1 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, QC, Canada H3A 2B4
2 Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada J2S 7C6
3 MDS Pharma Services, St Laurent, QC, Canada H4R 2N6
Correspondence
James W. Coulton
james.coulton{at}mcgill.ca
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ABSTRACT |
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Mario Jacques and James W. Coulton are affiliated to the Canadian Research Network on Bacterial Pathogens of Swine.
The GenBank accession number for the sequence of hgbA from Actinobacillus pleuropneumoniae reported in this paper is AF468020.
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INTRODUCTION |
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Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae, is the aetiological agent of porcine pleuropneumonia, a severe disease that causes worldwide economic loss to the swine industry (Fenwick & Henry, 1994). Different factors contribute to the virulence of A. pleuropneumoniae and a comprehensive review on its pathogenicity was published by Bossé et al. (2002)
. Potential iron sources for A. pleuropneumoniae include porcine transferrin (Niven et al., 1989
) for which A. pleuropneumoniae possesses specific receptors TbpA and TbpB (Gerlach et al., 1992
; Gonzalez et al., 1995
), and exogenous siderophores (Diarra et al., 1996
), including ferrichrome for which A. pleuropneumoniae has a specific receptor, FhuA (Mikael et al., 2002
). While both transferrin-binding proteins of A. pleuropneumoniae are upregulated in response to iron restriction (Gerlach et al., 1992
; Gonzalez et al., 1995
), the fhuA gene is regulated differently because it does not respond to iron restriction (Mikael et al., 2003
). A. pleuropneumoniae is also capable of using porcine Hb and haem compounds as sources of iron for growth (Bélanger et al., 1995
; Deneer & Potter, 1989
; D'Silva et al., 1995
). We therefore hypothesized that A. pleuropneumoniae has a specific OM protein that binds Hb and transports haem-associated iron into the cell. In preliminary experiments, OM proteins from A. pleuropneumoniae with both haemin- and Hb-binding activity were detected (Archambault et al., 2003
).
Exploiting Hb-agarose affinity interactions, we purified a protein (105 kDa, henceforth designated HgbA) from detergent-solubilized OM vesicles of A. pleuropneumoniae. Peptide sequences from HgbA were used to design oligonucleotides and to screen genomic libraries of A. pleuropneumoniae, leading to the complete 2838 bp ORF of hgbA. By using RT-PCR, the role of iron restriction on the expression and the transcript level of hgbA was investigated. An internally deleted hgbA mutant of A. pleuropneumoniae was created by a double-step transconjugation system; the mutant was still able to bind Hb as shown by flow cytometry, but was no longer able to use Hb as a source of iron.
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METHODS |
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Affinity chromatography for purification of Hb-binding protein(s).
Proteins were solubilized from OM vesicles by 1 % (w/v) Zwittergent 3,14 (Calbiochem) in 50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, pH 7·5, at 37 °C for 1 h. Centrifugation (12 000 g, 10 min, 4 °C) of this suspension yielded solubilized OM proteins. Following dialysis (2 h, 4 °C) against 50 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·5 % Zwittergent 3,14, OM proteins were mixed with Hb-agarose (Sigma). To remove non-specifically bound proteins, the protein-loaded gel was repeatedly washed with 50 mM Tris/HCl, pH 7·5, 1 M NaCl, 0·5 % Zwittergent 3,14, followed by boiling of the gel with its retained proteins in Laemmli sample buffer. Aliquots were loaded onto SDS-PAGE and the gels were either stained with Coomassie brilliant blue or transferred to a membrane for peptide sequencing.
N-terminal amino acid sequence determination.
The affinity-purified Hb-binding protein of A. pleuropneumoniae serotype 1, separated by SDS-PAGE, was electroblotted to a polyvinylidene difluoride (PVDF) microporous membrane (Immobilon-P; Millipore). A protein of 105 kDa, located on the membrane by Ponceau-S staining, was subjected to N-terminal amino acid sequencing (Sheldon Biotechnology Center, McGill University) by repetitive cycles of Edman degradation of the PVDF-bound protein followed by detection of the PTH-derived amino acid residue using an on-line C18 HPLC column with gas-phase/pulsed-liquid Procise Automated Sequence System (Applied Biosystems).
Trypsin digestion and peptide analysis.
