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The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: Role in heme utilization

Daniel J. Morton, Larissa L. Madore, Ann Smith, Timothy M. VanWagoner, Thomas W. Seale, Paul W. Whitby, Terrence L. Stull
DOI: http://dx.doi.org/10.1016/j.femsle.2005.09.016 193-199 First published online: 1 December 2005

Abstract

Haemophilus influenzae has an absolute growth requirement for heme and a heme binding lipoprotein (HbpA) has been implicated in the utilization of this essential nutrient. HbpA was identified by examining clones from an H. influenzae genomic library that caused Escherichia coli harboring the clone to bind heme. However, HbpA has not been shown to mediate heme acquisition in H. influenzae. We constructed an insertional mutation of hbpA in a nontypeable H. influenzae strain and demonstrated a role for the gene in utilization of multiple heme sources. This is the first report confirming a role for HbpA in utilization of heme.

Keywords
  • Haemophilus influenzae
  • Heme
  • Heme-binding protein

1 Introduction

Haemophilus influenzae are fastidious facultatively anaerobic Gram-negative bacteria that cause a range of human infections including otitis media, meningitis, epiglottitis and pneumonia [1]. While infections caused by encapsulated type b strains have been largely eradicated in the developed world following introduction of a vaccine based on the type b capsule [2], nontypeable H. influenzae (NTHi) strains continue to be a significant cause of otitis media and pneumonia. Since H. influenzae lacks all enzymes in the biosynthetic pathway for the porphyrin ring, it is unable to synthesize protoporphyrin IX (PPIX), the immediate precursor of heme [3,4], although most H. influenzae strains possess a gene encoding the enzyme ferrochelatase that mediates insertion of iron into PPIX to form heme [3,5,6]. Thus, H. influenzae has an absolute growth requirement for an exogenous source of PPIX or heme. The only known niche for H. influenzae is man and potential heme sources in the human host are limited. There is no significant source of free PPIX and heme is generally intracellular, in the form of hemoglobin or heme-containing enzymes [7]. Extracellular hemoglobin, derived from lysed erythrocytes, is bound by the serum protein haptoglobin, and the hemoglobin–haptoglobin complex is rapidly cleared by the reticuloendothelial cells of the liver, bone marrow or spleen [8,9]. Similarly, free heme, principally derived from the degradation of methemoglobin, is bound by the serum proteins hemopexin and albumin and cleared from the circulation [810]. Hemoglobin and the hemoglobin–haptoglobin, heme–hemopexin, and heme–albumin complexes as well as PPIX in the presence of an iron source can be utilized by H. influenzae as heme sources in vitro [11,12]. H. influenzae has evolved a complex multifunctional array of uptake mechanisms to ensure that it is able to utilize available porphyrin in vivo [13].

One protein implicated in heme utilization is the heme-binding lipoprotein (HbpA) [14,15]. HbpA was initially identified as a potential constituent of a heme acquisition pathway following transformation of an H. influenzae genomic DNA library into Escherichia coli and screening for recombinant clones with heme-binding activity [14]. Expression of heme-binding activity by E. coli correlated with the expression of a protein of approximately 51-kDa, sized on SDS–PAGE gels, that was subsequently purified in a heme-agarose affinity purification protocol, from both recombinant E. coli and H. influenzae, and shown to be a lipoprotein [14]. Additionally, HbpA was localized to the periplasmic space and shown to be associated with both the inner membrane and the outer membrane in H. influenzae [14,15]. The authors proposed that HbpA may serve to transport heme into the cytosol of H. influenzae subsequent to initial binding steps at the cell surface [14,15]. However, no direct evidence has been presented to date for the role of HbpA in heme utilization. The goal of this study was to determine if HbpA plays a role in heme acquisition in NTHi.

