1 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
2 Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona, Institut de Recerca i Tecnologia Agroalimentària (UAB-IRTA), Bellaterra, 08193 Barcelona, Spain
Correspondence
Jordi Barbé
jordi.barbe@uab.es Ignacio Badiola
ignacio.badiola{at}irta.es
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ABSTRACT |
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Present address: Centro de Bioactivos Químicos, Universidad Central de las Villas, Carretera Camajuani, km 5, Villa Clara, Cuba.
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INTRODUCTION |
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In bacteria, two families of global regulators involved in the control of genes whose products participate in several pathways of iron uptake have been described (Hantke, 2001). The Fur family has the Fur, PerR and Irr proteins as members, while the DtxR family includes the DtxR and IdeR proteins. Of these regulators, Fur seems to be present in all classes of proteobacteria (Radledge & Dover, 2000
; Hantke, 2001
). The irr gene has only been described so far in Bradyrhizobium japonicum (Hamza et al., 1998
), a member of the
-Proteobacteria, although in this organism the fur gene is also present (Hamza et al., 1999
). Likewise, the Fur protein has also been identified in some Gram-positive bacteria such as Bacillus subtilis (Bsat et al., 1998
). The rest of the regulatory proteins have so far only been described for Gram-positive bacteria (Hantke, 2001
), with the exception of PerR, whose presence has been reported in Campylobacter jejuni (van Vliet et al., 2002
).
The Fur protein has been studied in several bacterial species, whereas much less information is available about the product of the B. japonicum irr gene. The Fur protein, which has a size of about 17 kDa, exhibits Fe2+-dependent DNA-binding activity (Escolar et al., 1999). Genes under Fur control require the presence in their promoters of at least three contiguous NATA/TAT-like hexamers in either direct or inverse orientations to which this protein binds, repressing transcription, when the iron concentration is high (Escolar et al., 1999
). This sequence, known as the Fur box, is widespread in bacteria because it has been detected in the promoters of iron-regulated genes of several species belonging to families as diverse as the Enterobacteriaceae, Pseudomonadaceae, Neisseriaceae, Pasteurellaceae and Bacillaceae (Hantke, 2001
). Thus treatment of cultures of these organisms with iron-chelating agents like 2,2'-dipyridyl (DPD) induces expression of genes negatively regulated by the Fur protein. Nevertheless, it has also been demonstrated that Fur can act as a positive regulator, although the exact mechanism by which it stimulates gene expression has not been definitively established (Dubrac & Touati, 2000
).
Pasteurella multocida is responsible for causing diseases in many species of mammals and birds, originating important economic losses in farms. The presence of several iron-binding proteins regulated by the Fur protein has been reported for this organism (Bosch et al., 2001, 2002a
, b
). In this context, a fur-knockout mutant of P. multocida has been constructed in our laboratory (Bosch et al., 2001
). This mutant shows constitutive expression of high-molecular-mass proteins which have been associated with iron-uptake processes (Snipes et al., 1988
; Choi et al., 1991
). We noted the presence of two proteins that are strongly induced in DPD-treated cultures of P. multocida. In this work, we have shown that these two proteins are encoded by the same gene, hbpA, that both gene products bind haemin, and that expression of hbpA is regulated by iron in a Fur-independent manner.
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METHODS |
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Protein and immunoblot analysis.
Outer-membrane proteins from the P. multocida wild-type or fur strains were extracted from cultures grown under the desired conditions as described by Bosch et al. (2001). Briefly, cultures were centrifuged at 48 000 g and pellets were resuspended in 0·1 M acetate buffer/0·2 M lithium chloride at pH 5·8, incubated for 2 h at 45 °C in a shaking water-bath, and passed through a 21-gauge needle. These suspensions were then centrifuged at 10 000 g, and the pellets were discarded. Membrane fragments were obtained from the supernatant by centrifugation at 30 000 g for 2·5 h, and the pellet was resuspended in distilled water. The protein concentration of outer-membrane samples was determined by the Lowry method, and their profiles were examined by 12 % PAGE in the presence of SDS (Laemmli, 1970
).