The band on SDS-PAGE that corresponded to the 105 kDa protein was excised and digested with trypsin (Wilm et al., 1996). Gel particulates were dried by a Speed-Vac concentrator and then washed successively with water, acetonitrile and an ammonium bicarbonate/acetonitrile solution (50 : 50, v/v). Reduction was achieved by incubating gel particulates in a solution of 100 mM ammonium bicarbonate containing 10 mM dithiothreitol at 56 °C for 45 min; alkylation was in 100 mM ammonium bicarbonate containing 55 mM iodoacetamide. The gel particulates were dried completely and then incubated on ice with 0·1 % (w/v) trypsin in 100 mM ammonium bicarbonate for 45 min. Excess trypsin was decanted and gel particulates were incubated at 37 °C in 1 mM HCl for 12 h. Peptides were recovered by three serial extractions with 0·1 % trifluoroacetic acid (TFA), 0·1 % TFA/30 % (v/v) acetonitrile and 0·1 % TFA/60 % acetonitrile, separated by sonications of 30 min. Supernatants from all three steps were combined, freeze-dried and reconstituted in 20 µl 0·1 % TFA in 60 % methanol. The peptides were purified, concentrated and freed from detergent on ZipTip cartridges (Millipore) containing C18 resin, loaded onto a nanospray capillary (Protana) and infused into a QSTAR (MDS Sciex) mass spectrometer. Several double- and triple-charged peptides were selected for tandem mass spectrometry; peptides with triple charge were interpreted with Mascot (Matrix Science).
PCR amplification of hgbA fragments.
Based on amino acid sequences of N-terminal and internal peptides from the affinity-purified 105 kDa protein of A. pleuropneumoniae, oligonucleotide primers were designed for PCR amplification of genomic DNA. PCR products were sequenced and confirmed hgbA fragments were further used for screening the A. pleuropneumoniae genomic library. To establish the distribution of hgbA among the different serotypes, genomic DNA was isolated from reference strains representing serotypes 112 of A. pleuropneumoniae for use in PCR amplifications.
Southern blotting.
EcoRI-digested genomic DNA from A. pleuropneumoniae serotype 1 was separated on 0·8 % (w/v) agarose gels in TAE and transferred to a nylon membrane (Amersham Pharmacia Biotech) for DNA hybridization. Based on the N-terminal sequence (Table 2, P-1) and internal peptide sequence (Table 2
, P-3) of HgbA, two degenerate oligonucleotides, 5'-GARGARGARATGGARTTRGA-3' and 5'-GATGARCTNTAYTTYACNTTYAA-3', were synthesized and 3'-end labelled with DIG using the DIG Oligonucleotide 3'-End Labelling Kit (Roche) for use as probes in Southern blotting experiments. DNA hybridization was detected with phosphatase-labelled anti-DIG antibodies and NBT-BCIP as substrate.
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Construction of a deletion mutant of hgbA from A. pleuropneumoniae.
To introduce a deletion into A. pleuropneumoniae serotype 1 hgbA, a double-step transconjugation system using a sucrose sensitivity marker (Oswald et al., 1999) was used. The transconjugation vector pEMOC2 (Table 1
) carries chloramphenicol resistance and a sucrose sensitivity marker (Baltes et al., 2003
). Generating the A. pleuropneumoniae internal
hgbA strain involved several subcloning steps. Plasmid pRSC01 contained an 8·2 kb insert in pBK-CMV, including the entire 2·8 kb hgbA sequence with additional sequences 2·1 kb upstream and 3·3 kb downstream. The hgbA sequence harboured two BspEI restriction sites; pRSC01 was digested with BspEI thus releasing a 650 bp fragment and then religated to create pRSC04 with an in-frame hgbA deletion. The 4·3 kb NheI fragment from pRSC04 containing the internal deletion was subsequently cloned into the XbaI site of the transconjugation vector pBMK (Oswald et al., 1999
) to yield pRSC08. Finally, the 4·3 kb NotISalI fragment from pRSC08 was cloned into the NotI/SalI site of the transconjugation vector pEMOC2 to generate pRSC09. Plasmid pRSC09 containing an in-frame deletion of hgbA plus sequences 1·9 kb upstream and 2·4 kb downstream to facilitate allelic exchange was transformed into diaminopimelic-acid-auxotrophic E. coli
2155 cells as the donor for transconjugation into A. pleuropneumoniae. Transconjugation was achieved by mobilizing plasmid pRSC09 from E. coli
2155 to the recipient strain of A. pleuropneumoniae serotype 1 strain 4074 by conjugal transfer using a mating technique (Dehio & Meyer, 1997
) adapted by Oswald et al. (1999)
. Donor and recipient strains were grown on solid medium overnight; an aliquot of 750 µl donor cells was mixed with 250 µl recipient cells. The mixture was centrifuged (3000 g), resuspended in 200 µl TNM buffer (1 mM Tris/HCl, pH 7·2, 100 mM NaCl, 10 mM MgSO4), spotted onto BHI plates supplemented with 10 µg NAD ml1, 1 mM diaminopimelic acid and 10 mM MgSO4, and incubated at 37 °C for 6 h. Spots of bacterial cells were resuspended in 400 µl BHI supplemented with NAD and serial 10-fold dilutions were plated on BHI-NAD plates containing 2 µg chloramphenicol ml1. After incubation at 37 °C for 24 h, transconjugants appeared, carrying a copy of pRSC09 in the chromosome. Transconjugants were plated on MuellerHinton agar supplemented with 10 µg NAD ml1 and 10 % (w/v) sucrose and incubated at 30 °C for 48 h. This procedure yielded isolated colonies which had lost pEMOC2 sequences (chloramphenicol-sensitive) and which carried either an unaltered wild-type copy of hgbA or the internal hgbA deletion. Colonies from sucrose-containing MuellerHinton agar plates were streaked on BHI-NAD plates; candidates carrying the internal deletion in hgbA were analysed for their PCR profile as well as their capacity to use porcine Hb as a source of iron.
