Neisserial TonB-dependent outer-membrane proteins - CiteSeerX

Microbiology (2001), 147, 1277–1290

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Neisserial TonB-dependent outer-membrane proteins : detection, regulation and distribution of three putative candidates identified from the genome sequences Paul C. Turner,1, 4† Christopher E. Thomas,1 Igor Stojiljkovic,2 Christopher Elkins,1, 3 Goksel Kizel,4 Dlawer A. A. Ala’Aldeen4 and P. F. Sparling1,3 Author for correspondence : Paul C. Turner. Tel : j44 117 928 3241. Fax : j44 117 929 9162. e-mail : paul.turner!bristol.ac.uk

1, 3

Departments of Medicine1 and Microbiology and Immunology3, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA

2

Department of Microbiology and Immunology, 1510 Clifton Road, Emory University, AK 30322, USA

4

Meningococcal Research Group, Division of Microbiology and Infectious Diseases, University of Nottingham, University Hospital, Nottingham NG7 2UH, UK

Computer searches were carried out of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative noncontiguous genes were found and three of these (identified in gonococcal strain FA1090) were chosen for further study. Consensus motif analysis of the peptide sequences was consistent with the three genes encoding TonBdependent receptors. In view of the five previously characterized TonBdependent proteins of pathogenic neisseriae, the putative genes were labelled tdfF, tdfG and tdfH. TdfF had homology with the siderophore receptors FpvA of Pseudomonas aeruginosa and FhuE of Escherichia coli, whereas TdfG and TdfH had homology with the haemophore receptor HasR of Serratia marcescens. The aim of this project was to characterize these proteins and determine their expression, regulation, distribution and surface exposure. Strain surveys of iron-stressed commensal and pathogenic neisseriae revealed that TdfF is unlikely to be expressed, TdfG is expressed by gonococci only and that TdfH is expressed by both meningococci and gonococci. Expression of TdfH was unaffected by iron availability. Susceptibility of TdfH to cleavage by proteases in live gonococci was consistent with surface exposure of this protein. TdfH may function as a TonB-dependent receptor for a non-iron nutrient source. Furthermore, TdfH is worthy of future investigation as a potential meningococcal vaccine candidate as it is a highly conserved, widely distributed and surface-exposed outer-membrane protein.

Keywords : gonococcus, meningococcus, TonB-dependent proteins, iron-regulated proteins

INTRODUCTION

Iron is essential for nearly all bacteria and the ability of pathogens to obtain this and other nutrients is a major virulence determinant (Litwin & Calderwood, 1993 ; .................................................................................................................................................

† Present address : Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. 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. 0002-4749 # 2001 SGM

Payne, 1988). As the outer membrane of Gram-negative bacteria presents a major permeability barrier, the uptake of many macromolecules such as various iron complexes and some vitamins requires high affinity receptor and transport systems for delivery into the cytoplasm (Braun, 1995 ; Klebba et al., 1993). The receptor consists of an integral outer-membrane protein (OMP) (which may have an associated lipoprotein) that binds specific ligands (Guerinot, 1994). The ability to use these sources is normally dependent on the presence of inner-membrane-associated TonB, ExbB and ExbD proteins, giving rise to the description of a family of 1277

P. C. T U R N E R a n d O T H E R S

TonB-dependent outer-membrane receptors (Braun, 1995). Most of these receptors probably function as channels that open in the presence of ligand to allow access of the nutrient into the periplasmic space (Moeck & Coulton, 1998 ; Postle, 1999). 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 TonBdependent 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 microenvironments (Schryvers & Stojiljkovic, 1999). These receptors include transferrin-binding protein (TbpA ; Schryvers & Morris, 1988a), lactoferrin-binding protein (LbpA ; Schryvers & Morris, 1988b), two haemoglobinbinding 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 outermembrane 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. 1278