To identify both the 60 and 40 kDa proteins, SDS-PAGE gels were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad), and stained with Coomassie Brilliant Blue. Both proteins were then recovered from the membrane and their N-terminal amino acid sequences were determined by Edman degradation using Protein Sequencer 477A (Applied Biosystems).
The antigenicity of the 60 and 40 kDa proteins was determined by Western blot analysis. Crude extracts of E. coli BL21 cells overexpressing these proteins were subjected to SDS-PAGE. Gels were transferred to Immobilon-P membranes (Millipore) using a Hoefer miniVe (Amersham Pharmacia Biotech) TransBlot Cell. Membranes were air-dried for 20 min and blocked for 2 h in blocking solution (10 mM Tris/HCl pH 8, 150 mM NaCl, 0·4 g Block Reagent and 0·2 ml Tween 20 brought up to 200 ml H2O). Transferred proteins were immunostained overnight with specific antiserum at a dilution of 1/100 in blocking solution. Following this, membranes were washed three times (10 min each) with PBS and incubated in a 1/30 000 dilution in blocking solution of anti-mouse IgG, Fc-specific (Sigma), for 1 h. Afterwards, the membranes were washed three times with PBS and reactive polypeptides were visualized in alkaline phosphate buffer (100 mM NaCl, 50 mM Tris/HCl, 5 mM MnCl2) containing 4-nitro blue tetrazolium chloride and X-phosphate-5-bromo-4-chloro-3-indolyl phosphate (BCIP, 4-toluidine salt), as recommended by the supplier (Roche Diagnostics). All procedures were carried out at room temperature.
Protection studies.
Two groups of five female 3-week-old Swiss mice (obtained from Harlan Iberica; Barcelona, Spain) were injected intraperitoneally with either 3 µg HbpA protein recovered from polyacrylamide gels or 0·5 µg outer-membrane proteins from P. multocida wild-type cells. A third group of five mice was injected with PBS as the negative control. After 2 weeks, a second immunization was carried out. The challenge was made 3 weeks later by intraperitoneal inoculation of 100xLD50 of the P. multocida wild-type strain.
The number of animals which were alive 24, 48 and 72 h post-inoculation was recorded, and the virulence power was calculated as reported by Reed & Muench (1938). These animals were afterwards used to obtain serum for Western blot assays. To perform this, mice were bled from the vena cava and the blood was incubated at 37 °C for 2 h, and then kept overnight at 4 °C, to facilitate clot formation. Following this, the blood was centrifuged at 2000 g for 15 min and the serum was recovered and maintained at 4 °C.
To eliminate the antibodies against E. coli that could be present in recovered serum, this was incubated overnight at 4 °C with a sediment of E. coli cells harbouring the pET22-b vector alone.
Haemin binding of E. coli cells expressing the P. multocida wild-type and truncated hbpA genes.
Haemin binding was analysed as described by Genco et al. (1994). E. coli BL21(
DE3) cells carrying the pUA1035 or pUA1036 plasmid containing the whole or the truncated hbpA gene, respectively, were grown in LB medium and harvested after IPTG (1 mM) addition. The cells were washed with PBS and adjusted to an OD550 of 1·0, and 0·8 ml aliquots of the cell suspension in this buffer were mixed with 0·2 ml haemin to a concentration of 10 µM. Samples were incubated at 37 °C for 1 h and centrifuged. Afterwards, the A400 of the supernatant was measured. Haemin diluted in PBS was incubated under the same conditions as an appropriate control. The binding of haemin was determined by the decrease of the absorbance of the supernatant compared to that of control samples, which were set as being 100 %.
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RESULTS |
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Identification of PM0592 (HbpA) as a haemin-binding protein
The product of the PM0592 ORF includes the consensus amino acid sequence (D/E)TXXVXA(A/S) (where X is variable), which is characteristic of the TonB-dependent receptor proteins (Lundrigan & Kadner, 1986), which has been proposed to be a putative haemin-binding protein (May et al., 2001
). To confirm this possibility, a haemin-binding test was performed with E. coli cells carrying the pET22-b expression vector containing either the wild-type PM0592 gene or the PM0592 gene truncated at position 957 (Fig. 3
). The results indicate that E. coli cells expressing either of these two proteins can bind haemin, whereas those that only carry the pET22-b vector can not (Fig. 3
). For this reason, and since to our knowledge this is the first protein of P. multocida in which a haemin-binding activity has been experimentally demonstrated, the gene was redesignated hbpA (for haemin-binding protein).
