Timothy M. VanWagoner

The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon

BackgroundHaemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps in our knowledge remain. Unresolved issues include the identities of all genes exhibiting altered transcription in response to iron and heme availability, the fraction of such genes functioning in iron/heme acquisition, and the heterogeneity of this gene set among clinical isolates. Previously we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those observations by determining the iron/heme modulons of two clinical isolates, the type b isolate 10810 and the nontypeable isolate R2866. These data are used to begin to define the core iron/heme modulon of the species.ResultsMicroarray studies were performed to compare gene expression on transition from iron/heme-restricted to iron/heme-replete conditions for each isolate. Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed: 233 were maximally transcribed under iron/heme-replete conditions and 130 under iron/heme-restricted conditions. Of the 1883 ORFs representing genes of strain 10810, 353 were significantly differentially transcribed: 150 were preferentially transcribed under iron/heme-replete conditions and 203 under iron/heme-restricted conditions. Comparison of the data sets indicated that 163 genes exhibited similar regulation in both isolates and that 74 of these exhibited similar patterns of regulation in Rd KW20. These comprise the putative core iron/heme modulon.ConclusionThis study provides evidence for a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and/or heme in the growth environment. Elucidation of this modulon provides a means to identify genes with unrecognized roles in iron/heme acquisition or homeostasis, unanticipated responsiveness to environmental levels of the micronutrients or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

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.

Complex Role of Hemoglobin and Hemoglobin-Haptoglobin Binding Proteins in Haemophilus influenzae Virulence in the Infant Rat Model of Invasive Infection

ABSTRACT Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.

Transcriptional Profile of Haemophilus influenzae: Effects of Iron and Heme

Haemophilus influenzae requires either heme or a porphyrin and iron source for growth. Microarray studies of H. influenzae strain Rd KW20 identified 162 iron/heme-regulated genes, representing approximately 10% of the genome, with > or =1.5-fold changes in transcription in response to iron/heme availability in vitro. Eighty genes were preferentially expressed under iron/heme restriction; 82 genes were preferentially expressed under iron/heme-replete conditions.

Characterization of Three New Competence-Regulated Operons in Haemophilus influenzae

Haemophilus influenzae is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized. To date, several operons involved in transformation in this organism have been described. These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media. The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in H. influenzae. In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis. These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes. The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in Pseudomonas aeruginosa. An insertion in HI0939 abolished both DNA binding and uptake. The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in Klebsiella oxytoca.

Characterization of the Haemophilus influenzae tehB gene and its role in virulence

The Haemophilus influenzae ORF designated HI1275 in the Rd KW20 genomic sequence encodes a putative S-adenosyl methyltransferase with significant similarity to tellurite-resistance determinants (tehB) in other species. While the H. influenzae tehB can complement an Escherichia coli tehB mutation, thus restoring tellurite resistance, its role in H. influenzae is unknown. In a previous study defining the iron and haem modulon of H. influenzae, we showed that transcription of this gene in H. influenzae Rd KW20 increases during growth in iron- and haem-restricted media. Since iron and haem uptake genes, and other known virulence factors, constitute the majority of the iron- and haem-regulated gene set, we postulated that tehB may play a role in nutrient acquisition and/or the virulence of H. influenzae. A tehB mutant was constructed in the H. influenzae type b strain 10810 and was evaluated for growth defects in various supplemented media, as well as for its ability to cause infection in rat models of infection. Deletion of tehB leads to an increase in sensitivity both to tellurite and to the oxidizing agents cumene hydroperoxide, tert-butyl hydroperoxide and hydrogen peroxide. The tehB mutant additionally showed a significantly reduced ability to utilize free haem as well as several haem-containing moieties including haem-human serum albumin, haemoglobin and haemoglobin-haptoglobin. Examination of the regulation kinetics indicated that transcription of tehB was independent of both tellurite exposure and oxidative stress. Paired comparisons of the tehB mutant and the wild-type H. influenzae strain 10810 showed that tehB is required for wild-type levels of infection in rat models of H. influenzae invasive disease. To our knowledge this is the first report of a role for tehB in virulence in any bacterial species. These data demonstrate that H. influenzae tehB plays a role in both resistance to oxidative damage and haem uptake/utilization, protects H. influenzae from tellurite exposure, and is important for virulence of this organism in an animal model of invasive disease.