2 Materials and methods

2.1 Bacterial strains and growth conditions

NTHi strain HI1388 is a clinical ear isolate representing electrophoretic type (ET) 43 and has been previously described [16,17]. H. influenzae were routinely maintained on chocolate agar with bacitracin (BBL, Becton-Dickinson, Sparks, MD, USA) at 37 °C. When necessary, H. influenzae were grown on brain heart infusion (BHI) agar (Difco, Becton-Dickinson, Sparks, MD, USA) supplemented with 10 μg ml−1 heme and 10 μg ml−1β-NAD (supplemented BHI; sBHI) and the appropriate antibiotic(s). Heme-deplete growth was performed in BHI broth supplemented with 10 μg ml−1β-NAD alone (heme-deplete BHI; hdBHI). E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) was used for cloning experiments and was routinely grown on LB agar supplemented with the appropriate antibiotics. Spectinomycin was used at 200 μg ml−1 in both H. influenzae and E. coli, kanamycin was used at 50 μg ml−1 in E. coli and chloramphenicol was used at 1.5 μg ml−1 in H. influenzae and 50 μg ml−1 in E. coli.

2.2 Heme sources

Human hemoglobin, human haptoglobin, human serum albumin (HSA), and bovine hemin were purchased from Sigma. Stock heme solutions were prepared at 1 mg ml−1 heme in 4% v/v triethanolamine as previously described [18]. Hemoglobin was dissolved in water immediately before use. Hemoglobin–haptoglobin complexes were prepared as previously described [19]. Heme–albumin complexes were made by mixing 100 μg heme and 20 mg HSA per ml of water as previously described [11].

Rabbit hemopexin was prepared as described previously and the heme–hemopexin complexes were characterized by the typical features of their absorption spectra, which include the prominent shoulder at 290 nm that appears upon heme binding [20,21].

2.3 DNA methodology

Restriction endonucleases were obtained from New England Biolabs (Beverly, MA, USA) and used as directed by the manufacturer. Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA.) as directed by the manufacturer. Plasmid DNA was isolated using Wizard Plus Minipreps DNA purification system (Promega, Madison, WI, USA) according to the manufacturer's directions. Sequencing of double-stranded template DNA was performed by automated sequencing on an ABI Prism Model 3700 DNA Analyzer at the Recombinant DNA/Protein Resource Facility, Oklahoma State University, Stillwater, OK, USA. Oligonucleotides were synthesized by Qiagen.

2.4 Construction of hbpA insertion mutants

Insertional mutations of hbpA were constructed as follows. A pair of primers was designed for use in the polymerase chain reaction (PCR) based on the available H. influenzae strain Rd KW20 genomic sequence [22] to amplify a 2776-bp region encompassing the hbpA gene. Primers were designated HBPA-1 and HBPA-2 and had the respective sequences 5′-GGACGAATTTTAAATCG-3′ and 5′-CCATCAAAGAAAATAATTGG-3′. PCRs were performed in a 50 μl volume using 100 ng of NTHi strain HI1388 chromosomal DNA as template, and the reactions contained 2 mM MgCl2, 200 μM each deoxynucleoside triphosphate (New England Biolabs), 10 pmol of each primer and 2 U of FastStart Taq DNA Polymerase (Roche, Indianapolis, IN, USA). PCR was carried out for 30 cycles, with each cycle consisting of denaturation at 95 °C for 1 min, annealing for 1 min at 51 °C and primer extension at 72 °C for 1 min with one final extension of 30 min. PCR products of the expected size were obtained and were successfully cloned into the TA cloning vector pCR2.1-TOPO (Invitrogen). Cloned amplicons were confirmed as correct by automated DNA sequencing, and a plasmid harboring the correct insert was designated pDJM1. The spectinomycin resistance marker from pSPECR [23] was excised with EcoRV and cloned into the unique PmlI site (internal to hbpA) of pDJM1 to yield pDJM346. Competent H. influenzae were transformed to spectinomycin resistance with pDJM346, using the static aerobic method as previously described [24], and selected on sBHI agar containing spectinomycin. Correct chromosomal recombinations were confirmed by the molecular size of a PCR product resolved on an agarose gel (data not shown).

