Antigenic and genetic characterization of aputative hybrid transferrin-binding protein Bfrom Neisseria meningitidis

Tamara Menéndez, Mairet Pérez, and Anabel Alvarez

Abstract: The transferrin-binding protein Bs (TbpBs) from the bacterium Neisseria meningitidis have been divided intotwo families according to genetic and antigenic features. TbpB from meningococcal strain B385 showed a molecularmass similar to that exhibited by TbpBs belonging to the high molecular mass family of TbpBs. TbpB was recognizedby immunoassay using a specific serum directed against the TbpB of the reference strain for this family (strain M982).It was also recognized by a serum elicited against the TbpB of the reference strain for the low molecular mass family(strain B16B6). The tbpB gene from strain B385 was cloned and sequenced. The highest degree of sequence homologywas found to be with the TbpBs belonging to the high molecular mass family, although a region of 14 amino acidsthat is only present in the TbpB from strain B16B6 was also found. This report illustrates a TbpB that shows hybridantigenic and genetic behaviour.

Key words: Neisseria meningitidis, transferrin-binding proteins, TbpB families.

Résumé : Les protéines B de liaison à la transferrine (TbpBs) du Neisseria meningitidis ont été classées en deuxfamilles d’après leurs propriétés génétiques et antigéniques. La TbpB de la souche de méningocoque B385 avait lamême masse moléculaire que celui des TbpBs appartenant à la famille des TbpBs de masse moléculaire élevée. Lorsd’immunoessais, la TbpB était reconnue par un sérum spécifique dirigé contre la TbpB de la souche de référence decette famille (souche M982). La TbpB était aussi reconnue par un immunsérum produit contre la TbpB de la souche deréférence de la famille de masse moléculaire faible (souche B16B6). Le gène tbpB de la souche B385 a pu être clonéet séquencé. Le plus fort degré d’homologie séquentielle a été observé avec les TbpBs appartenant à la famille demasse moléculaire élevée, mais il y avait une région de 14 acides aminés qui se retrouvait uniquement dans la TbpBde la souche B16B6. Ces résultats décrivent une TbpB qui présente un comportement génétique et antigénétiquehybride.

Mots clés : Neisseria meningitidis, protéines de liaison à la transferrine, familles de protéines TbpBs.

[Traduit par la Rédaction] Notes 1054

When grown in conditions of iron restriction, Neisseriameningitidis expresses several new proteins, two of which,TbpA and TbpB, are transferrin-binding proteins (Tbps).These Tbps are components of the meningococcaltransferrin receptor, which is involved in the acquisition ofiron from human transferrin during growth in vivo(Schryvers and Morris 1988; Legrain et al. 1993; Gorringe etal. 1995). As recently reviewed (Ala’Aldeen 1996), TbpBappears to be an attractive vaccine candidate, since it consti-tutes a surface-exposed molecule (Schryvers and Morris1988; Irwin et al. 1993) that induces antibodies in animalsand humans (Gorringe et al. 1995; Ferreirós et al. 1993;Danve et al. 1993; Ala’Aldeen et al. 1994; Lissolo et al.

1995; Ala’Aldeen and Borriello 1996). These antibodies arebactericidal and protective, and also inhibit bacterial growth(Lissolo et al. 1995; Ala’Aldeen and Borriello 1996; Pintoret al. 1996).

Most clinical meningococcal isolates express TbpB pro-teins in the molecular mass range of 85–88 kDa. Few iso-lates, however, express a low molecular mass TbpB (68–70 kDa) (Ferreirós et al. 1991; Rokbi et al. 1993, 1997).These two groups also have different antigenic properties(Rokbi et al. 1993). These differences form the basis for theclassification of TbpBs from N. meningitidis into two fami-lies, the high and low molecular mass families. The strainsM982 and B16B6 have been designated as reference strainsfor each of these two families, respectively (Legrain et al.1993; Rokbi et al. 1993).

According to recent studies (Johnson et al. 1997), it isworth noting that for a TbpB or an outer membrane proteinbased vaccine against N. meningitidis to be broadly cross-protective, at least one TbpB of each family needs to be in-cluded in the formulation. Therefore, it was important for usto know to which family belonged the TbpB derived fromthe strain B385. The meningococcal serogroup B strainB385 is the most frequent clinical isolate in our country.