Growth in iron-deficient conditions.
Kinetics of growth of wild-type A. pleuropneumoniae and A. pleuropneumoniae internal hgbA mutant were compared in iron-sufficient and iron-deficient conditions. Overnight bacterial cultures were diluted 100-fold and grown until the culture reached exponential phase. A. pleuropneumoniae strains carrying either wild-type hgbA or the internal
hgbA mutant were then diluted 100-fold by inoculating into (i) NAD-supplemented BHI broth, (ii) NAD-supplemented BHI broth in the presence of 80 µM EDDHA, and (iii) NAD-supplemented BHI broth in the presence of 80 µM EDDHA and 20 µg pig Hb ml1. Growth kinetics were monitored by measurement of OD600 at time zero and at 1 h intervals; assays for each condition were performed in triplicate. Strains of A. pleuropneumoniae carrying wild-type hgbA, the internal
hgbA mutant and the internal
hgbA mutant plus plasmids (complementation, see below) were also tested in growth promotion assays in the presence of various sources of iron as follows. Twofold serial dilutions of EDDHA were made in BHI-NAD broth supplemented with various sources of iron, including Hb from pig, cow, horse, sheep, goat, human and turkey, haemin, ferricrocin and ferric chloride. The highest and lowest concentrations of EDDHA in the assay were 1280 and 20 µM, respectively. Overnight bacterial cultures were used as inocula at a final concentration of 1x106 c.f.u. ml1, approximately 1000-fold dilution of overnight culture. The total volume of the assay was 2·5 ml and each assay was set up in triplicate. Cultures were grown overnight at 37 °C with agitation and the concentration of EDDHA that inhibited visible growth in each condition was reported.
Expression of recombinant A. pleuropneumoniae proteins in E. coli.
Both wild-type hgbA and internal hgbA recombinant proteins of A. pleuropneumoniae were expressed in E. coli and analysed by SDS-PAGE. These strains were also assessed for binding of fluorescein-labelled Hb by flow cytometry. The expression vector pET24b (Novagen) was used for cloning recombinant A. pleuropneumoniae proteins to which were appended a C-terminal His tag, followed by expression in E. coli BL21(DE3) cells, the host for pET vector derivatives. To generate pRSC06, two primers were synthesized: Sal1-Hgb [5'-GAGGTCGACA(159)ACAGTGCATTGGCACAAGAGC-3'; SalI site underlined and number corresponds to the nucleotide in GenBank AF468020] and Xho1-Hgb [5'-GCGCTCGAGG(4)AAAGTAACCTCTGCGGTTAAC-3'; XhoI site underlined and number corresponds to the nucleotide in GenBank AF468020] specific to the 5' and 3' region of hgbA. Using chromosomal DNA from A. pleuropneumoniae as template and primers Sal1-Hgb and Xho1-Hgb, a 2·8 kb fragment containing hgbA sequence was amplified and cloned into pGEM-T Easy to yield pHGBA-14. This fragment contained hgbA sequences corresponding to the codons of the mature sequence of HgbA, but did not contain codons for the entire signal sequence or the stop codon. The 2·8 kb fragment was recovered from pHGBA-14 as a SalIXhoI fragment and cloned into pET24b(+) digested with SalI and XhoI to yield pRSC06. To generate pRSC05, pRSC06 was first digested with BamHI (within hgbA) and XhoI, and the 0·9 kb fragment containing the codons for the C-terminal portion of HgbA was ligated to pET24b(+) digested with BamHI and XhoI; the resulting plasmid was named pRSC03. The 1·9 kb NheIBamHI fragment from pRSC01 containing the codons for the N-terminal portion of HgbA, including its signal sequence and ribosome-binding site, was then cloned into pRSC03 digested with NheI and BamHI to yield pRSC05. To generate pRSC14, pRSC05 was first digested with NheI and XhoI and the 2·8 kb fragment containing hgbA was ligated to pBK-CMV digested with NheI and XhoI; the resulting plasmid was named pRSC12. Plasmid pRSC12 was then digested with BspEI, releasing a 0·65 kb fragment, and religated. This construct, termed pRSC13, contained an in-frame deletion in hgbA. Next, the 2·15 kb NheIXhoI fragment from pRSC13 was cloned into pET24b(+) to generate pRSC14 which contained sequences of hgbA with an internal in-frame deletion, its signal sequence and a C-terminal His tag.