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DISCUSSION |
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The existence of Fur-independent iron-regulatory mechanisms has been demonstrated in a few Gram-negative bacterial species. Thus 2-D gel electrophoresis analysis of a Yersinia pestis fur strain revealed the presence of several unidentified proteins, either iron-repressible or -inducible (Staggs et al., 1994). Likewise, a catalase and an alkyl hydroperoxide reductase (encoded by the katA and ahpC genes, respectively) are negatively regulated by iron in C. jejuni fur cells through the PerR protein (van Vliet et al., 1998
, 2002
). Moreover, the ptxR gene of Pseudomonas aeruginosa, which encodes a regulator of exotoxin A production, seems to be under iron control through a fur-independent mechanism in cells growing under aerobic but not under microaerobic conditions (Vasil et al., 1998
). However, transcription of this ptxR gene requires the presence of an alternative sigma factor encoded by pvdS, which is directly regulated by Fur (Vasil et al., 1998
). In this way, it cannot be definitively confirmed that iron-mediated control of ptxR is absolutely independent of the Fur protein. On the other hand, the iron regulation of the Bradyrhizobium japonicum hemB gene, whose product participates in the haem biosynthesis pathway, is not mediated by Fur, but rather by Irr (Hamza et al., 2000
). In fact, this is the only protein different from Fur for which a role in iron-regulated gene expression has been demonstrated. It must be noted that a TBLASTN search in the P. multocida genome sequence using the B. japonicum Irr protein as a query has not revealed the presence of any Irr-like protein. Therefore, an orthologue of this protein is not responsible for hbpA regulation in P. multocida.
Recently, a novel iron regulator, RirA, has been identified as controlling iron assimilatory gene function in Rhizobium leguminosarum (Todd et al., 2002). No protein showing similarity to RirA has yet been identified in the genome of P. multocida.
An unexpected result obtained in this work is the negative effect that the presence of Mn2+ has on the transcription of the P. multocida hbpA gene. The importance of this cation in the virulence of Salmonella typhimurium has recently been demonstrated (Boyer et al., 2002). This bacterial species has a Mn2+-dependent gene network which is regulated by the product of the mntR gene (Patzer & Hantke, 2001
). Nevertheless, a mntR-like gene does not seem to be present in P. multocida, as shown by TBLASTN analysis carried out by us. Therefore, the participation of Mn2+ in P. multocida hbpA control must be through an unknown regulatory protein different from MntR.
Furthermore, we have also found that a programmed translational frameshift modulates termination of hbpA mRNA translation in vivo. It has been proposed that these kinds of strategies are often used by pathogenic bacteria to allow escape from the host defence system (Dorman & Smith, 2001) or to adapt to variations in the supply and amount of various iron sources (Lewis et al., 1997
; Schryvers & Stojiljkovic, 1999
). However, this seems unlikely to be the case with P. multocida HbpA, since both the wild-type and the truncated HbpA proteins bind haemin (Fig. 3
) and are recognized by serum obtained from animals previously infected with this organism (Fig. 7b
).
Moreover, it should be noted that use of the HbpA haemin-binding protein in immunization assays is not able to protect mice against a challenge with virulent P. multocida cells. These data are in agreement with the existence in P. multocida of several putative haem- or haemoglobin-binding proteins (May et al., 2001). In accord with this prediction, PM0040, PM0236, PM0741, PM1081, PM1282 and PM1428 ORFs from P. multocida have been cloned in our laboratory, and, after overexpression in E. coli, we have found that all of them bind haem in vitro (unpublished observations). Finally, from the perspective of using iron-binding proteins for vaccination, our data on HbpA suggest that strategies other than the inoculation of a single type of protein should be used in those bacteria which, like P. multocida, present more than one kind of receptor.
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ACKNOWLEDGEMENTS |
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Received 27 March 2003;
revised 8 May 2003;
accepted 12 May 2003.
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