METHODS Strains, plasmids and culture conditions. The strains used in the Western blot surveys were chosen to represent a diverse group of commensal and pathogenic neisseriae. The gonococcal strains used in the strain survey consisted of 31 clinical strains collected from East Africa (kind gift of R. Brunham, University of British Columbia, Vancouver, Canada), 24 clinical strains collected from North Carolina (Hobbs et al., 1999) and 3 laboratory strains, FA1090, MS11 and FA19 (Dempsey et al., 1991 ; Meyer et al., 1982). The meningococcal strains used in the survey consisted of 21 strains collected from CDC, Atlanta, USA (Richardson & Stojiljkovic, 1999), 6 clinical strains collected from the University of Nottingham and FAM20 (Dyer et al., 1987). These strains included meningococci from serogroups A, B, C, Y and 29E. The commensal neisseria strains consisted of Neisseria lactamica (6 strains), Neisseria cinerea, Neisseria flava (2 strains), Neisseria flavescens (2 strains), Neisseria mucosa, Neisseria perflava, Neisseria subflava, Neisseria polysaccharea and Neisseria sicca (2 strains). Most of the commensal strains were supplied by Janne Cannon and JoAnn Dempsey (University of North Carolina, USA). All the other bacterial strains and plasmids used for this study are listed in Table 1.

E. coli strains were grown at 37 mC in Luria broth (LB) medium containing appropriate antibiotics for selection. Additional haem or 5-aminolevulinic acid (ALA) was added to a final concentration of 8 µM and 150 mM, respectively, where appropriate. Antibiotics were used at the following concentrations (µg ml−") : ampicillin, 100 ; kanamycin, 60 ; chloramphenicol, 30 ; streptomycin, 80 ; spectinomycin, 80. N. gonorrhoeae and N. meningitidis were cultured at 37 mC under 5 % CO on gonococcal base (GCB) agar medium # (Difco) containing Kellogg’s supplement I (Kellogg et al., 1963). Additional haem, ALA or Kellogg’s 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−" were prepared by dissolving haemin (Sigma) in 0n1 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 mC in a shaker incubator with 5 % CO . 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  (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 ( ; 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

Neisserial TonB-dependent receptors Table 1. Strains and plasmids used in this study Strain or plasmid

Genotype

Source or reference

Pathogenic neisseriae FA1090 MS11 FAM20 NalR spontaneous mutant of FAM18 FA6969 FA1090 tdfG : : Ω (Smr Spr) FA6970 FA1090 tdfH : : Ω (Smr Spr) FA6972 FA1090 tdfF : : Ω (Smr Spr) FA6977 MS11 tdfF : : Ω (Smr Spr) FA6976 MS11 tdfH : : Ω (Smr Spr) FA7031 FAM20 tdfF : : Ω (Smr Spr) FA7030 FAM20 tdfH : : Ω (Smr Spr)

Dempsey et al. (1991) Meyer et al. (1982) Dyer et al. (1987) This study This study This study This study This study This study This study

E. coli strains DH5αMCR BL21 (DE3) IR1532 IR1583

hemA mutant IR1532 with pLAFR cosmid expressing meningococcal TonB, ExbB and ExbD

IR1173

IR1583 with pHmbR

Plasmids pCRII pHP45Ω pUNCH1301 pUNCH1302 pUNCH1307 pUNCH1308 pUNCH1313 pUNCH1314 pUNCH1315 pUNCH1309 pUNCH1320 pET30a pUNCH1310 pUNCH1316 pUNCH1321 pUNCH1333 pMCL210 pUNCH1327

Vector for inserting PCR product Source for Ω cassette pCRII with tdfG PCR insert using TDFG1 and TDFG2 primers pUNCH1301 with Ω cassette from pHP45Ω pCRII with tdfH PCR insert using TDFH1 and TDFH2 primers pUNCH1307 with Ω cassette from pHP45Ω pCRII with tdfF PCR insert using TDFF1 and TDFF2 primers pUNCH1313 with Ω cassette from pHP45Ω pCRII with tdfG PCR insert using TDFG3 and TDFG4 primers pCRII with tdfH PCR insert using TDFH3 and TDFH2 primers pCRII with tdfF PCR insert using TDFF3 and TDFF4 primers Vector for expressing recombinant proteins pET30a expressing recombinant TdfH pET30a expressing recombinant TdfG pET30a expressing recombinant TdfF pET30a expressing recombinant TdfF minus hexahistidine tag Vector used for expression of TdfH pMCL210 with TdfH insert including promoter region