Plasmids pET24b, pRSC05, pRSC06 and pRSC14 were each transformed into E. coli BL21(DE3) cells. Cultures containing the pET24b-derived recombinant plasmids or vector alone were grown in LB broth plus 50 µg kanamycin ml1 for 2 h until cells reached an OD600 of 0·40·6. Samples were removed for uninduced control; IPTG (final concentration of 0·4 mM) was added to the remainder of the culture and incubated for 24 h at 25 °C (Mikael et al., 2002). Bacteria were harvested by centrifugation and whole-cell lysates were analysed by SDS-PAGE and Coomassie blue staining. Cell envelope proteins were also prepared as described above and similarly analysed by SDS-PAGE and Coomassie blue staining.
Complementation.
A 6·7 kb EcoRI fragment from pRSC01 carrying the entire hgbA sequence was cloned into the EcoRI site of the E. coliA. pleuropneumoniae shuttle vector pJF224-XN (Frey, 1992) to yield pRSC15. To assess complementation, this plasmid was introduced into the A. pleuropneumoniae internal
hgbA strain by electroporation (Oswald et al., 1999
). Briefly, a 50 ml culture of A. pleuropneumoniae internal
hgbA mutant was grown to an OD600 of 0·5 in BHI. Cells were kept on ice for 30 min and then centrifuged for 10 min at 3000 g at 4 °C, and washed thrice in 30 ml ice-cold GYTT medium. Cells were resuspended in 0·5 ml GYTT (1/100) and kept at 70 °C. A 125 µl sample of cell suspension was mixed with 12 µg plasmid pRSC15. Electroporation was performed using a Gene Pulser (Bio-Rad) at U=2·5 kV, C=10 µF, R=600 W and a pulse length of 5·0 ms in a 2 mm cuvette. After electroporation, 1 ml NAD-supplemented BHI broth was added and the cells were incubated for 3 h prior to spreading on BHI-NAD plates containing 0·5 µg chloramphenicol ml1. A. pleuropneumoniae with a chromosomal deletion in
hgbA and harbouring either vector pJF224-XN or pRSC15 was inoculated into NAD-supplemented BHI broth in the presence of various concentrations of EDDHA and 20 µg pig Hb ml1, and growth was monitored.
Labelling of pig Hb with fluorescein.
Labelling of pig Hb with fluorescein was achieved using a FLUOS labelling kit (Roche Diagnostics) (Archambault et al., 1999). The molar ratio was set at 1 : 100 after standardization; 1 mol Hb was mixed with 100 mol FLUOS. Briefly, 39·5 µl of a FLUOS solution (20 mg ml1) was added to 1 ml of a pig Hb solution (1 mg ml1 in PBS, pH 7·4) and incubated in the dark for 2 h at room temperature. The HbFLUOS reaction mixture was then applied to a Sephadex G-25 column and unbound FLUOS was separated by gel filtration.
Flow cytometry and analysis.
Bacterial cultures of both wild-type and A. pleuropneumoniae internal hgbA mutant were grown in NAD-supplemented BHI broth (iron-sufficient) and NAD-supplemented BHI broth in the presence of 40 µM EDDHA (iron-deficient). E. coli BL21 cells harbouring pRSC05, pRSC06, pRSC14 or vector pET24b alone were grown with induction by 0·5 mM IPTG for 2 h. Cultures were harvested, washed in 1 ml PBS, centrifuged and resuspended in 90 µl PBS. Ten microlitres of the FLUOS-Hb (final dilution 1/100) was added to the cells and incubated at 37 °C for 30 min with agitation. After washing, E. coli and A. pleuropneumoniae cells were suspended in 12 ml fresh PBS or fresh PBS containing 2 % (w/v) paraformaldehyde, respectively. Green fluorescence in the samples was measured by subjecting cells to flow cytometry using a FACStar flow cytometer (Becton Dickinson Immunocytometry Systems) equipped with a water-cooled 2 W argon ion laser operating at 488 nm and with a 200 mW light output. Negative controls included bacterial cells not incubated with FLUOS-Hb and cells incubated with unlabelled pig Hb. Results were expressed as a percentage of gated fluorescent events where a total of 30 000 events were analysed for each sample. All experiments were performed at least twice; samples were always run in duplicate.
RT-PCR.
