Departments of Medicine1 and Microbiology and Immunology3, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
Department of Microbiology and Immunology, 1510 Clifton Road, Emory University, AK 30322, USA2
Meningococcal Research Group, Division of Microbiology and Infectious Diseases, University of Nottingham, University Hospital, Nottingham NG7 2UH, UK4
Author for correspondence: Paul C. Turner. Tel: +44 117 928 3241. Fax: +44 117 929 9162. e-mail: paul.turner{at}bristol.ac.uk
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ABSTRACT |
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Keywords: gonococcus, meningococcus, TonB-dependent proteins, iron-regulated proteins
Abbreviations: HmBP, haemin-binding protein; OMP, outer-membrane protein
The GenBank accession number for the sequence of tdfH from meningococcal strain IR1074 reported in this paper is AF227418.
a Present address: Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
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INTRODUCTION |
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The best characterized TonB-dependent OMPs are siderophore receptors (Braun & Killmann, 1999 ; Moeck & Coulton, 1998
). Many bacteria and fungi can secrete siderophores, which chelate exogenous ferric iron. The siderophores bind to specific outer-membrane receptors and are shuttled to the cytoplasm via periplasmic and inner-membrane transporter systems. Examples of siderophore receptors include the FepA receptor for the phenolate siderophore enterochelin of Escherichia coli (Fiss et al., 1982
) and the FpvA receptor for the hydroxamate siderophore pyoverdin of Pseudomonas aeruginosa (Poole et al., 1993
). Although Neisseria gonorrhoeae and Neisseria meningitidis are unable to synthesize siderophores, they can use the siderophore enterochelin as an iron source through the TonB-dependent receptor FetA (previously known as FrpB) (Carson et al., 1999
) and the hydroxamate siderophore aerobactin by a still uncharacterized mechanism (Beucher & Sparling, 1995
).
Pathogenic neisseriae, which consist of the human pathogens N. gonorrhoeae and N. meningitidis, have evolved at least five TonB-dependent receptors enabling them to obtain iron from a diverse range of host micro-environments (Schryvers & Stojiljkovic, 1999 ). These receptors include transferrin-binding protein (TbpA; Schryvers & Morris, 1988a
), lactoferrin-binding protein (LbpA; Schryvers & Morris, 1988b
), two haemoglobin-binding proteins (HpuB and HmbR; Chen et al., 1996
; Lewis & Dyer, 1995
; Stojiljkovic et al., 1996
) and FetA (West & Sparling, 1985
). TbpA, LbpA and HpuB have associated lipoproteins: TbpB, LbpB and HpuA, respectively. With the exception of TbpA/TbpB all these receptors undergo phase variation in vitro and all are regulated by the iron-dependent transcriptional repressor Fur (Schryvers & Stojiljkovic, 1999
; Thomas & Sparling, 1996
).
In a computer search for further neisserial outer-membrane receptors, seven putative non-contiguous genes were identified in the gonococcal (FA1090 Gonococcal Genome Sequencing Project; http://dna1.chem.ou.edu/gono.html) and meningococcal genome (Parkhill et al., 2000 ; Tettelin et al., 2000
) databases encoding proteins with sequences similar to the TonB-dependent family of proteins (Tdf). Three of these putative genes, which had been initially identified in gonococcal strain FA1090, were chosen for further study. In view of the five previously characterized neisserial TonB-dependent receptors the genes were labelled tdfF, tdfG and tdfH. Here we report on the analysis, expression, regulation and distribution of the gene products among commensal and pathogenic neisseriae, examine possible functional roles for these proteins and discuss the merits of one of them as a potential vaccine candidate.
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METHODS |
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N. gonorrhoeae and N. meningitidis were cultured at 37 °C under 5% CO2 on gonococcal base (GCB) agar medium (Difco) containing Kelloggs supplement I (Kellogg et al., 1963 ). Additional haem, ALA or Kelloggs supplement II (iron nitrate) were added where appropriate. To induce iron stress, bacteria were also grown in chemically defined medium (CDM), which was rendered low in iron by treatment with Chelex-100 (Bio-Rad) (West & Sparling, 1985
). Iron-replete controls were grown with 10 µM ferric nitrate. Stock solutions of 5 mg haem ml-1 were prepared by dissolving haemin (Sigma) in 0·1 M NaOH. For iron-limiting conditions, desferrioxamine B (Desferal; Ciba-Geigy) was added to a final concentration of 50 µM for GCB medium and 10 µM for CDM.
