genes mediate the utilization of heme from hemoglobin-haptoglobin. These genes are also involved in the use of heme from hemoglobin, although additional gene products independently mediate the acquisition of heme from this substrate. Different strains of H. influenzae possess one to four hgp genes. A nontypeable H. influenzae mutant lacking all the hgp genes was constructed and compared to the wild-type strain in a chinchilla (Chinchilla lanigera) model of otitis media. Compared to the wild-type strain, the hgp-deficient mutant exhibited a significantly delayed onset of detectable middle ear infection and significantly reduced duration of infection as assessed by both video otoscopy and tympanometry and as evidenced by viable bacterial counts in middle ear effusions. In addition, the maximum bacterial load in the middle ears of chinchillas infected with the mutant strain was significantly reduced when compared to the parent. These data indicate that the hemoglobin/hemoglobin-haptoglobin binding proteins are required for bacterial proliferation during H. influenzae-induced otitis media in chinchillas.     

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.     

Fetal and neonatal transmission of type-b anti-haemophilus influenzae antibodies protecting the newborn rat

Haemophilus influenzae type b is the most common cause of bacterial meningitis in children. Intranasal or intraperitoneal inoculation of infant rats with Haemophilus influenzae type b results in bacteremia and meningitis and has been proved to be a reproductible model of the human disease. For these reasons, it was of interest to use rats as experimental model for the study of anti-Haemophilus influenzae type b vaccine. Immune serum against Haemophilus influenzae type b cell surface extracts was prepared on rabbit. In a first experiment, pregnant rats were passively immunized with either immune rabbit serum or normal rabbit serum or saline. In a second experiment, suckling rat mothers were passively immunized with either immune rabbit serum or normal rabbit serum or saline. All infant rats were inoculated intraperitoneally at 6 days of age with Haemophilus influenzae type b. Bacteremia was determined in all infected rats 24 h after challenge. Only infant rats from immune serum-treated mothers were protected. This protection may be transferred from mother to young rat either before of after the birth.     

Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b.

Chemical mutagenesis techniques and genetic transformation methods were used to construct isogenic mutants of Haemophilus influenzae type b (Hib) defective in the ability to synthesize lipooligosaccharide (LOS). A mutant (17B) which expressed a LOS molecule with an altered oligosaccharide was less virulent than the wild-type parent strain, as determined by measurement of the ability of these strains to produce bacteremia in infant rats after intranasal challenge. Further mutagenesis of this mutant strain yielded two new mutants with different LOS phenotypes. Mutant 7A was slightly sensitive to the bactericidal activity present in normal infant rat serum and was even less virulent than its immediate parent strain (17B) in the intranasal challenge model. However, both mutants 17B and 7A could produce bacteremia and meningitis when introduced into infant rats by the intraperitoneal route. The other LOS mutant (14A) derived from mutant 17B exhibited a level of virulence equivalent to that of the original wild-type strain. Genetic transformation of wild-type chromosomal DNA into the essentially avirulent mutant 7A and selection of transformants on the basis of their LOS antigenic characteristics resulted in the sequential restoration of full virulence to this mutant. These findings suggest that LOS is involved on at least two different levels in the ability of Hib to produce invasive disease in the infant rat model. Changes in LOS phenotype can independently affect the ability of Hib to produce bacteremia after intranasal challenge and the sensitivity of Hib to killing by normal infant rat serum. These results reinforce the significance of Hib LOS in the expression of virulence by this pathogen.     

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.     

Genetic and phenotypic variation in the capsulation and virulence of culture collection strains of Haemophilus influenzae type b

Haemophilus influenzae type b strains deposited in the National Reference Collections of the United States, United Kingdom and Sweden are derived from strains isolated in the United States during the 1940s. With respect to both the genetic locus for capsulation and to virulence, assessed in the infant rat bacteremia model, these strains are no longer representative of clinical isolates which currently cause meningitis and other serious infections in children all over the world. Alternative well-characterized strains resembling current common clinical isolates may often be more suitable for the study of Haemophilus influenzae pathogenicity.     

Role of adherence in the pathogenesis of Haemophilus influenzae type b infection in infant rats.

We evaluated the role of pili in the pathogenesis of disease due to Haemophilus influenzae type b (HiTb), using the infant rat model. Piliated and nonpiliated HiTb strains were isolated from the nasopharynx and cerebrospinal fluid, respectively, of three children. Infant rats inoculated intranasally with nonadherent HiTb developed bacteremia and meningitis more frequently (P = 0.005) than animals inoculated with companion adherent HiTb strains. When analyzed separately, only one HiTb pair (884/880) demonstrated significant differences in the incidence of bacteremia and meningitis between the adherent and nonadherent strains. Blood or cerebrospinal isolates recovered from infant rats inoculated with piliated adherent HiTb strains were not piliated and were not adherent in vitro. Adherent and nonadherent HiTb colonized the nasopharynx of infant rats equally. The piliated strains of HiTb were not adherent in vivo or in vitro to rat nasal or buccal epithelial cells, respectively. Piliated strains of HiTb have no apparent advantage over nonpiliated HiTb strains for colonization or invasion of infant rats. Furthermore, the loss of piliation is noted for cerebrospinal fluid, blood, and nasal isolates of HiTb cultured from infant rats inoculated with an adherent piliated HiTb strain. Thus, the loss or suppression of pili may be an important prerequisite for the invasion of the host by HiTb strains that are highly piliated.     

Role of lipooligosaccharide in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats.

Clonally related strains of Haemophilus influenzae biogroup aegyptius have recently been associated with Brazilian purpuric fever (BPF), a fulminant, systemic disease in children. Using an infant rat bacteremia model for BPF, we found that a rat blood-passaged BPF isolate of H. influenzae biogroup aegyptius was more virulent than the original strain was. When compared with the original strain, the animal-passaged variant was found to display an altered lipooligosaccharide (LOS) phenotype and to lack pili. To examine the role of LOS phenotype and pili in virulence, we isolated isogenic variants differing in LOS phenotype or expression of pili. The virulence of variants was compared by examining the results of blood cultures obtained 24 h after intraperitoneal inoculation with 10(5) CFU. Our results indicate that the LOS phenotype is a critical determinant of BPF clone virulence for infant rats. To a lesser extent, the absence of piliation and an undefined additional factor(s) contribute to virulence.     

The periplasmic disulfide oxidoreductase DsbA contributes to Haemophilus influenzae pathogenesis

Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and causes disease when it invades the bloodstream, lungs, or middle ear. Proteins that mediate critical interactions with the host during invasive disease are likely to be secreted. Many secreted proteins require addition of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability. In this study, we evaluated the role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models, and complemented strains exhibited virulence equivalent to that of the parental strains. Comparison of predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was examined because of the importance of exogenous heme for aerobic growth of H. influenzae. The presence of a dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and complementation restored its in vivo phenotype to that of the parental strain. These results indicate that dsbA is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H. influenzae pathogenesis.     

Protein sources of heme for Haemophilus influenzae.

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.     

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.     

Aerobic growth deficient Haemophilus influenzae mutants are non-virulent: implications on metabolism

We investigated aerobic metabolism in Haemophilus influenzae to better understand its essential physiological growth pathways. We describe the isolation and characterization of transposon insertions leading to knockout mutations in lpdA, encoding dihydrolipoamide dehydrogenase. H. influenzae Rd lpdA::Tn10d-cat mutants were unable to grow aerobically and an H. influenzae type b lpdA::Tn10d-cat mutant was significantly attenuated in an infant rat infection model. Since LpdA is a functional subunit of both pyruvate dehydrogenase (aceEF) and alpha-ketoglutarate dehydrogenase (sucAB) the phenotype of the lpdA mutant was further explored by creating separate knockout mutants in the sucAB and aceEF loci. DeltaaceEF and deltasucAB mutants were both significantly attenuated in virulence in the infant rat, but only the sucAB mutant was able to grow aerobically. We therefore conclude that the ability for aerobic growth is critical for invasive disease, and furthermore that a TCA cycle enzyme, alpha-ketoglutarate dehydrogenase, appears to contribute a key metabolic function in vivo, but is not required for growth under laboratory conditions.     