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\seqItools\promoter. html). Putative ribosome-binding sites and transcriptional terminators were identified manually and by using the DNA Strider software program available from Christian Marc

Bethesda Research Laboratories Novagen Stojiljkovic et al. (1996) Prince et al. (1988) ; Stojiljkovic et al. (1996) Stojiljkovic et al. (1996) Invitrogen Corporation Prentki & Kirsch (1984) This study This study This study This study This study This study

Novagen This study This study This study This study Nakano et al. (1995) This study

(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  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 5h-TATGAGCGCGTAGAAGTCGT-3h (TDFF1) and 5h-ACGAGCCGTAAAGCGACAGG3h(TDFF2), 5h-AAAAAGCCCCGCCCTCACG-3h (TDFG1) and 5h-TTTGCCTGCATTCATTGGA-3h (TDFG2), 5h-GG1279


ATCCTATAGTTATCCCGAAGATGC-3h (TDFH1) and 5h-AAGCTTGGCGTGGGAATGAAATGGAT-3h (TDFH2), respectively. To construct clones for expression in pET30a, DNA from tdfF, tdfG and tdfH was amplified with primers 5hCCCAAACCGCAGGAAAGCA-3h (TDFF3) and 5h-ATGCGTGTACCTCTGGTGTTCC-3h (TDFF4), 5h-AAGCTTGCGCGGACGACGTGTATTAC-3h (TDFG3) and 5h-CTCGAGGGGTACGCGTTGCAGGTA-3h (TDFG4), 5h-GGATCC TATAGTTATGCCGAAGATGC-3h (TDFH3) and 5h-AAGCTTGGCGTGGGAATGAAATGGAT-3h (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 5hGGATTCTTGATGCACCTGCCGTTTA-3h (TDFH6) and 5h-AAGCTTGGCGTGGGAATGAAATGGAT-3h (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 mC at 3 min for 1 cycle ; 94 mC for 45 s, 55–58 mC for 45 s and 72 mC for 3 min for 30 cycles ; and 72 mC 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 DH5α (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 2n1 kb, SmaI-digested Ω DNA cassette containing antibiotic resistance genes to streptomycin and spectinomycin was derived from pHP45Ω (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 Ω cassette was ligated to linearized pUNCH1313, pUNCH1301 and pUNCH1307 to generate pUNCH1314, pUNCH1302 and pUNCH1308, respectively, following transformation into E. coli DH5α (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 : : Ω), FA6970 (FA1090 tdfH : : Ω) and FA6972 (FA1090 tdfF : : Ω) were digested with MluI (MluI sites were absent from all three ORFs and the Ω cassette). Following electrophoresis and Southern blotting of restriction-enzyme-digested DNA, the blot was probed with a digoxigenin (DIG)-labelled probe 1280

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 DH5α (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 mC. At mid-exponential growth (Klett–Summerson 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−" (Qi et al., 1994). This had the effect of inhibiting synthesis of nonrecombinant host proteins. After further incubation for 2 h, E. coli cells were harvested by centrifugation at 4000 g for 15 min and stored at k20 mC 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, 0n1 M sodium phosphate, 0n01 M Tris\HCl, pH 8n0) at 5 ml (g wet wt)−" and stirred for 1 h at room temperature prior to centrifugation at 10 000 g for 15 min at 4 mC. The supernatant lysate was loaded onto a nickel-nitrolotriacetic acid (NiNTA) column, pre-equilibrated in buffer A, at a rate of 10–15 ml h−" and the column was washed with at least 10 column vols of buffer B (8 M urea, 0n1 M sodium phosphate, 0n01 M Tris\HCl, pH 9n0) and 10 column vols of buffer C (0n01 M Tris\HCl, pH 6n3) (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 7n4). 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−" in 1 M urea. Polyclonal antibodies against the denatured recombinant proteins were then raised

Neisserial TonB-dependent receptors in Elite-New Zealand white rabbits. Rabbits at Covance (Denver, PA, USA) were initially immunized with 250 µg recombinant protein in Freund’s 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 7n5 % 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. Haeminbinding 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 gelpurified PCR product was then ligated into EcoRI- and HindIII-digested, gel-purified plasmid vector pMCL210 to generate pUNCH1327, following transformation into competent E. coli DH5α (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−" for 0–40 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.