The growth phenotype was assessed by resuspending gonococcal and meningococcal strains in 10 ml GCB broth containing 50 µM Desferal (with and without various amounts of haem) followed by incubation at 37 °C in a shaker incubator with 5% CO2. The growth phenotype on agar was assessed by adding haem to a final concentration of 8 µM on GCB medium containing Desferal and supplement I.
Genome analysis.
Putative non-contiguous TonB-dependent receptor genes were identified using BLAST (Altschul et al., 1990 ) programs comparing amino acid sequences from a panel of characterized TonB-dependent receptors in GenBank with DNA from the N. gonorrhoeae FA1090 (http://dna1.chem.ou.edu/gono.html), N. meningitidis Z2491 (Group A) (Parkhill et al., 2000
) and N. meningitidis MC58 (Group B) (Tettelin et al., 2000
) genome databases translated in all six reading frames (TBLASTN; National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/blast/). The panel included receptors for iron citrate (FecA from E. coli; AAA23760) (Pressler et al., 1988
), phenolate siderophores (FepA from E. coli; AAA65994) (Lundrigan & Kadner, 1986
), hydroxamate siderophores (FpvA from P. aeruginosa; AAA25819) (Poole et al., 1993
), haem (HemR from Yersinia enterocolitica; CAA48250) (Stojiljkovic & Hantke, 1992
) and haemophores (HasR from Serratia marcescens; CAA70172) (Letoffe et al., 1994
). Three of the putative genes, tdfF, tdfG and tdfH (initially identified in gonococcal strain FA1090), were chosen for further study.
DNA and peptide analyses were performed using a combination of methods that included the use of web-based programs and commercially available software. Putative promoter regions were identified using the Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter predictor (http://www.fruitfly.org/seq_tools/promoter.html). Putative ribosome-binding sites and transcriptional terminators were identified manually and by using the DNA Strider software program available from Christian Marc (marck@jonas.saclay.cea.fr). Prediction of signal sequence within the predicted coding sequences was made using the software program SignalP (version 1.1) available at the web site of the Centre for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/). Peptide alignments and similarity/identity scores were performed by using the GeneJockey II PAM 250 CLUSTAL alignment program (Biosoft).
PCR.
The design of the PCR primers was based on analysis of sequencing contiguities released from the University of Oklahoma FA1090 Gonococcal Genome Sequencing Project web site (http://dna1.chem.ou.edu/gono.html). To construct isogenic mutants, the ORFs for tdfF, tdfG and tdfH were amplified with primer pairs 5'-TATGAGCGCGTAGAAGTCGT-3' (TDFF1) and 5'-ACGAGCCGTAAAGCGACAGG-3'(TDFF2), 5'-AAAAAGCCCCGCCCTCACG-3' (TDFG1) and 5'-TTTGCCTGCATTCATTGGA-3' (TDFG2), 5'-GGATCCTATAGTTATCCCGAAGATGC-3' (TDFH1) and5'-AAGCTTGGCGTGGGAATGAAATGGAT-3' (TDFH2), respectively. To construct clones for expression in pET30a, DNA from tdfF, tdfG and tdfH was amplified with primers 5'-CCCAAACCGCAGGAAAGCA-3' (TDFF3) and 5'-ATGCGTGTACCTCTGGTGTTCC-3' (TDFF4), 5'-AAGCTTGCGCGGACGACGTGTATTAC-3' (TDFG3) and 5'-CTCGAGGGGTACGCGTTGCAGGTA-3' (TDFG4), 5'-GGATCCTATAGTTATGCCGAAGATGC-3' (TDFH3) and 5'-AAGCTTGGCGTGGGAATGAAATGGAT-3' (TDFH2), respectively. Due to limitations in availability of the FA1090 genome sequence at the time, amplification involving the tdfG primers represented approximately 84% of the complete ORF.
To construct a clone expressing TdfH from its own putative promoter, DNA from tdfH was amplified with primers 5'-GGATTCTTGATGCACCTGCCGTTTA-3' (TDFH6) and 5'-AAGCTTGGCGTGGGAATGAAATGGAT-3' (TDFH2).