2.5 Complementation of the hpbA insertion mutation

Complementation of the hbpA insertion mutants was achieved as follows. The insert of pDJM1 was excised in a SacI/XbaI double digest and ligated to SacI/XbaI digested pASK5 to yield pDJM383. pASK5 allows for complementation of gene disruptions in H. influenzae by insertion of a gene in a nonessential locus, in this case the outer membrane protein OmpP1 [25]. The plasmid pDJM383 was linearized with HindIII and transformed into the H. influenzae hbpA insertion mutant made competent using MIV media as described by Poje and Redfield [26] and selected on sBHI containing chloramphenicol. A chloramphenicol resistant colony was identified and the correct chromosomal rearrangements were confirmed by the molecular size of PCR products from PCRs with two different primer pairs. HBPA-1 and HBPA-2 gave two products corresponding respectively to the mutated gene and the complementing gene in the OmpP1 locus (data not shown). The second PCR utilized primers 5′ ompP1 and 3′ ompP1 [25] and gave a single band corresponding to insertion of pDJM383 in the OmpP1 locus (data not shown). Competent H. influenzae were similarly transformed to chloramphenicol resistance using pASK5 and chromosomal rearrangements of chloramphenicol resistant isolates confirmed by sizing of PCR products on agarose gels.

2.6 Growth studies with H. influenzae

Growth studies were performed using the Bioscreen C Microbiology Reader (Oy Growth Curves AB Ltd., Helsinki, Finland). H. influenzae were grown for 12–14 h on chocolate agar with bacitracin, and these 12–14 h cultures were used to inoculate 10 ml hdBHI cultures that were incubated for 4 h at 37 °C with shaking (175 rpm; Lab-Line, Enviro Shaker). The 4 h cultures were pelleted by centrifugation, washed once in PBS containing 0.1% w/v gelatin and resuspended to an optical density at 605 nm of 0.5 (Shimadzu UV-1201S spectrophotometer) in the same buffer. One millilitre of the bacterial suspension was diluted in 5 ml of 0.1% w/v gelatin in PBS, and the final bacterial suspension was used to inoculate fresh hdBHI (0.1% v/v inoculum to give an approximate initial concentration of 200,000 cfu ml−1) supplemented as appropriate. Growth curves were performed in 300 μl volumes with five replicates for each growth condition in each individual experiment. Experiments were performed at least twice. Optical density measurements were taken at 600 nm at 30 min intervals with the Bioscreen C set to incubate at 37 °C with constant shaking (machine setting “low”).

2.7 Statistics

Statistical comparisons of growth between strains under the same growth conditions in vitro were made using the Kruskal–Wallis test. Analyses were performed using Analyse-It for Microsoft Excel v1.71 (Analyze-It Software Inc., Leeds, England). A P value < 0.01 was taken as statistically significant.

3 Results and discussion

The heme-binding protein HbpA of H. influenaze has been previously proposed as a component of a periplasmic heme transport system [14,15], however, no role for HbpA in heme utilization by H. influenzae has been demonstrated to date. In order to investigate the possible role of HbpA in the utilization of heme from various potential in vivo heme sources we constructed an hbpA insertion mutant of NTHi strain HI1388. Initially a 2776-bp DNA fragment encompassing the hbpA gene and approximately 600-bp on either side of the coding sequence was successfully amplified from strain HI1388 and sequenced (GenBank Accession No. DQ022304). Clones from two independent PCR reactions were sequenced with any sequence discrepancies being resolved by sequencing of a third independent clone if necessary. The predicted protein encoded by the HI1388 hbpA gene is 547 amino acids in length with a leader sequence of 17 amino acids predicted using neural networks and hidden Markov Models with Signal P 3.0 (http://www.cbs.dtu.dk/services/SignalP). The predicted molecular mass of the mature HbpA protein of HI1388 is 59 kDa. HbpA from HI1388 exhibits 99.5% identity to HbpA from the strain used in the original H. influenzae genome sequencing project, Rd KW20 [22], and 98.4% identity to HbpA from the type b strain DL42 from which hbpA was first sequenced [15] (identities calculated using the sequence alignment application AlignX of the Vector NTI suite v. 8, Invitrogen). HbpA from HI1388 additionally has 99.6% and 99.3% identity to the predicted HbpA proteins from two recently sequenced nontypeable H. influenzae strains R2866 and R2846 (http://www.genome.washington.edu/uwgc/). HbpA from strain HI1388 additionally shows significant homology to additional putative proteins derived from the following members of the Pasteurellaceae: Actinobacillus pleuropneumoniae 4074 (identity 79.5%), Mannheimia succiniciproducens MBEL55E (identity 78.8%), Pasteurella multocida Pm70 (identity 78.6%) and H. ducreyi 35000HP (identity 76.6%). No function has been experimentally determined for the HbpA homologs in any of these species. HbpA also shows significant homology to DppA of E. coli (identity 64.1%); DppA is a periplasmic dipeptide binding protein and is a component of the dipeptide permease system of E. coli which is involved in transport of peptides consisting of two or three l-amino acids [27,28]. It is interesting to note that DppA has also been shown to be involved in the transport of the heme precursor 5-aminolevulinic acid in E. coli [27,29]. HbpA additionally cross reacts with polyclonal antisera raised against DppA [27]. However, while HbpA is highly homologous to DppA, genes encoding proteins homologous to additional proteins of the dipeptide transport system, i.e. DppBCDE, are located in a region of the H. influenzae chromosome unlinked to HbpA (ORFs HI1184-HI1187 in the Rd KW20 genomic sequence [22]). HbpA itself is encoded by the ORF designated HI0853 in strain Rd KW20; searches of the Conserved Domain Database (CDD v2.03) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [30] show that HbpA contains domains characteristic of bacterial extracellular solute binding proteins (Pfam00496) and ABC-type transport systems (COG0747, COG4166 and COG4533). Upstream of HbpA (HI0853) is an ORF (HI0852) encoding an uncharacterized putative permease, while downstream of HI0853 is an ORF (HI0854) encoding a putative protein exhibiting limited homology to HugZ (19.5% identity, 32.8% similarity) and HuvZ (23.3% identity, 34.4% similarity) of Pleisiomonas shigelloides and Vibrio anguillarum, respectively [31,32]. hugZ and huvZ both comprise part of a heme uptake gene cluster, however in neither case is the function of the gene product known although some data indicate that HugZ may be involved in protection against heme toxicity [31]. While the hbpA loci in all sequenced H. influenzae strains are similarly arranged, in other members of the Pasteurellaceae the locus does not follow the same pattern, for example the sequenced H. ducreyi, P. multocida and M. succiniciproducens strains do not have a hugZ homolog. Although both P. multocida and M. succiniciproducens have homologs of HI0852 in the former it is not contiguous with hbpA and in the latter it is, and H. ducreyi contains no HI0852 homolog. These observations may indicate that neither HI0852 nor the hugZ homolog is related to HbpA function.

In order to mutate hbpA in HI1388 a spectinomycin resistance was successfully inserted into a unique Pml I site within the hbpA coding sequence and the resulting construct was used to transform strain HI1388 to spectinomycin resistance. A strain HI1388 derivative with an insertional mutation of the hbpA gene was confirmed and designated HI1883. Utilizing the delivery vector pASK5 [25] we also constructed a complementation of the mutant strain, a strain containing both the mutated hbpA locus and an intact hpbA inserted in the OmpP1 locus was identified and designated HI2098. We additionally constructed a derivative of HI1883 transformed to chloramphenicol resistance with pASK5, thus resulting in a mutation of OmpP1 but without the complementing hbpA gene, this strain (designated HI2097) acted as a control for the impact of OmpP1 mutation on heme utilization. Strain HI1388 and its derivative with the hbpA insertion mutation (strain HI1883), the complemented hbpA insertion mutant (HI2098) and the OmpP1 mutant derivative of the hbpA insertion mutant (strain HI2097) were compared for their ability to grow on various heme sources. Heme sources utilized in growth assays were heme, heme–hemopexin complexes, heme–human serum albumin complexes, hemoglobin and hemoglobin–haptoglobin complexes; all of the tested heme sources have previously been shown to be utilized by H. influenzae in vitro [11]. Fig. 1 compares growth of the wildtype strain HI1388 and its hbpA insertion mutant derivative strain HI1883, the complemented hbpA mutant HI2098, and strain HI2097 in heme. At heme concentrations of either 10 or 1 μg ml−1 strain HI1883 exhibited a delayed onset of growth and grew to the lower maximal optical density than the wildtype strain (Fig. 1; Table 1); similar results were obtained for growth at heme concentrations of 5 or 2 μg ml−1 (data not shown). The complemented hbpA insertion mutant (HI2098) gave the same growth pattern as the wildtype strain at 10 μg ml−1 heme and at 1 μg ml−1 heme grew significantly better than the hbpA insertion mutant although it was not completely restored to wildtype levels (Fig. 1). The hbpA insertion mutant derivative harboring the OmpP1 mutation (HI2097) was indistinguishable from the hbpA mutant in its growth in heme (Fig. 1). Growth comparisons were additionally performed using the heme–hemopexin complex, heme–human serum albumin complexes, hemoglobin and the hemoglobin–haptoglobin complex as heme sources; results from growth in these additional heme sources were comparable to those obtained with heme and are summarized in Table 1. Growth of the hbpA insertion mutant in heme–hemopexin was significantly reduced compared to the wildtype strain at 10 μg ml−1 of the heme–hemopexin complex (approximately 100 ng ml−1 heme equivalent), while at 5 μg ml−1 heme–hemopexin complex (50 ng ml−1 heme equivalent) the mutant strain did not grow over the course of the experiment (Table 1). Growth with the heme–human serum albumin complex as the sole heme source gave similar results; at both 200 and 100 ng ml−1 heme equivalent growth of the mutant strain was significantly delayed in onset and attained a reduced final cell density and at 50 ng ml−1 heme equivalent the mutant showed no detectable growth (Table 1). At hemoglobin concentrations of 10 or 5 μg ml−1growth of the mutant strain was both significantly delayed in onset and reached a lower final optical density compared to the wildtype strain and at 1 μg ml−1 hemoglobin the mutant strain did not grow at all (Table 1); the complemented hbpA insertion mutant grew as well as the wildtype strain in hemoglobin (data not shown). With hemoglobin–haptoglobin as the sole heme source at all tested concentrations (10, 5, and 2 μg ml−1 hemoglobin equivalent) growth of the mutant was both delayed in onset and attained a lower final density (Table 1). For all of the examined heme sources growth of the hbpA insertion mutant was significantly delayed and/or reduced compared to the wildtype strain (P < 0.0001 for all growth conditions). These data clearly demonstrate that HbpA is involved in heme utilization by H. influenzae, and based on previous reports localizing HbpA to the periplasmic space the protein likely functions to transport heme across the periplasmic space from outer membrane receptor proteins to the inner membrane. However, since the hbpA mutant strain retains the ability to utilize all tested heme sources at reduced levels it is apparent that additional periplasmic heme transport systems are present. The availability of complete and partial genomic sequences from four nontypeable H. influenzae strains (strains R2866 and R2846 [http://www.genome.washington.edu/uwgc/]; strain 86-028NP [http://www.microbial-pathogenesis.org/site/] and strain 3224A [http://www.micro-gen.ouhsc.edu]) will facilitate identification and molecular analysis of additional potential periplasmic heme transport systems. Further studies will characterise potential additional periplasmic heme transport mechanisms and their interactions with HbpA.