Participation of complement in the nonimmune host defense against experimental Haemophilus influenzae type b septicemia and meningitis.

This study was undertaken to determine whether the terminal complement components (C3-9) are involved in the nonimmune host defense against Haemophilus influenzae type b septicemia and meningitis. Using cobra venom factor, infant rats were depleted of C3 and C5. After intranasal challenge with H. influenzae type b, the complement-depleted rats developed a greater incidence and magnitude of bacteremia and a higher mortality rate. In contrast to the effects on bacteremia, complement depletion did not directly influence either the occurrence of meningitis or bacterial multiplication within the cerebrospinal fluid. These experiments provide evidence that the complement system may be an important mechanism of natural immunity to H. influenzae type b.     

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin–haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.     

Role of the 145-kilodalton surface protein in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats.

Brazilian purpuric fever (BPF) is a fulminant infection associated with bacteremia with clonally related strains of Haemophilus influenzae biogroup aegyptius. Case-associated clone strains are more virulent for infant rats than are non-BPF case-associated H. influenzae biogroup aegyptius isolates. I sought to determine the possible role of P145, a 145-kDa surface protein of BPF case H. influenzae biogroup aegyptius clone isolates, in virulence. First, I compared the virulence of two case-associated clone isolates from the blood of children with BPF from Serrana, Brazil, which differed in P145 expression but were identical in all other phenotypic and genotypic characteristics studied. Twenty-four hours after intraperitoneal inoculation of infant rats, there was a significantly higher incidence (51 versus 26%; P = 0.035) and magnitude (2.9 +/- 5.8 versus 0.7 +/- 2.0 CFU/0.01 ml; P = 0.024) of bacteremia in rats inoculated with the P145-expressing strain. I next compared the virulence of a P145-expressing case-associated clone strain with two P145-nonexpressing phase variants of this strain. One variant exhibited a lower mean magnitude of bacteremia and one displayed a similar magnitude of bacteremia compared with that displayed the P145-expressing parental strain. P145-expressing revertants of the P145-nonexpressing strains exhibited the same virulence as the P145-negative variants from which they were derived. Colonies grown from blood cultures maintained the P145 phenotype of the inoculated strain. These results suggest that P145 expression does not contribute to the virulence of the BPF case clone strain for infant rats following intraperitoneal inoculation.     

The molecular basis of pathogenicity in Haemophilus influenzae: comparative virulence of genetically-related capsular transformants and correlation with changes at the capsulation locus cap

Serotype b strains of Haemophilus influenzae are strikingly more highly associated with episodes of invasive, life-threatening infection in young children than are strains of other serotypes, but the role that the capsule itself plays in determining this virulence has not been dissected away from that of possibly linked virulence determinants such as lipopolysaccharide (LPS). Using DNA from clinical isolates of all six serotypes (a-f) and a genetically-defined capsule-deficient recipient strain Rb-: 02, we constructed a series of capsular transformants otherwise identical with respect to outer-membrane protein and LPS subtype, biotype, and electrotype. Cloned DNA was also used to create type a and b transformants isogenic outside the capsulation locus to provide the most rigorous test to determine whether capsule alone modulates pathogenicity. Capsular transformants showed the same spectrum of virulence in an infant rat bacteremia/meningitis assay as wild-type strains, thus implicating the capsule polysaccharide as an independent determinant of virulence. Experiments in intact and splenectomised rats identified a critical role for type b capsule in enabling organisms to evade splenic clearance.     