A. pleuropneumoniae serotype 1 reference strain 4074 was grown on BHI-NAD solid medium overnight. Colonies were inoculated into 25 ml cultures of (i) BHI-NAD broth for iron-sufficient conditions and (ii) BHI-NAD broth supplemented with 50 µM EDDHA for iron-deficient conditions. Growth was monitored by turbidity; organisms were harvested in exponential phase (OD6000·8), washed immediately with sterile PBS and total bacterial RNA was isolated with the RNeasy Mini Kit (Qiagen). The eluate containing RNA was treated with DNaseI (RNase-free, FPLC purity; Amersham) in a solution of 40 mM Tris, pH 7·5, 6 mM MgCl2, and incubated at 37 °C for 10 min. RNA samples were purified once more with the RNeasy cleanup protocol and concentrations were determined spectrophotometrically at 260 nm. RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) as recommended by the manufacturer, with 500 ng RNA sample and a primer pair, F2 (5'-GAGTTACGTCATACGAATG-3') and B5 (5'-TGATCGCTACCGGTCGCA-3'), to amplify an internal fragment of A. pleuropneumoniae hgbA. Controls included a primer pair specific for A. pleuropneumoniae fhuA and another primer pair specific to the tbp locus in A. pleuropneumoniae. All primer pairs were chosen to amplify a product of approximately 800 bp. The same primers used with genomic DNA as template served as a positive control for the RT-PCR. Control experiments for DNA contamination were performed in which no reverse transcriptase was added to the RNA samples prior to the PCR step. RT-PCR was performed with an equal amount of template RNA and equal volumes of the amplicon were loaded for all samples.
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RESULTS |
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N-terminal and internal peptide sequences of HgbA from A. pleuropneumoniae
By Edman degradation, the N-terminal sequence was determined (Table 2, P-1) from HgbA of A. pleuropneumoniae serotype 1 reference strain 4074. A BLASTP search for this peptide found no homologies with proteins in the NCBI GenBank database. HgbA was then digested with trypsin followed by liquid chromatography and peptide fragments were subjected to de novo sequencing by electrospray ionization mass spectrometry. Two internal peptide sequences (P-2 and P-3) were identical to sequences from a known TonB-dependent Hb-binding OM protein of P. multocida (accession no. AF237932) and from H. influenzae (accession no. U51922). A third internal peptide (P-4) displayed 70 % identity with the same protein from P. multocida. We conclude that HgbA protein from A. pleuropneumoniae is most highly related to the TonB-dependent Hb-binding protein from P. multocida (Bosch et al., 2002
).
Cloning and analysis of hgbA from A. pleuropneumoniae
Synthetic oligonucleotides based on the N-terminal (P-1) and internal peptide (P-3) sequences of HgbA were DIG-labelled as probes in Southern hybridization experiments with genomic A. pleuropneumoniae DNA. Both probes hybridized to a 6 kb EcoRI fragment of A. pleuropneumoniae DNA. The positions of the internal peptide sequences of HgbA of A. pleuropneumoniae (Table 2) were compared to homologous peptides in HgbA of P. multocida. Three pairs of degenerate oligonucleotide primers were then used for PCR amplification of genomic A. pleuropneumoniae DNA, yielding three fragments. Their nucleotide sequences were assembled into an incomplete ORF of 2355 bp (785 aa). The predicted protein showed similarities with the TonB-dependent family of Hb-binding OM proteins involved in haem transport: HgbA of P. multocida (69 %), HgbA of H. influenzae (68 %), HupA of H. ducreyi (64 %) and HgbA of Actinobacillus actinomycetemcomitans (75 %). To isolate larger genomic fragments from A. pleuropneumoniae containing the entire hgbA, a Sau3AI-digested genomic DNA library from A. pleuropneumoniae serotype 1 was screened with DIG-labelled PCR products. Following three rounds of purification, sequencing of the insert from clone pC157A showed a partial hgbA sequence, interrupted by a BamHI restriction site that was attributed to the design of the library. Parallel screening of an EcoRI-digested genomic library yielded a positive clone, pA111. These clones contained sequences of hgbA that were overlapping, yet individually neither had the entire hgbA sequence. By ligating a 4·2 kb EcoRVXhoI fragment from pA111 with a fragment from pC157A digested with EcoRV (within hgbA) and SalI (multiple cloning site), we generated a plasmid (pRSC01) that had pBK-CMV vector as backbone and 8·2 kb from A. pleuropneumoniae. Plasmid pRSC01 contained the full-length 2·8 kb hgbA sequence with additional sequences: 2·1 kb upstream and 3·3 kb downstream (Fig. 2
a). The complete ORF (2838 bp) encoded HgbA of 946 aa; the N-terminal sequence and the three internal peptide sequences (P-1, P-2 and P-3) were positioned within the predicted HgbA, confirming its identity. Upstream of hgbA was a complete ORF for a potential haemin-binding protein (174 aa) homologous to HugZ in Plesiomonas shigelloides. The promoter sequences (Fig. 2b
) revealed a putative ShineDalgarno sequence (AAGGAG) 7 bases upstream of the proposed GTG start codon; 10 (TAGTTT) and 35 (ATAATTAA) boxes, respectively, were located 40 and 60 bases upstream of the translation start codon. A potential Fur-binding sequence (ATAATTAAACATTTAC) was identified 60 bases upstream from the proposed start codon.