Haemophilus influenzae OxyR: Characterization of Its Regulation, Regulon and Role in Fitness

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae

BackgroundHaemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae.ResultsComparative genomics revealed a putative four gene operon in the recently sequenced nontypeable H. influenzae strain R2846 that encodes predicted proteins exhibiting significant identity at the amino acid level to proteins involved in the utilization of the siderophore ferrichrome in other bacterial species. No siderophore biosynthesis genes were identified in the R2846 genome. Both comparative genomics and a PCR based analysis identified several additional H. influenzae strains possessing this operon. In growth curve assays strains containing the genes were able to utilize ferrichrome as an iron source. H. influenzae strains lacking the operon were unable to obtain iron from ferrichrome. An insertional mutation in one gene of the operon abrogated the ability of strains to utilize ferrichrome. In addition transcription of genes in the identified operon were repressible by high iron/heme levels in the growth media.ConclusionsWe have identified an iron/heme-repressible siderophore utilization locus present in several nontypeable H. influenzae strains. The same strains do not possess genes encoding proteins associated with siderophore synthesis. The siderophore utilization locus may enable the utilization of siderophores produced by other microorganisms in the polymicrobial environmental niche of the human nasopharynx colonized by H. influenzae. This is the first report of siderophore utilization by H. influenzae.

Comparison of transcription of the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear.

BackgroundHaemophilus influenzae is a significant cause of childhood otitis media, and also has an absolute growth requirement for heme. Recent microarray studies using three H. influenzae isolates were used to propose a putative core of genes responsive to iron and heme levels. Included in the core modulon were thirty seven genes that are preferentially expressed under iron/heme limitation, most of which are directly involved with iron and or heme acquisition. In this report, the core iron/heme modulon was further refined following microarray analysis of two additional nontypeable H. influenzae isolates from patients with otitis media. The transcriptional status of the genes comprising the refined iron/heme core modulon was then assessed in vivo, in a chinchilla model of otitis media. These in vivo experiments were performed to address the hypothesis that iron and heme regulated genes are both highly expressed in vivo and important, during clinical infection.ResultsMicroarray analysis of two additional H. influenzae strains resulted in the definition of a core of iron/heme responsive genes. This core consisted of 35 genes maximally expressed under heme restriction and a further 20 genes maximally expressed in heme replete conditions. In vivo studies were performed with two nontypeable H. influenzae strains, 86-028NP and HI1722. The majority of operons identified as members of the core modulon by microarray were also actively upregulated in the chinchilla ear during otitis media. In 86-028NP, 70% of the operons were significantly upregulated while in HI1722 100% of the operons were upregulated in samples recovered from the chinchilla middle ear.ConclusionThis study elucidates a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and heme in the growth environment, and further assesses transcription of these genes in vivo. Elucidation of this modulon allows for identification of genes with unrecognized roles in iron/heme acquisition or homeostasis and/or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization

BackgroundHaemophilus influenzae requires a porphyrin source for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. This porphyrin requirement may be satisfied by either heme alone, or protoporphyrin IX in the presence of an iron source. One protein involved in heme acquisition by H. influenzae is the periplasmic heme binding protein HbpA. HbpA exhibits significant homology to the dipeptide and heme binding protein DppA of Escherichia coli. DppA is a component of the DppABCDF peptide-heme permease of E. coli. H. influenzae homologs of dppBCDF are located in the genome at a point distant from hbpA. The object of this study was to investigate the potential role of the H. influenzae dppBCDF locus in heme utilization.FindingsAn insertional mutation in dppC was constructed and the impact of the mutation on the utilization of both free heme and various proteinaceous heme sources as well as utilization of protoporphyrin IX was determined in growth curve studies. The dppC insertion mutant strain was significantly impacted in utilization of all tested heme sources and protoporphyin IX. Complementation of the dppC mutation with an intact dppCBDF gene cluster in trans corrected the growth defects seen in the dppC mutant strain.ConclusionThe dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae.