The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization
© Morton et al; licensee BioMed Central Ltd. 2009
Received: 30 April 2009
Accepted: 24 August 2009
Published: 24 August 2009
Haemophilus 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.
An 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.
The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae.
Haemophilus influenzae are fastidious facultatively anaerobic Gram-negative bacteria that cause a range of human infections including otitis media, meningitis, epiglottitis and pneumonia [1, 2]. H. influenzae lacks all enzymes in the biosynthetic pathway for the porphyrin ring and as a result is unable to synthesize protoporphyrin IX (PPIX), the immediate precursor of heme. Since H. influenzae cannot synthesize PPIX the organism has an absolute growth requirement for an exogenous source of PPIX or heme [3, 4]. As a result of this growth requirement H. influenzae has evolved a complex multifunctional array of uptake mechanisms to ensure that it is able to utilize available porphyrin in vivo . One protein shown to be involved in utilization of heme by H. influenzae is the heme binding lipoprotein HbpA [6–8]. 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 . 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 . 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 [7, 8]. Subsequently HbpA was shown definitively to have a role in heme utilization in multiple H. influenzae strains and to be important in virulence in both mouse and rat models of H. influenzae bacteraemia [6, 9, 10].
HbpA exhibits significant homology to the periplasmic dipeptide binding protein DppA of Escherichia coli (for example in comparing HbpA from nontypeable H. influenzae strain HI1388 [Genbank Accession No. AAY87900] and DppA from E coli K12 substrain MG1655 [Genbank Accession No. AAC76569] the two proteins exhibit 51.3% identity and 64.1% consensus as determined using the AlignX tool of Vector NTI 10.3.0). In E. coli DppA functions with the dipeptide ABC transporter DppBCDF to transport both peptides and heme across the periplasmic space [11–13]. This E. coli DppABCDF peptide-heme permease is encoded by the consecutive genes dppABCDF. In H. influenzae the gene encoding HbpA is not located near the genes encoding the H. influenzae DppBCDF proteins. In the H. influenzae strain Rd KW20 genomic sequence hbpA has the locus tag HI0853 while the dppBCDF homologs are located at HI1184-1187 . Although it has not been experimentally established bioinformatic analyses indicate that there is a promoter upstream of dppB and that the dppBCDF gene cluster in H. influenzae is transcribed on a polycistronic message. The H. influenzae DppBCDF proteins exhibit significant homology to the DppBCDF proteins of E. coli; in pairwise comparisons of the proteins from H. influenzae strain Rd KW20 and from E. coli K12 substrain MG1655 identities were respectively 59.6% for DppB, 61.3% for DppC, 73.1% for DppD and 74.6% for DppF. Since the dppBCDF locus in E. coli is known to be involved in heme utilization we examined the potential role of the homologous H. influenzae locus in the utilization of this essential growth factor.
Bacterial strains and growth conditions
H. influenzae Rd KW20 (ATCC 51907) is the strain used in the original H. influenzae genome sequencing project and was obtained from the ATCC. 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). Kanamycin was used at 20 μg ml-1 and chloramphenicol was used at 1.5 μg ml- for growth of H. influenzae.
Human hemoglobin, human haptoglobin from pooled human sera, human serum albumin (HSA), and heme (as hemin) and PPIX were purchased from Sigma. Stock heme solutions (1 mg ml-1 heme in 4% v/v triethanolamine) were prepared as previously described  (heme is correctly defined as ferrous PPIX while hemin is ferric PPIX; however for the purposes of this manuscript heme is used as a general term and does not indicate a particular valence state). PPIX stock solutions at 1 mg ml-1 were made in water and autoclaved prior to use. Hemoglobin was dissolved in water immediately before use. Hemoglobin-haptoglobin complexes and heme-albumin complexes were prepared as previously described [16, 17].
Construction of a dppC insertional mutant
An insertional mutation in dppC was constructed as part of an unrelated study . A chromosomal library of H. influenzae strain Rd KW20 was constructed as follows: H. influenzae chromosomal DNA was digested with Pvu II and phosphorylated Asc I linkers were ligated to the digested DNA at 15°C overnight. Fragments were separated by agarose gel electrophoresis and fragments in excess of approximately 2000-bp were purified. The purified fragments were digested with Asc I and ligated to Asc I digested pASC15 (pASC15 is a minimalized vector containing a unique Asc I site that was constructed as part of the previous unrelated study ). The ligation mixture was transformed into electrocompotent E. coli DH5α and recombinant plasmids were recovered. The recombinant H. influenzae library was mutagenized using the EZ::Tn<KAN-2> kit (Epicentre technologies) as directed by the manufacturer. Transposon insertion sites were mapped by sequencing out from the transposon unit into the flanking DNA. A plasmid was identified with a transposon insertion within the coding sequence of dppC disrupting codon 290 out of a total of 295 codons. The mutated plasmid was designated pASC1262 and was used to transform H. influenzae Rd KW20 to kanamycin resistance using a modification of the static-aerobic method as previously described . A kanamycin-resistant transformant with the correct chromosomal rearrangement was identified using the PCR and designated as H. influenzae strain TMV1262.