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Distribution of hgbA among A. pleuropneumoniae serotype reference strains
To determine whether the hgbA gene was unique to the A. pleuropneumoniae serotype 1 reference strain or widely distributed among different serotypes of this organism, DNA from serotypes 212 was tested by PCR and Southern blotting. Two pairs of primers were used to amplify fragments of 750 and 350 bp, corresponding to an internal and a C-terminal region of hgbA, respectively. All 12 serotypes tested positive for both amplicons by PCR and all gave positive hybridization signals (data not shown). These collective results indicate that hgbA is present among all serotypes 112 of A. pleuropneumoniae.
Deletion mutant of A. pleuropneumoniae hgbA
To construct a hgbA strain of A. pleuropneumoniae serotype 1, a double-step transconjugation system was used to introduce an unmarked in-frame deletion. The mutant strain had an internally deleted fragment (648 bp) extending from Asp592 to Pro807 that maintained the reading frame of hgbA. Using a pair of primers specific to the coding sequences of hgbA, both first and second crossover events were analysed by PCR. The single crossover event, integration of pRSC09, carried both wild-type and the internal
hgbA; successful counter selection with sucrose yielded the mutant strain carrying the chromosomal deletion in hgbA (Fig. 3
). To investigate whether the A. pleuropneumoniae internal
hgbA strain was compromised for use of Hb as iron source, growth promotion tests were performed with various sources of iron on both wild-type and internal
hgbA strains under iron-deficient conditions. Wild-type A. pleuropneumoniae and the internal
hgbA strain required the same concentration of EDDHA (40 µM) to inhibit growth. At this concentration of EDDHA, the internal
hgbA strain could not grow in NAD-supplemented BHI medium in the presence of any source of Hb tested: pig, cow, horse, sheep, goat, human and turkey, all at 20 µg ml1. When other sources of iron (haemin at 20 µg ml1, ferricrocin at 40 µM, ferric chloride at 80 µM) were tested, the wild-type and the internal
hgbA strain behaved alike; both strains were able to use these alternative sources of iron for growth. Growth characteristics of both wild-type and internal
hgbA strains were also assessed (Fig. 4
). While the internal
hgbA strain lagged slightly behind the wild-type strain in NAD-supplemented BHI medium, the wild-type strain recovered from 80 µM EDDHA iron deprivation by addition of 20 µg pig Hb ml1; the mutant strain could not recover.
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Complementation
To assess whether utilization of pig Hb could be restored in the internal hgbA strain of A. pleuropneumoniae by complementation, a growth promotion assay was performed on this mutant strain that had been transformed with pRSC15 carrying a wild-type hgbA. Results (Table 3
) show that the complemented strain could use 20 µg pig Hb ml1 in the presence of up to 640 µM EDDHA; the internal
hgbA strain transformed with vector pJF224-XN alone could not use pig Hb. These data confirmed successful complementation of the A. pleuropneumoniae internal
hgbA strain by a single gene, hgbA.
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DISCUSSION |
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Our most sensitive assay for recognition and binding of Hb by HgbA employed flow cytometry and confirmed (Fig. 6) that Hb bound to native HgbA in the OM of A. pleuropneumoniae, to the internal
HgbA protein on intact cells and to the two homologous recombinant HgbA species expressed in E. coli. Hence, A. pleuropneumoniae expressing HgbA with 216 aa, Asp592 to Pro807, deleted remains proficient for Hb binding but unable to use Hb as its iron source. It is noteworthy that this large deletion covers the FRAP motif (HgbA Phe655 to Pro658 plus His669), a region reported (Bracken et al., 1999
) to be essential for haem utilization by the HemR receptor of Yersinia enterocolitica. Conversely, these authors showed that the FRAP motif was not essential for binding of HemR to an Hb-agarose affinity column. We now show concordance between the properties of HemR from Y. enterocolitica and the activity of HgbA of A. pleuropneumoniae. Bacterial growth kinetics (Fig. 4
) substantiated these observations, leading to the conclusion that, following Hb binding, the sequence Asp592 to Pro807 of HgbA contributes to haem import. The molecular determinants of the release of haem from Hb and its traverse across HgbA are presently under investigation.
What structural elements might be affected by a deletion in HgbA that spans 216 aa? We recently completed analyses of HgbA by comparative modelling and by a Hidden Markov model (P. D. Pawelek & J. W. Coulton, unpublished data), identifying the topological similarities of HgbA to bacterial OM receptors BtuB, FepA, FhuA and FecA of E. coli. Our HgbA model has a globular N-terminal cork domain contained within a 22-stranded barrel domain, its folds being similar to the structures of OM receptors that have been solved by X-ray crystallography. HgbA residue Asp592 in our model was assigned to surface-exposed loop 6; Pro807 was positioned near the end of
strand 18 as it emerged on the periplasmic face. The net deletion therefore removes
strands 1217 and most of
strand 18, meaning that an odd number of strands would be missing. Although unusual, such structural reorganizations are not impossible, especially since the mutant protein appeared to be remarkably stable and was not subject to endogenous proteolysis.
Might HgbA merit consideration as a vaccine candidate against A. pleuropneumoniae? From our Southern blotting survey for the presence of hgbA among 12 serotypes of this organism, we determined that the gene is widely distributed in this species. The bacterium shows a clear preference for the source of Hb (swine) over other mammalian sources, suggesting that the determinants for interaction of HgbA with Hb are conserved in surface-located loops of the receptor at the OM. Preliminary investigations into the virulence of the A. pleuropneumoniae internal hgbA mutant compared to infection of swine by wild-type A. pleuropneumoniae are in progress. Continuing studies on a recombinant, purified HgbA will establish its utility as a swine vaccine.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Archambault, M., Labrie, J., Rioux, C., Dumas, F., Thibault, T., Elkins, C. & Jacques, M. (2003). Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1. Can J Vet Res 67, 271277.[Medline]
Baltes, N., Tonpitak, W., Hennig-Pauka, I., Gruber, A. D. & Gerlach, G. F. (2003). Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence. FEMS Microbiol Lett 220, 4148.[CrossRef][Medline]
Bélanger, M., Bégin, C. & Jacques, M. (1995). Lipopolysaccharides of Actinobacillus pleuropneumoniae bind pig hemoglobin. Infect Immun 63, 656662.[Abstract]
Bosch, M., Garrido, M. E., Llagostera, M., de Rozas, M. P., Badiola, I. & Barbé, J. (2002). Characterization of the Pasteurella multocida hgbA gene encoding a hemoglobin-binding protein. Infect Immun 70, 59555964.
Bossé, J. T., Janson, H., Sheehan, B. J., Beddek, A. J., Rycroft, A. N., Kroll, S. & Langford, P. R. (2002). Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect 4, 225235.[CrossRef][Medline]
Bracken, C. S., Baer, M. T., Abdur-Rashid, A., Helms, W. & Stojiljkovic, I. (1999). Use of heme-protein complexes by the Yersinia enterocolitica HemR receptor: histidine residues are essential for receptor function. J Bacteriol 181, 60636072.
Buchanan, S. K., Smith, B. S., Venkatramani, L., Xia, D., Esser, L., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat Struct Biol 6, 5663.[CrossRef][Medline]
Byers, B. R. & Arceneaux, J. E. L. (1998). Microbial iron transport: iron acquisition by pathogenic microorganisms. In Metal Ions in Biological Systems, Iron Transport and Storage in Microorganisms, Plants and Animals, vol. 35, pp. 3766. Edited by A. Sigel & H. Sigel. New York: Marcel Dekker.
Dehio, C. & Meyer, M. (1997). Maintenance of broad-host-range incompatibility group P and Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal transfer from Escherichia coli. J Bacteriol 179, 538540.[Abstract]
Deneer, H. G. & Potter, A. A. (1989). Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae. Infect Immun 57, 798804.[Medline]
Diarra, M. S., Dolence, J. A., Dolence, E. K., Darwish, I., Miller, M. J., Malouin, F. & Jacques, M. (1996). Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catecholate siderophores. Appl Environ Microbiol 62, 853859.[Abstract]
D'Silva, C. G., Archibald, F. S. & Niven, D. F. (1995). Comparative study of iron acquisition by biotype 1 and biotype 2 strains of Actinobacillus pleuropneumoniae. Vet Microbiol 44, 1123.[CrossRef][Medline]
Eddy, S. R., Sonnhammer, E. L. L., Bateman, A., Birney, E., Durbin, R. & Howe, K. L. (2000). The Pfam protein families database. Nucleic Acids Res 28, 263266.
Elkins, C., Chen, C. J. & Thomas, C. E. (1995). Characterization of hgbA locus encoding a hemoglobin receptor from H. ducreyi. Infect Immun 63, 21942220.[Abstract]
Fenwick, B. & Henry, S. (1994). Porcine pleuropneumonia. J Am Vet Med Assoc 204, 13341340.[Medline]
Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K. & Welte, W. (1998). Siderophore-mediated iron transport: crystal structure of the FhuA with bound lipopolysaccharide. Science 282, 22152220.
Frey, J. (1992). Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae. Res Microbiol 143, 263269.[CrossRef][Medline]
Genco, C. A. & Dixon, D. W. (2001). Emerging strategies in microbial haem capture. Mol Microbiol 39, 111.[CrossRef][Medline]
Gerlach, G. F., Anderson, C., Potter, A. A., Klashinsky, S. & Willson, P. J. (1992). Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae. Infect Immun 60, 892898.[Abstract]
Gonzalez, G. C., Yu, R.-Y., Rosteck, P. R. J. & Schryvers, A. B. (1995). Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes. Microbiology 141, 24052416.[Abstract]
Hanson, M. S. & Hansen, E. J. (1991). Molecular cloning of a haemin-binding lipoprotein from Haemophilus influenzae type b. Mol Microbiol 5, 267278.[Medline]
Lee, B. C. (1992). Isolation of an outer membrane haemin-binding protein of Haemophilus influenzae type b. Infect Immun 60, 810816.[Abstract]
Lee, B. C. (1995). Quelling the red menace: haem capture by bacteria. Mol Microbiol 18, 383390.[Medline]
Litwin, C. M. & Calderwood, S. B. (1993). The role of iron in the regulation of virulence genes. Clin Microbiol Rev 6, 137149.[Abstract]
Locher, K. P., Rees, B., Koebnik, R., Mitschler, A., Moulinier, L., Rosenbusch, J. P. & Moras, D. (1998). Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 95, 771778.[Medline]
Maciver, I., Latimer, J. L., Liem, H. H., Muller-Eberhard, U. Z., Hrkal, Z. & Hansen, E. J. (1996). Identification of an outer membrane protein involved in utilization of haemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae. Infect Immun 64, 37033712.[Abstract]
Mikael, L. G., Pawelek, P. D., Labrie, J., Sirois, M., Coulton, J. W. & Jacques, M. (2002). Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae. Microbiology 148, 28692882.
Mikael, L. G., Srikumar, R., Coulton, J. W. & Jacques, M. (2003). fhuA of Actinobacillus pleuropneumoniae encodes a ferrichrome receptor and is not regulated by iron. Infect Immun 71, 29112915.
Morton, D. J., Whitby, P. W., Jin, H., Ren, Z. & Stull, T. L. (1999). Effect of multiple mutations in hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b. Infect Immun 67, 27292739.
Niven, D. F., Donga, J. & Archibald, F. S. (1989). Responses of Haemophilus pleuropneumoniae to iron restriction; changes in the outer membrane protein profile and the removal of iron from porcine transferrin. Mol Microbiol 3, 10831089.[Medline]
Oswald, W., Walaiporn, T., Ohrt, G. & Gerlach, G. F. (1999). A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker. FEMS Microbiol Lett 179, 153160.[CrossRef][Medline]
Otto, B. R., Verweij-Van Vught, A. M. J. J. & Maclaren, D. M. (1992). Transferrins and heme compounds as iron sources for pathogenic bacteria. Crit Rev Microbiol 18, 217233.[Medline]
Palmer, K. L., Grass, S. & Munson, R. S. (1994). Identification of a hemolytic activity elaborated by Haemophilus ducreyi. Infect Immun 62, 30413043.[Abstract]
Simpson, W., Olczak, T. & Genco, C. A. (2000). Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis. J Bacteriol 182, 57375748.
Stojiljkovic, I., Larson, J., Hwa, V., Anic, S. & So, M. (1996). HmbR outer membrane receptors of pathogenic Neisseria spp. iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation. J Bacteriol 178, 46704678.[Abstract]
Vézina, G., Sirois, M., Clairoux, N. & Boissinot, M. (1997). Cloning and characterization of the groE locus from Actinobacillus pleuropneumoniae. FEMS Microbiol Lett 147, 1116.[CrossRef][Medline]
Wandersman, C. & Stojiljkovic, I. (2000). Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. Curr Opin Microbiol 3, 215220.[CrossRef][Medline]
Williams, P. & Griffiths, E. (1992). Bacterial transferrin receptors: structure, function and contribution to virulence. Med Microbiol Immunol 181, 301322.[Medline]
Wilm, M., Shevchenko, A., Houthaeve, T., Brelt, S., Schwelgerer, L., Fotsis, T. & Mann, M. (1996). Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466469.[CrossRef][Medline]
Received 21 January 2004;
revised 25 February 2004;
accepted 5 March 2004.
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