Primers with nucleotides that are underlined had incorporated restriction enzyme recognition sequences. The template used for PCR was chromosomal DNA extracted from strain FA1090 (Genomic DNA kit; Qiagen). The PCR reaction conditions were as follows: 94 °C at 3 min for 1 cycle; 94 °C for 45 s, 5558 °C for 45 s and 72 °C for 3 min for 30 cycles; and 72 °C for 3 min for 1 cycle.
Mutagenesis and transformation.
Plasmids containing gonococcal DNA insert were constructed as follows (Table 1). The tdfF, tdfG and tdfH PCR products were ligated into the plasmid vector pCRII (TA cloning kit; Invitrogen) to generate pUNCH1313, pUNCH1301 and pUNCH1307, respectively. All the plasmids were transformed into competent E. coli DH5
(MCR) cells (Bethesda Research Laboratories) and positive clones were selected by the presence of a white phenotype on IPTG/X-Gal LB medium containing ampicillin and kanamycin.
A 2·1 kb, SmaI-digested DNA cassette containing antibiotic resistance genes to streptomycin and spectinomycin was derived from pHP45
(Prentki & Kirsch, 1984
). The cassette was inserted into the PCR-derived, cloned gonococcal DNA by linearizing the plasmids as follows: pUNCH1313 was linearized with NarI (partial digest), pUNCH1301 with BspEI and pUNCH1307 with NruI. Since BspEI and NarI digestion does not produce blunt ends, linearized pUNCH1313 and pUNCH1301 were treated with Klenow enzyme plus deoxynucleoside triphosphates to blunt the ends.
The cassette was ligated to linearized pUNCH1313, pUNCH1301 and pUNCH1307 to generate pUNCH1314, pUNCH1302 and pUNCH1308, respectively, following transformation into E. coli DH5
(MCR) and selection on LB agar with ampicillin, streptomycin and spectinomycin (Table 1
). The orientation and size of the cloned DNA were confirmed by restriction endonuclease analysis prior to transformation into N. gonorrhoeae or N. meningitidis. Transformation, using plasmid DNA, to mutate the chromosomes of FA1090, MS11 and FAM20 by allelic exchange was performed as described previously (Turner et al., 1998
; Table 1
).
Southern blot analysis and preparation of hybridization probes.
Phenol/chloroform-extracted chromosomal DNA from FA1090, MS11, FAM20, FA6969 (FA1090 tdfG::), FA6970 (FA1090 tdfH::
) and FA6972 (FA1090 tdfF::
) were digested with MluI (MluI sites were absent from all three ORFs and the
cassette). Following electrophoresis and Southern blotting of restriction-enzyme-digested DNA, the blot was probed with a digoxigenin (DIG)-labelled probe derived from either the antibiotic cassette or PCR-derived DNA using the TDFF1/TDFF2, TDFG1/TDFG2 or TDFH1/TDFH2 primers. The antibiotic cassette probe was labelled using the DIG High Prime Kit and the PCR products were labelled using the PCR DIG Labelling Mix (Boehringer Mannheim).
Generation of recombinant proteins.
Expression clones of tdfF, tdfG and tdfH were constructed as follows. PCR products containing gonococcal ORFs but lacking signal sequence were cloned into plasmid vector pCRII to generate pUNCH1320, pUNCH1315 and pUNCH1309, respectively. Each plasmid was transformed into competent E. coli DH5 (MCR) cells and positive clones were selected by the presence of a white phenotype on IPTG/X-Gal LB medium containing ampicillin and kanamycin. Inserts containing the ORF of interest were removed from the plasmids by relevant double restriction endonuclease digestion, followed by gel purification. pUNCH1320, pUNCH1315 and pUNCH1309 were digested with BamHI/XhoI, HindIII/XhoI and BamHI/HindIII, respectively. The inserts from tdfF, tdfG and tdfH were directionally ligated into similarly digested, gel-purified expression vector pET30a to generate pUNCH1321, pUNCH1316 and pUNCH1310, respectively (Table 1
). The pET30a-derived expression clones were then electroporated into competent E. coli BL21 (DE3) containing the plasmid pLysS (Novagen). The orientation and size of the cloned DNA were confirmed by restriction endonuclease analysis. Partial sequencing analysis using T7 promoter and T7 terminator oligonucleotides as primers was also used to confirm that each plasmid contained the correct insert.