RESULTS Identification of putative TonB-dependent receptor genes

Three putative non-contiguous TonB-dependent receptor genes (tdfF, tdfG and tdfH) were initially identified in the N. gonorrhoeae FA1090 genome database (http :\\dna1.chem.ou.edu\gono.html) by using  (Altschul et al., 1990) programs comparing amino acid sequences from a panel of characterized TonB-dependent receptors in GenBank with gonococcal DNA translated in all six reading frames (http :\\ www.ncbi.nlm.nih.gov\blast\). Searches of the N. meningitidis Z2491 (Group A) and N. meningitidis

MC58 (Group B) genome databases with tdfF (FA1090 CDS co-ordinates 19 500–17 326), tdfG (527 873–531 487) and tdfH (926 140–928 905) revealed orthologues of tdfF and tdfH with identities of 93 and 95 %, respectively (Table 2). Similarly, sequence analysis of tdfH from meningococcal Group B strain IR1074 (Richardson & Stojiljkovic, 1999) showed an identity on translation of 96 % compared to tdfH from FA1090. No homologue for tdfG was found in either of the meningococcal databases. As further sequence information became available, searches of the gonococcal and meningococcal genome databases revealed the presence of four additional putative genes that shared homology on translation to TonB-dependent proteins. One of these (NMA1161\ NMB0964\FA1090 CDS co-ordinates 1 151 414– 1 153 705) shared homology to putative TonB-dependent receptor proteins of unknown function in P. aeruginosa (AAG04170) and the plant pathogen Xylella fastidiosa (AAF83194). The other three (NMB1829, NMA2193\ NMB0293 and NMA1558, respectively) shared homology to hydroxamate siderophore receptors (Pizza et al., 2000 ; Tettelin et al., 2000). No homologue for NMB1829 was present in meningococcal strain Z2491, whereas a predicted coding sequence (co-ordinates 79 794–80 334) that contained a frame-shift mutation and homology to the C-terminal region part of NMB1829 was present in the FA1090 genome database. Conversely, no homologue for NMA2193\NMB0293 (Klee et al., 2000) or NMA1558 was present in the FA1090 gonococcal genome database. Two probable pseudogenes (NMA1663–1662\NMB1449 and NMB 1346), which encode TonB-dependent proteins with homology to siderophore receptors, were also found in the genome databases of meningococcal strains Z2491 and MC58. These predicted coding sequences contained authentic frame-shift mutations, which make it unlikely that functional gene products are expressed by these strains (Pizza et al., 2000 ; Tettelin et al., 2000). 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 B (BtuB ; Heller & Kadner, "# 1985) utilization. 1281


Table 2. Amino acid sequence analysis of TdfF, TdfG and TdfH from gonococcal strain FA1090 Putative protein

Homologue

Function*

Similarity/identity (%)†

TdfF

NMA0575 NMB1882 FhuE FpvA PupB HI1217 HasR NMA1700 NMB1497 IR1074 AA ORF HI1217 CJ0178 HasR

Meningococcal strain Z2491 CDS orthologue Meningococcal strain MC58 CDS orthologue Siderophore receptor of Escherichia coli Siderophore receptor of Pseudomonas aeruginosa Siderophore receptor of Pseudomonas putida Unknown CDS of Haemophilus influenzae Haemophore receptor of Serratia marcescens Meningococcal strain Z2491 CDS orthologue Meningococcal strain MC58 CDS orthologue Meningococcal strain IR1074 CDS orthologue Unknown CDS of Actinobacillus actinomycetemcomitans Unknown CDS of Haemophilus influenzae Unknown CDS of Campylobacter jejuni Haemophore receptor of Serratia marcescens

94\93 94\93 47\29 44\29 46\30 28\14 20\11 97\95 97\95 97\96 75\59 70\54 48\32 31\16

TdfG TdfH

* Predicted coding sequences (CDS) are derived from bacterial genome sequence databases. † Similarity and identity are based upon entire length alignment across the predicted mature protein sequences of TdfF, TdfG or TdfH.