Figure 1

Growth of the H. influenzae nontypeable strain HI1388, the hbpA insertion mutant strain HI1883, the complemented hbpA insertion mutant strain HI2098, and strain HI2097 (the HI1883 derivative transformed with pASK5 containing an OmpP1 mutation) in hdBHI supplemented with heme as the sole heme source. Wildtype strain HI1388 at 10 μg ml−1 heme (closed diamonds), and at 1 μg ml−1 (closed circles). The hbpA insertion mutant strain HI1883 at 10 μg ml−1heme (open diamonds), and at 1 μg ml−1 (open circles). The complemented hbpA insertion mutant strain HI2098 at 10 μg ml−1heme (closed squares), and at 1 μg ml−1 (closed triangles). Strain HI2097 at 10 μg ml−1heme (open squares), and at 1 μg ml−1 (open triangles). Results are mean ± SD for quintuplicate results from representative experiments. Using the Kruskal–Wallis test for HI1388 versus HI1883 at both 10 μg ml−1and 1 μg ml−1 heme P < 0.0001, for HI1388 versus HI2098 at 10 μg ml−1P= 0.0135 over the entire growth curve and P= 0.2924 over the first 18 h of the growth curve.

View this table:
Table 1

Growth characteristics of H. influenzae strain HI1388, the hbpA insertion mutant HI1883 and the complemented hpbA insertion mutant strain HI2098 in various heme sources

StrainHeme sourceaDoubling time (hours)bTime to onset of growth (hours)cMaximum OD attainedTime maximum OD attained (hours)
HI1388Heme 10 μg ml−11.212.51.38
HI1883Heme 10 μg ml−11.965.50.8913
HI2098Heme 10 μg ml−11.162.51.38
HI1388H-Hpx 100 ng ml−12.315.50.5711.5
HI1883H-Hpx 100 ng ml−119.1112.50.2423
HI1388H-Hpx 50 ng ml−15.425.50.3613
HI1883H-Hpx 50 ng ml−1NGdNGNGNG
HI1388HSA 200 ng ml−1161.312
HI1883HSA 200 ng ml−12.1911.51.0519.5
HI1388HSA 100 ng ml−11.1260.9311
HI1883HSA 100 ng ml−13.73130.4420
HI1388HSA 50 ng ml−12.6260.6411
HI1883HSA 50 ng ml−1NGNGNGNG
HI1388Hgb 10 μg ml−10.995.51.1511
HI1883Hgb 10 μg ml−14.029.50.6816.5
HI1388Hgb 5 μg ml−11.65.50.6410.5
HI1883Hgb 5 μg ml−15.55100.3315
HI1388Hgp–Hapt 10 μg ml−10.995.51.312
HI1883Hgp–Hapt 10 μg ml−11.8971.215.5
HI1388Hgp–Hapt 5 μg ml−10.995.51.211
HI1883Hgp–Hapt 5 μg ml−11.8971.0315
HI1388Hgp–Hapt 2 μg ml−11.7160.7411
HI1883Hgp–Hapt 2 μg ml−13.1890.4816.5
  • aHeme source used in growth curve. H- Hpx is the heme–hemopexin complex with concentration given as the heme equivalent, HSA is the heme–human serum albumin complex with concentration given as the heme equivalent, Hgb is hemoglobin, Hgp-Hapt is the hemoglobin–haptoglobin complex with concentration given as the hemoglobin equivalent.

  • bDoubling time was calculated from the exponential growth phase of the growth curve.

  • cTime of first increase in optical density.

  • dNG, no growth detected.

Acknowledgements

This work was supported by Public Health Service Grant AI29611 from the National Institutes of Allergy and Infectious Diseases to T.L.S., D.J.M. and P.W.W. We gratefully acknowledge the support of the Children's Medical Research Institute. We thank Scott D. Mills for kindly providing pASK5.

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