Expression and purification of recombinant proteins.
E. coli BL21 (DE3) strains containing pLysS and each of the expression plasmids for recombinant tdfF, tdfG or tdfH were inoculated into 500 ml LB broth containing kanamycin and chloramphenicol and incubated at 37 °C. At mid-exponential growth (KlettSummerson colorimeter reading of 75), IPTG was added to a final concentration of 3 mM. The bacterial RNA polymerase inhibitor rifampicin was then added 30 min later to give a final concentration of 200 µg ml-1 (Qi et al., 1994 ). This had the effect of inhibiting synthesis of non-recombinant host proteins. After further incubation for 2 h, E. coli cells were harvested by centrifugation at 4000 g for 15 min and stored at -20 °C prior to purification.
Purification was performed under denaturing conditions as follows (Ni-NTA Spin Handbook; Qiagen). The cells were resuspended in buffer A (6 M guanidine hydrochloride, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 8·0) at 5 ml (g wet wt)-1 and stirred for 1 h at room temperature prior to centrifugation at 10000 g for 15 min at 4 °C. The supernatant lysate was loaded onto a nickel-nitrolotriacetic acid (Ni-NTA) column, pre-equilibrated in buffer A, at a rate of 1015 ml h-1 and the column was washed with at least 10 column vols of buffer B (8 M urea, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 9·0) and 10 column vols of buffer C (0·01 M Tris/HCl, pH 6·3) (Qiagen). The hexahistidine tagged recombinant protein, which had remained bound to the Ni-NTA column was then eluted with 10 ml buffer C containing 250 mM imidazole and collected in 1 ml fractions. The fractions containing the recombinant proteins were pooled and excess urea and imidazole were removed by slow dialysis against PBS (pH 7·4). Removal of the urea caused the proteins to precipitate. The proteins were recovered by centrifugation and resuspended in PBS to give a final concentration of 1 mg ml-1 in 1 M urea. Polyclonal antibodies against the denatured recombinant proteins were then raised in Elite-New Zealand white rabbits. Rabbits at Covance (Denver, PA, USA) were initially immunized with 250 µg recombinant protein in Freunds complete adjuvant and boosted with 125 µg recombinant protein at 3-weekly intervals for 9 to 12 weeks.
Western blot analysis.
OMPs and whole-cell lysates were separated by electrophoresis on 7·5% polyacrylamide gels (West & Sparling, 1985 ). Transfer and development were performed as described by Towbin et al. (1979)
. To determine if TdfH could be affinity-purified by haem, haem-affinity purification on total membranes and outer-membrane preparations of FA1090 was performed under the conditions described by Lee (1992
, 1994
) prior to SDS-PAGE. Haemin-binding protein (97 kDa HmBP) and ferric-binding protein (FbpA) polyclonal antibodies were gifts from B. C. Lee (University of Calgary, Canada) and T. Mietzner (University of Pittsburgh, USA), respectively. The FbpA, FetA and HmBP antibodies were used at dilutions of 1:5000, 1:3000 and 1:1000 respectively (Beucher & Sparling, 1995
). The polyclonal antibodies to TdfF, TdfG and TdfH were used at a dilution of 1:1000.
Expression of TdfH in E. coli hemA mutants.
An expression clone of tdfH was made as follows. A PCR product containing the putative gonococcal tdfH promoter region, complete ORF and primer-incorporated restriction enzyme sites (TDFH6 and TDFH2) was digested with EcoRI and HindIII. The gel-purified PCR product was then ligated into EcoRI- and HindIII-digested, gel-purified plasmid vector pMCL210 to generate pUNCH1327, following transformation into competent E. coli DH5 (MCR) cells. Positive clones were selected by the presence of a white phenotype on IPTG/X-Gal LB medium containing chloramphenicol. Plasmid clones expressing TdfH were purified and electroporated into competent E. coli strains IR1532 (hemA mutant) and IR1583 [IR1532 with pLAFR cosmid (Prince et al., 1988
) expressing meningococcal TonB, ExbB and ExbD] (Table 1
). Expression of TdfH was confirmed by analysis of Western blots containing E. coli whole-cell lysates probed with antibody to TdfH (data not shown).