.................................................................................................................................................................................................................................................................................................................

Fig. 1. Analysis of TdfF, TdfG and TdfH peptides. The regions are based on the seven TonB consensus regions described previously (Cornelissen et al., 1992). TbpA, N. gonorrhoeae transferrin-binding protein A ; BtuB, E. coli vitamin B12 receptor ; FepA, E. coli ferric enterochelin receptor ; FecA, E. coli ferric citrate receptor ; PupA, Pseudomonas putida pseudobactin receptor. TonB boxes and the C-terminal membrane-spanning domains are denoted by the boxed area in regions 1 and 7. Identical amino acids are marked by an asterisk ; similar amino acids are marked in grey blocks.

TdfF, TdfG and TdfH contained typical putative signal sequences and peptidase 1 cleavage sites (consistent with export through the inner membrane) and the mature 1282

proteins were predicted to have molecular masses of 78 kDa (705 aa), 134 kDa (1180 aa) and 101 kDa (897 aa), respectively. All three proteins contained C-terminal

Neisserial TonB-dependent receptors 1

2

3

4

kDa 116

TdfG TdfH

97 TdfF

66

.................................................................................................................................................

Fig. 2. Coomassie-stained 7n5 % SDS-PAGE gel of recombinant fusion proteins. Fusion proteins of TdfF, TdfG and TdfH were expressed in E. coli strain BL21 (DE3) in the presence of IPTG and rifampicin using the expression vector pET30a. Lanes : 1, recombinant TdfG (pUNCH1316) ; 2, recombinant TdfH (pUNCH1310) ; 3, recombinant TdfF (pUNCH1321) ; 4, pET30a vector-only control.

localization motifs consistent with outer-membrane localization (Struyve et al., 1991). TdfG and TdfH both had a C-terminal phenylalanine (a feature typical for OMPs), whereas TdfF had a C-terminal lysine that was preceded by phenylalanine. Homopolymeric or tandem DNA repeats, which are required for phase variation of many neisserial OMPs, were not detected in any of the regions in or around tdfF, tdfG or tdfH (Saunders et al., 2000).

them. The ORF directly upstream of tdfF in gonococcal 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).  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).

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\).

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).

DNA sequence flanking tdfF, tdfG and tdfH

Expression of 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

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 1283


1

2

3

4

kDa TdfH

97

FetA

70

.................................................................................................................................................

Fig. 3. Western blot of OMPs probed sequentially with antibodies to TdfH and the iron-regulated protein FetA (FrpB). Lanes : 1, FA1090 ; 2, FA6970 (FA1090 tdfH : : Ω) ; 3, FA1090 (ironreplete) ; 4, FA1090 (iron-depleted). The presence of increased amounts of FetA in lane 4 compared to lane 3 is evidence that this culture was iron-starved.

1

2

3

4

5 kDa

TdfG

fusion proteins (Fig. 2) were expressed in E. coli BL21 using the pET30a vector (which contained an Nterminal hexahistidine tag) and affinity-purified in a NiNTA 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 : : Ω) and FA6970 (FA1090 tdfH : : Ω) 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. Analysis of Coomassie- (Fig. 4) and silver-stained (data not shown) SDS-PAGE gels containing outer-membrane preparations showed an iron-regulated protein band of approximately 130 kDa (predicted size 134 kDa) for FA1090 that was absent in the tdfG isogenic mutant (FA6969). Examination of Coomassie- and silverstained SDS-PAGE gels of OMPs from FA1090, FA6970 (FA1090 tdfH : : Ω), FAM20 and FA7030 (FAM20 tdfH : : Ω) failed to identify a TdfH-specific band (data not shown) due to the presence of several co-migratory bands that gave a similar apparent molecular mass (e.g. TbpA, the 97 kDa HmBP).

116

Strain survey 97

66

.................................................................................................................................................

Fig. 4. Detection and regulation of TdfG in gonococcal strain FA1090. Coomassie-stained 12 % SDS-PAGE gel containing gonococcal strains FA1090 and FA6969 (FA1090 tdfG : : Ω) OMP preparations. Lanes : 1, FA1090 (iron-replete) ; 2, FA1090 (irondepleted) ; 3, FA6969 (iron-depleted) ; 4, FA6969 (iron-replete) ; 5, high molecular mass marker.

were constructed by insertional inactivation of the respective ORFs with an Ω (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 Ω 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 1284

Panels of iron-stressed, whole-cell lysates from commensal and pathogenic neisseriae were also assessed for expression and distribution of TdfF, TdfG and TdfH on Western blots. Although DNA sequence homologous to tdfF was present in all three sequenced neisserial genomes, no bands specific to TdfF were seen on Western blots for any of the strains tested (Table 3). As it was possible that the polyclonal antibodies failed to recognize the TdfF portion of the fusion protein, confirmation that the polyclonal antibody could bind to TdfF was obtained by observing an immunoreactive band on Western blots with recombinant TdfF that lacked the original vector-derived N-terminal part of the coding region of the fusion protein including the histidine tag (data not shown). Sequence analysis of the plasmid expressing the recombinant TdfF protein (pUNCH1321) confirmed that it contained the correct sequence. Thus, on the basis of the Western blots (which included analysis of isogenic mutants) TdfF could not be detected in Neisseria spp. TdfG was expressed in 4 of 23 (17 %) gonococcal strains in the Western blot survey and in none of 28 meningococcal nor 8 commensal neisserial strains (Table 3). Unlike tdfF and tdfH, tdfG was not detected in gonococcal strain MS11 and meningococcal strain FAM20 on Southern blots (data not shown). Western blots of neisserial whole-cell lysates showed that TdfH was expressed in 28 of 28 (100 %) meningococcal strains and 47 of 58 (81 %) gonococcal strains and, with the

Neisserial TonB-dependent receptors Table 3. Prevalence of TdfF, TdfG and TdfH among Neisseria spp. .................................................................................................................................................................................................................................................................................................................

Data are based on the number of whole-cell lysates from strains that gave immunoreactive bands of the predicted molecular mass on Western blots probed with polyclonal antibodies to TdfF, TdfG or TdfH. Species

N. meningitidis

N. gonorrhoeae

Group/serotype

No. of strains expressing TdfF

No. of strains expressing TdfG

No. of strains expressing TdfH

Group A Group B Group C Group Y Group E All groups Serotype 1A Serotype 1B All serotypes

0\10 0\3 0\9 0\5 0\1 0\28 0\10 0\13 0\23 0\1 0\1 0\1 0\1 0\1 0\1 0\1

0\10 0\3 0\9 0\5 0\1 0\28 1\10 3\13 4\23 0\1 0\1 0\1 0\1 0\1 0\1 0\1

0\1 0\8

0\1 0\8

10\10 3\3 9\9 5\5 1\1 28\28 15\17 32\41 47\58 1\6 0\1 0\2 0\2 0\1 0\1 0\1 0\1 0\2 1\17

N. lactamica N. cinerea N. flava N. flavescens N. mucosa N. perflava N. subflava N. polysaccharea N. sicca All commensals

(a)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

kDa 97

(b)

18

19

20

21

22

23

24

25

26

27

97

.................................................................................................................................................................................................................................................................................................................