Complementation of the dppC insertional mutant
To complement the dppC mutation a plasmid was constructed carrying the entire dppBCDF operon. A 4100-bp PCR product, encompassing the entire dppBCDF gene cluster, as well as 100-bp upstream of the start codon of dppB and 110-bp downstream of the stop codon of dppF, was amplified from H. influenzae strain Rd KW20 chromosomal DNA using the primers dppBCDF-1 and dppBCDF-2 having the sequences 5'-GGATCCTCCGATAGGATCTGTG-3' and 5'-GGATCCGTGCGGTAGAATTCAAGAG-3' respectively. The primers dppBCDF-1 and dppBCDF-2 were designed to add Bam HI sites to each end of the PCR product in order to facilitate subsequent subcloning. The PCR was performed in a 50 μl volume using 100 ng of H. influenzae Rd KW20 chromosomal DNA as template, and the reactions contained 2 mM MgCl2, 200 μM of each deoxynucleoside triphosphate (New England Biolabs), 10 pmol of each primer and 2 U of Taq DNA Ploymerase (Roche). PCR was carried out for 30 cycles, with each cycle consisting of denaturation at 95°C for 1 min, annealing for 1 min at 56°C and primer extension at 72°C for 4 min with one final extension of 30 min. An amplicon of the expected size was cloned into pCR2.1-TOPO to yield pMB26 and confirmed by automated DNA sequencing. pMB26 was digested with Bam HI and the band corresponding to the chromosomally derived insert was ligated to Bam HI digested pACYC184, a shuttle vector with the p15a origin of replication that allows establishment of the plasmid in H. influenzae, to yield pDJM137. pDJM137 was confirmed by automated DNA sequencing, and was electroporated into the H. influenzae dppC mutant strain to yield the corresponding merodiploid strain. Electoporation of H. influenzae was carried out as previously described  and transformants selected on chloramphenicol. A transformant containing pDJM137 was identified and designated HI2208.
Statistical comparisons of growth between strains under the same growth conditions in vitro were made using the Kruskal-Wallis test. Some analyses were made over selected periods of growth as specified in the results. Analyses were performed using Analyse-It for Microsoft Excel v1.71 (Analyze-It Software Inc., Leeds, England). A P value < 0.05 was taken as statistically significant.
Results and discussion
That the growth defects reported in this manuscript result from mutation of dppC is supported by the observation that complementation of the mutant strain with an intact dppBCDF gene cluster corrected the growth defect reported for the mutant strain (data is shown for growth in hemoglobin at 10 μg ml-1 in Figure 3 and in hemoglobin-haptoglobin at both 10 and 5 μg ml-1 in Figure 4).
The data reported herein indicate that the dppBCDF operon constitutes part of the H. influenzae periplasmic heme/porphyrin transport system(s). However, since heme utilization is not completely abrogated, it is clear that an additional periplasmic system(s) must be available to transport heme. Several potential candidates for such a system(s) have been identified . One additional locus potentially involved in periplasmic heme transport is the sap operon. The sap operon comprises the genes SapABCDFZ (HI1638-HI1643 in strain Rd KW20) and is involved in resistance to antimicrobial peptides . The SapABCDF proteins show significant homology to HbpA and the H. influenzae DppBCDF proteins, and preliminary studies indicate a potential role for the sap operon in heme utilization . Two additional putative periplasmic proteins are homologous to both HbpA and SapA and may be involved in heme acquisition; these two proteins are encoded by the ORFs designated HI0213 and HI1124 in the H. influenzae strain Rd KW20 genomic sequence . In a microarray study of the response of Rd KW20 to iron and heme levels in the growth media the ORF HI0213 was maximally transcribed under conditions of iron/heme restriction, supporting a potential role in heme acquisition , although in two additional strains HI0213 transcript levels were not affected by iron/heme levels . In Rd KW20 the locus HI0213 is a stand alone gene encoding a putative permease component of an ABC transporter, which could potentially interact with the DppBCDF proteins. The locus HI1124 is the permease component of an ABC transporter encoded by the operon HI1120-HI1124 and designated OppABCDF in the original Rd KW20 sequencing project . Although there is as yet no empirical data for a role of either HI0213 or OppABCDF in heme utilization based on homology to HbpA and DppBCDF they warrant further investigation.
In conclusion a role for the dppBCDF locus of H. influenzae in periplasmic heme/porphyrin transport has been identified. Further studies will seek to elucidate additional periplasmic heme/porphyrin transport systems, and clarify the precise roles of HbpA and DppBCDF.
This work was supported in part by Public Health Service Grant AI29611 from the National Institute of Allergy and Infectious Disease to TLS and by health research contract HR-06-080 from The Oklahoma Center for the Advancement of Science and Technology to DJM. The authors gratefully acknowledge the support of the Children's Medical Research Institute. The authors thank Michelle Bailey for technical assistance.
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