Protease susceptibility of TdfH.
Gonococci and meningococci were grown to mid-exponential growth phase (Klett reading of 75) and trypsin or chymotrypsin was added to give a final concentration of 2 µg ml-1 for 040 min. Protease treatment was stopped by adding PMSF to a final concentration of 500 µM, followed by centrifugation, resuspension of the bacterial pellet in loading buffer and boiling for 3 min. Western blot analysis of the whole-cell lysates was performed by probing with the rabbit polyclonal antibodies to TdfH or FbpA.
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RESULTS |
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Identification of homology with other TonB-dependent receptors
Detailed comparisons of the mature proteins of TdfF, TdfG and TdfH from gonococcal strain FA1090 with characterized TonB-dependent receptors revealed extensive areas of homology within all seven previously described conserved domains of TonB-dependent proteins (Fig. 1) (Cornelissen et al., 1992
; Kadner, 1990
; Klebba et al., 1993
; Struyve et al., 1991
). The domains included a TonB box located near the N terminus and a C-terminal membrane-spanning domain. The characterized TonB-dependent receptors used in the comparison were chosen to represent a diverse array of functions, including iron citrate (FecA; Pressler et al., 1988
), hydroxamate siderophore (PupA; Bitter et al., 1991
), phenolate siderophore (FepA; Lundrigan & Kadner, 1986
), human transferrin (TbpA; Cornelissen et al., 1992
) and vitamin B12 (BtuB; Heller & Kadner, 1985
) utilization.
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Alignment of the mature protein of TdfF to the ferric pyoverdin receptor (FpvA) of P. aeruginosa (Poole et al., 1993 ) and the coprogen and rhodotorulic acid receptor (FhuE) of E. coli (Sauer et al., 1990
) showed amino acid identities of 29 and 30%, respectively. Alignments of TdfG and TdfH to the haemophore receptor (HasR) of S. marcescens (Letoffe et al., 1994
) showed amino acid identities of 11 and 16%, respectively (Table 2
). Probable orthologues for TdfH were identified in the complete and incomplete genome sequence databases for the mucosal pathogens Haemophilus influenzae (identity 54%), Actinobacillus actinomycetemcomitans (identity 59%) and Campylobacter jejuni (identity 32%), based on alignment along the entire length of the predicted mature proteins (Table 2
) (Fleischmann et al., 1995
; http://www.ncbi.nlm.nih.gov/blast/).
DNA sequence flanking tdfF, tdfG and tdfH
As genes involved in similar functions are often arranged together on the chromosome, we examined the DNA sequence flanking tdfF, tdfG and tdfH to determine what functional relationship, if any, may exist between them. The ORF directly upstream of tdfF in gono coccal strain FA1090 and meningococcal strain Z2491 showed homology (identity 31%) to the enterochelin periplasmic transporter protein CeuE of C. jejuni (Richardson & Park, 1995 ). CeuE is responsible for transporting the phenolate siderophore, enterochelin, through the periplasmic space. No obvious promoter sequence was present between the two ORFs, which suggested that they may be transcriptionally linked. Upstream and in the opposite orientation to the ceuE homologue was an ORF with homology to transcriptional regulators of siderophore receptor genes, including the pyochelin receptor regulator (PchR) of P. aeruginosa (Heinrichs & Poole, 1996
). Downstream to tdfF was an ORF with homology (E. coli amino acid identity 37%) to the enzyme isospartate methyltransferase and located between them was an inverted repeat consistent with a putative transcriptional terminator (Li & Clarke, 1992
).
Upstream of tdfG in gonococcal strain FA1090 was an ORF with homology to a potential membrane protein of unknown function in H. influenzae (HI1376; Fleischmann et al., 1995 ). Downstream to tdfG were two ORFs with homology to the enteropathogenic E. coli autotransporter AIDA-I adhesin and AIDA-I adhesin precursor protein, respectively (Suhr et al., 1996
). BLAST searches of the two meningococcal genome databases with gonococcal tdfG failed to find any significant homology for this gene and, unlike tdfF and tdfH, tdfG could not be detected in gonococcal strain MS11 and meningococcal strain FAM18 on Southern blots (data not shown).
Downstream to tdfH were ORFs with homology to the housekeeping enzyme aspartokinase of E. coli (amino acid identity 29%; Li & Clarke, 1992 ). Similarly, an ORF with homology on translation to the enzyme proline dehydrogenase of E. coli (amino acid identity 51%; Ling et al., 1994
) was located upstream of tdfH in gonococcal strain FA1090. Examination of the meningococcal Z2491 and MC58 genome databases revealed that the predicted coding sequences upstream of tdfH showed homology on translation to a probable integral membrane protein, CstA (carbon starvation protein, amino acid identity 62%) of unidentified function in E. coli (Schultz & Matin, 1991
) and a conserved hypothetical protein, Psu-I (pseudouridine synthase, amino acid identity 36%) in E. coli (Kammen et al., 1988
), respectively. TdfH probably does not form part of an operon since it had its own putative promoter and ribosome-binding site and located just downstream was an inverted repeat consistent with a transcriptional terminator (Tinoco et al., 1973
).
Expression of TdfF, TdfG and TdfH
To act as a control for testing antibodies against the protein of interest and to help determine function, isogenic tdfF, tdfG or tdfH mutants (FA6972, FA6969 and FA6970, respectively) of gonococcal strain FA1090 were constructed by insertional inactivation of the respective ORFs with an (Spr Smr) antibiotic resistance gene cassette. Single copies of all three genes and appropriate cassette insertion were confirmed using PCR-generated DNA probes to tdfF, tdfG or tdfH and the
cassette in Southern blots (data not shown). Expression, distribution and regulation of TdfF, TdfG and TdfH were then determined on Western blots using rabbit polyclonal antibodies to recombinant proteins containing heterologous TdfF, TdfG or TdfH. The fusion proteins (Fig. 2
) were expressed in E. coli BL21 using the pET30a vector (which contained an N-terminal hexahistidine tag) and affinity-purified in a Ni-NTA column prior to immunization of rabbits. Western blots of whole-cell lysates and outer-membrane preparations from gonococcal strain FA1090, grown under either iron-limiting or iron-replete conditions, showed immunoreactive bands when probed with the polyclonal antibodies to TdfG (data not shown) or TdfH (Fig. 3
). These bands were absent when FA6969 (FA1090 tdfG::
) and FA6970 (FA1090 tdfH::
) were probed with the TdfG or TdfH antibodies, respectively. No specific immunoreactive bands were detected in gonococcal strain FA1090 when probed with the TdfF polyclonal antibody.
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Since it is possible that more than one receptor may be involved in haem utilization by pathogenic neisseriae, we also tested an E. coli hemA mutant that expressed TdfH and neisserial TonB, ExbB and ExbD (IR1583) for ability to grow in the presence of haem. We introduced a compatible plasmid (pUNCH1327), expressing neisserial TdfH from its own putative promoter, into IR1583 (Table 1), but found it was unable to utilize exogenous haem for growth. Heterologous expression of a neisserial TonB-dependent receptor and TonB, ExbB and ExbD in an E. coli hemA mutant (IR1173) has been used previously to demonstrate a haemoglobin phenotype for the N. meningitidis haemoglobin receptor HmbR (Stojiljkovic et al., 1995
). As the amino acid sequences of TdfH, TonB, ExbB and ExbD in N. meningitidis and N. gonorrhoeae were very similar (98, 79, 98 and 99% identity, respectively), we assumed that a cosmid expressing meningococcal tonB, exbB and exbD genes would permit function of gonococcal TdfH. Expression of TdfH was confirmed in IR1532 and IR1583 by detecting an immunoreactive band of appropriate size in whole-cell lysates on Western blots probed with antibody to TdfH (data not shown). DNA sequence of the tdfH gene in pUNCH1327 revealed one silent base substitution and one conserved valine for isoleucine amino acid change in the predicted protein sequence, when compared to DNA sequence from the FA1090 gonococcal genome database.
TdfH is not the 97 kDa HmBP
The 97 kDa HmBP described by Lee has been postulated as a putative haem receptor/transporter based on haem affinity purification (Lee, 1992 ) and inhibition of growth with haem as sole iron source in the presence of a monoclonal antibody to HmBP (Lee & Levesque, 1997
). To determine if TdfH was the same as the 97 kDa HmBP, total membrane protein preparations made from gonococcal strain FA1090 grown under iron-depleted conditions were haem affinity-purified within a haem agarose matrix (Lee, 1992
). Unlike HmBP, TdfH could not be affinity-purified with haemin (data not shown). Furthermore, HmBP antibodies failed to give an immunoreactive band with recombinant TdfH in Western blots. As a control, the HmBP antibodies gave immunoreactive bands from total membrane preparations and whole-cell lysates of FA1090 and FA6970 (FA1090 tdfH::
) (data not shown).
Surface exposure of TdfH
Surface exposure of TdfH was determined by susceptibility of live bacterial suspensions of gonococcal strain FA1090 and meningococcal strain FAM20 undergoing exponential growth in broth to cleavage by proteases. Multiple breakdown products of TdfH were detected on Western blots of whole-cell lysates taken from live cell suspensions at various time points following exposure to trypsin (Fig. 6a) and chymotrypsin (data not shown). As a control, the same whole-cell lysates failed to show protease susceptibility of the periplasmic ferric-binding protein (FbpA) when probed with polyclonal antibody to FbpA (Fig. 6b
and data not shown). The FbpA antibody functioned as a control, since periplasmic FbpA has multiple trypsin cleavage sites but is not susceptible to this enzyme in live intact gonococci (Cornelissen & Sparling, 1996
).
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DISCUSSION |
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Homology of TdfF, TdfG and TdfH to the TonB-dependent family of proteins was confirmed by amino acid consensus of the translated gene products with seven TonB-dependent receptor conserved domains. One of the genes, tdfF, had principal homology with hydroxamate siderophore receptors, whereas the other two, tdfG and tdfH, had principal homology with haem/haemophore receptors. Confirmation of the presence of tdfF, tdfG and tdfH in FA1090 and construction of the respective isogenic mutants were confirmed by PCR and Southern hybridization experiments. Similarly, tdfF and tdfH, but not tdfG, were detected in gonococcal strain MS11 and meningococcal strain FAM20 using DNA probes.
Although a homologous DNA sequence for tdfF was present in all three sequenced strains of pathogenic neisseriae, TdfF did not appear to be expressed by gonococci, meningococci or commensal neisseriae. One possible explanation is that other factors are required for expression as occurs with the ferric pyochelin receptor (FptR) and the pyochelin regulator (PchR) (Heinrichs & Poole, 1996 ) of P. aeruginosa. A putative gene with homology on translation to the C terminus of PchR (Heinrichs & Poole, 1993
) was located upstream of tdfF, adjacent to the putative enterobactin periplasmic transporter (CeuE) homologue of C. jejuni (Richardson & Park, 1995
). It is possible that the PchR homologue may play a critical role in regulating expression of TdfF in the presence of an unidentified siderophore. However, further analysis also revealed an Inouye/Correia type repeat (Correia et al., 1986
, 1988
) between the upstream ceuE homologue and the putative ribosome-binding site of tdfF in FA1090. Similarly, another repeat was present in N. meningitidis strain Z2491 but in a different location, in the middle of the ceuE homologue. Inouye/Correia repeats are frequently found flanking other neisserial OMP genes, including pilC, opa and hmbR (Parkhill et al., 2000
; Tettelin et al., 2000
). These repeats can vary in length but characteristically contain 26 bp inverted repeats at both ends. tdfF may have originally formed part of an operon which has become insertionally inactivated by insertions upstream of the ORF. Apparent lack of expression was not due to mutation within tdfF, since the ORF was intact without frame shifts or premature stop codons based on our own sequence data, the gonococcal and meningococcal genome sequence data as well as recombinant expression of the protein in E. coli. Analysis of tdfF messenger RNA by reverse transcriptase PCR and/or Northern blot analysis will help to determine if tdfF can be transcribed and if it is transcriptionally linked to the ceuE homologue; these experiments were not attempted in this study.