The replication of type A influenza viruses in the infant rat: a marker for virus attenuation

Twenty recombinant influenza virus strains bearing HSw1N1, H1N1 or H3N2 surface antigens, together with their respective wild-type or laboratory-propagated parent viruses, were inoculated into 2 day-old infant rats and their replication in the turbinates and lungs of these animals observed over a period of 5 days. In addition, the ability of each of the recombinant and parent viruses to enhance a subsequent infection of these infant rats by Haemophilus influenzae type b was determined. The results showed that both parent and recombinant viruses replicated less well in the lungs than in the turbinates of infant rats, but the titres in both tissues were generally lower for the recombinant strains. The capacity of the majority of the recombinant influenza viruses to promote bacterial infection of the infant rats, as determined by the incidence of H. influenzae bacteraemia and meningitis, was also markedly less than that of their parent viruses. A correlation between virulence for man and both the replication in infant rat turbinates and the ability to enhance H. influenzae infection, was established for the virus strains studied. The data are discussed in relationship to the value of the infant r-H influenzae system as a laboratory marker for the determination of the virulence of influenza virus strains.     

hgpB, a Gene Encoding a Second Haemophilus influenzae Hemoglobin- and Hemoglobin-Haptoglobin-Binding Protein

Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by H. influenzae. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to hgpA, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced Escherichia coli harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of H. influenzae at the newly identified locus, designated hgpB, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An hgpA hgpB double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of H. influenzae has been identified and characterized, and the presence of an additional protein of similar function has been revealed.     

Monoclonal anti-LPS inner core antibodies protect against experimental hematogenous Haemophilus influenzae type b meningitis

This study tested the protective activity of antibodies to the LPS core of Haemophilus influenzae (Borrelli et al., Infect. Immun. 1995;63: 3683-92) in a hematogenous meningitis model. Meningitis was established by intraperitoneal inoculation of infant rats with H. influenzae type b (Hib). The severity of infection was determined by daily assessment of mortality, symptoms of disease and weight changes. Mortality occurred rapidly after infection with 10(5)cfu/rat and most animals died within 24 h. At a lower infection dose (10(4)cfu/rat) the rats survived, but developed symptoms of disease such as tremor, hypothermia, lethargy and anorexia within 12-72 h post challenge. Surviving animals showed decreased weight gain. Bacteremia was detected by daily blood-cultures in 10/10 rats and cleared 6 days after inoculation. The monoclonal anti-LPS antibody MAHI 3 was used in passive protection studies. MAHI 3 increased the survival in the high inoculum group (10(5)cfu/rat) from 10-17% in control animals to 60-90%. At the lower inoculum concentration (10(4)cfu/rat) MAHI 3 treatment reduced the symptoms and blood counts. Intraperitoneal injection of MAHI 3 was more effective than intranasal injection as shown by the effect on bacteremia. We conclude that anti-LPS antibodies can protect against mortality caused by hematogenous Hib infections in infant rats.     

Characterization and virulence analysis of catalase mutants of Haemophilus influenzae.

In addition to detoxifying peroxides generated by aerobic metabolism, the catalases of pathogenic bacteria have also been hypothesized to serve as virulence factors by enabling microorganisms to resist the oxidative bursts of host inflammatory cells. Using transposon mutagenesis of the hktE gene, encoding the Haemophilus influenzae structural gene for catalase, we constructed defined catalase mutants of H. influenzae strains Rd- and Eagan b+. These mutants show no detectable catalase production during exponential or stationary phases or following induction with hydrogen peroxide or ascorbic acid, indicating that hktE is the only functional hydroperoxidase gene present in these two strains of H. influenzae. Exponential-phase cultures of hktE mutants are 8- to 25-fold more sensitive to hydrogen peroxide than the wild type. Using the infant rat model, hktE mutants of strain Eagan b+ were 2.3-fold less virulent than the wild type following intraperitoneal inoculation (P = 0.07). When administered intranasally, the Eagan b+ hktE mutant produced wild-type levels of bacteremia and nasal colonization. The results of this study show that while the H. influenzae hktE gene is important for survival in the presence of peroxides, deletion of the gene produces only a modest reduction in ability to cause lethal sepsis following parenteral challenge and no change in ability to colonize following intranasal inoculation in the infant rat model of infection.