Fig. 5. Western blots showing expression of TdfH among Neisseria spp. (a) Whole-cell lysates of gonococci and commensal neisseriae probed with polyclonal rabbit antiserum to recombinant TdfH. Lanes : 1, FA1090 (positive control) ; 2, FA6970 (FA1090 tdfH : : Ω) (negative control) ; 3, N. subflava ; 4, N. flava; 5, N. flavescens; 6, N. lactamica; 7–17, strains of N. gonorrhoeae. (b) Lanes : 18, FA1090 (positive control) ; 19–23, strains of N. meningitidis ; 24–27, strains of N. lactamica.

exception of one out of six strains of N. lactamica, in none of eight different species of commensal neisseriae (Table 3 and Fig. 5). Unlike FetA (FrpB), which was used as a control for evidence of iron regulation, expression of TdfH in FA1090 and FAM20 outer-membrane preparations and whole-cell lysates was not affected by availability of iron during growth (Fig. 3 and data not shown).

Role of TdfG and TdfH in haem utilization

As TdfG and TdfH showed identities of 11 and 16 %, respectively, to the haemophore receptor (HasR ; Letoffe et al., 1994), the isogenic mutants FA6969 (FA1090 tdfG : : Ω), FA6970 (FA1090 tdfH : : Ω) and FA7031 (FAM20 tdfH : : Ω) were tested for growth in the presence of haem as a sole iron source using step dilution plates or 1285


growth in broth. All three mutants grew as well as the parent strains (FA1090 and FAM20) with haem as sole iron source (data not shown). 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 : : Ω) (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 fol1286

(a) kDa

1

2

3

4

10

11

12

5

6

7

8

97

44 (b) 9

37

.................................................................................................................................................

Fig. 6. Western blot of trypsin-exposed whole bacteria probed with polyclonal antibody to TdfH (a) or FbpA (b). Suspensions of gonococcal strain FA1090 or meningococcal strain FAM20 in exponential growth phase were exposed to 2 mg trypsin ml−1 at 37 mC for either 0, 10, 20 or 40 min. Whole-cell lysates from these suspensions were probed on Western blots with polyclonal antibody to TdfH or FbpA. (a) Lanes : 1, FA1090 at 0 min ; 2, FA1090 at 10 min ; 3, FA1090 at 20 min ; 4, FA1090 at 40 min ; 5, FAM20 at 0 min ; 6, FAM20 at 10 min ; 7, FAM20 at 20 min ; 8, FAM20 at 40 min. (b) Lanes : 9, FAM20 at 0 min ; 10, FAM20 at 10 min ; 11, FAM20 at 20 min ; 12, FAM20 at 40 min. Unlike TdfH (97 kDa), no tryptic fragments were seen with the periplasmic protein FbpA (37 kDa) following exposure to trypsin.

lowing 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). DISCUSSION

We carried out computer searches of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative noncontiguous genes were found and three of these (identified in gonococcal strain FA1090) were examined for expression, regulation and distribution among both pathogenic and commensal neisseriae. Partial characterization of one of the proteins, TdfH, was also undertaken. Homology of TdfF, TdfG and TdfH to the TonBdependent family of proteins was confirmed by amino

Neisserial TonB-dependent receptors

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  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. 1287


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 TonBdependent vitamin B receptor (BtuB) of E. coli which is "# (Heller et al., 1988 ; Heller & also not iron-regulated 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 surfaceexposed 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. ACKNOWLEDGEMENTS This work was supported by the Wellcome Trust (044338\Z\95\Z to P. C. T.) and the National Institute of Health (AI 26837 and AI 31496 to P. F. S.). We are also indebted to the FA1090 gonococcal, MC58 meningococcal and Z2491 meningococcal genome sequencing projects. We thank A. Rountree for expert technical assistance and all the members of the Sparling laboratory for helpful comments and suggestions. We also thank T. Mietzner for the polyclonal antibody against FbpA, B. C. Lee for the polyclonal antibody to HmBP, Janne Cannon and Jo-Ann Dempsey for providing most of the commensal strains and Marcia Hobbs and R. Brunham for providing most of the gonococcal strains.

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Received 24 January 2001 ; accepted 2 February 2001.

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