TdfG is an iron-regulated OMP of approximately 130 kDa that was detected in only 17% (4/23) gonococcal strains and none of the meningococcal or commensal strains examined. The true numbers of strains expressing TdfG may have actually been higher than this survey indicated due to the limits of detection of this protein in Western blots. However, further evidence for lack of expression in at least some strains of meningococci was obtained by searches of the two meningococcal genome databases (Group A and Group B strains) and in Southern hybridization experiments. TdfG is unlikely to be widely distributed among pathogenic neisseriae and the reasons for limitation of expression to only a few strains of gonococci remain unclear. Although it is possible that TdfG may no longer be functionally important, analysis of TdfG expression in strains associated with particular clinical syndromes such as pelvic inflammatory disease or disseminated gonococcal infection may help to elucidate a role for this protein in pathogenesis.
In contrast, TdfH was expressed by all the meningococcal serogroups and serotypes (28 of 28) tested in this study. Similarly over 80% (47 of 58) of the gonococcal strains expressed TdfH and, with the exception of N. lactamica (1 of 6 strains), TdfH was not detected in any of eight different species of commensal neisseriae. N. lactamica is closely related to N. meningitidis and colonization with some strains of N. lactamica has been implicated in protection against invasive disease (Griffiss et al., 1991 ). As expression of TdfH occurs primarily in meningococci and gonococci, this protein may play an important role in pathogenesis. This hypothesis is further supported by the presence of probable orthologues for this protein in other mucosal pathogens, including H. influenzae, A. actinomycetemcomitans and C. jejuni.
Homology with conserved regions of TonB-dependent proteins and BLAST searches of translated protein products in GenBank are consistent with TdfH being a TonB-dependent outer-membrane receptor. We demonstrated that TdfH is present in outer-membrane preparations of gonococci and meningococci and that this protein is surface-exposed, based upon protease susceptibility of the native protein in live intact bacteria. Surface exposure is required for a ligand to bind to an OMP receptor. Conclusive proof of whether TdfH is a TonB-dependent protein would require its function to be known and demonstration that this function is lost in a TonB mutant, neither of which was determined in this study.
Despite extensive efforts, the functional attributes of TdfH were not determined. We found no evidence that TdfH is a haem receptor and established that TdfH was not the 97 kDa HmBP (a putative haem receptor) based on lack of antibody cross-reactivity and failure of enrichment of TdfH by haem affinity purification. An E. coli hemA mutant expressing heterologous TdfH, TonB, ExbB and ExbD also failed to use exogenous haem as a porphyrin source. As there was a single conserved amino acid substitution in the cloned and expressed TdfH protein, when compared with the predicted amino acid sequence in the FA1090 genomic database, we cannot exclude the possibility that TdfH without such a presumed PCR mistake would function as a haem receptor. However, as the FA1090 tdfH mutant still grew normally on haem as sole source of iron, we concluded that TdfH was unlikely to be directly involved in haem uptake.
Interestingly, unlike the previously characterized neisserial TonB-dependent receptors, TdfH did not appear to be regulated by iron. Furthermore, sequence analysis failed to identify a putative Fur box for this gene. Fur is the major transcriptional regulator involved in iron-regulation (Thomas & Sparling, 1996 ). TdfH may function as a receptor for a ligand that is not involved in iron utilization as occurs with the TonB-dependent vitamin B12 receptor (BtuB) of E. coli which is also not iron-regulated (Heller et al., 1988
; Heller & Kadner, 1985
). TdfH might function as a vitamin receptor or alternatively it may be a receptor for a trace metal such as copper (e.g. from caeruloplasmin) or zinc. Recently a zinc transporter system (ZnuABC) has been described for E. coli and Haemophilus ducreyi (Lewis et al., 1999
; Patzer & Hantke, 1998
). Similarly, zinc periplasmic transporters are likely to be present in the gonococci (Chen & Morse, 2000
) and meningococci. Further work is currently being undertaken to establish the functional role of TdfH and these putative transporter proteins among pathogenic neisseriae and other mucosal pathogens.
Even without an identified function, TdfH remains a possible meningococcal vaccine candidate as it is a commonly expressed, highly conserved and surface-exposed OMP. In addition to antibodies that may be produced following natural infection, studies on whether antibodies to the native protein are bactericidal and/or protect in a meningococcal animal challenge model are required to determine the potential of TdfH for inclusion in a future vaccine.
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Received 24 January 2001;
accepted 2 February 2001.