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Received March 9, 1999. Revision received August 19, 1999.Accepted August 20, 1999.

T. Menéndez,1 M. Pérez, and A. Alvarez. División deVacunas, Centro de Ingeniería Genética y Biotecnología,Ave 31, entre 158 y 190 Apartado 6162, CP 10600,Cubanacán, La Habana, Cuba.

1Author to whom all correspondence should be addressed(e-mail: aalvarez@cigb.edu.cu).

The development of outer membrane protein based vac-cines constitutes an attractive strategy against the diseasecaused by serogroup B strains of N. meningitidis, becauseserogroup B capsular polysacharide is poorly immunogenicin humans (Mandrell and Zollinger 1982). For example, thecurrent available Cuban antimeningococcal vaccine VA-MENGOC-BC is composed by outer membrane vesiclesfrom the strain B385, in which outer membrane proteins aremajor components (Sierra et al. 1991).

The N. meningitidis B385 (B:4:P1.19,15) used in thisstudy was kindly provided by colleagues of the Finlay Insti-tute (Havana, Cuba). The N. meningitidis B16B6 (B:2a:P1.2)and M978 (B:8:P1.1,7) were kindly provided by Dr. MarkAchtman (Berlin, Germany). They were grown in brain heartinfusion (BHI) overnight at 37°C with the candle jarmethod. The BHI contained 50 µM of ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA).

The outer membrane vesicles (OMVs) from N. meningi-tidis were obtained using the LiCl extraction method (Moccaand Frasch 1982). In every case, 20 µg of proteins, as deter-mined following the method of Lowry et al. (1951), wereloaded per lane and subjected to 10% SDS–PAGE as de-scribed by Laemmli (1970). The proteins were transferredfrom acrylamide gels to 0.45-µm pore size nitrocellulosemembranes (Amersham, England). The membranes wereblocked with 5% w/v skimmed milk in phospate-buffered sa-line (PBS) with 0.05% v/v Tween 20 (blocking solution),washed with PBS – 0.05% v/v Tween 20 (PBST), and incu-bated for 1 h at 37°C with blocking solution containing hu-man transferrin (hTf) conjugated with horseradishperoxidase (HRP) or rabbit sera directed against the TbpA–TbpB complex of N. meningitidis M982 or B16B6. The rab-bit sera were kindly provided by Dr. Quentin-Millet (Marcyl’Étoile, France). The membranes incubated with antibodieswere washed with PBST and incubated for 1 h at 37°C withdonkey anti-rabbit immunoglobulins conjugated with HRP(Amersham, U.K.). Finally, all the membranes were washedagain and the reactions developed with 50 mM NaAc buffer

– 0.2 mg 3-amino-9-ethyl-carbazole/mL – 0.015% v/v hy-drogen peroxide for 5 min.

Polymerase chain reaction (PCR) was used for the ampli-fication of tbpB gene from strain B385, and it was per-formed under the following conditions: 30 cycles of 95ºC for1 min, 60ºC for 1 min, and 72ºC for 2 min. The reactioncomponents were 1 µg of N. meningitidis total chromosomalDNA, 50 pmol of each primer (5′GCTCTAGATTGTCTGG-GTGGCGGCGG3′ (sense) and 5′CGGGATCCTTATTGCA-CAAGCTG TTGGCG3′ (antisense)), 200 µM of eachdeoxynucleoside triphosphate (dNTP), PCR buffer (10 mMKCl – 20 mM Tris–HCl (pH 8.8) – 10 mM (NH4)2SO4 –2 mM MgSO4 – 0.1% v/v Triton X-100), double-distilledwater up to a final volume of 75 µL, and 1 U per reaction ofThermus aquaticus DNA polymerase. The amplified DNAfragment was purified from low-melting-point agarose gelsas described (Sambrook et al. 1989) and cloned, unmodified,into a thymidine tailed vector (Marchuck et al. 1991), usingthe pMOS-Blue T-vector kit (Amersham, U.K.), followingthe instructions of the manufacturer.

The DNA sequence of the tbpB gene was performed fol-lowing the dideoxy-chain termination method (Sanger et al.1977) using the Sequenase 2.0 kit (Amersham-USB,U.S.A.), according to the manufacturer’s instructions. Rou-tine recombinant DNA techniques were performed as de-scribed (Sambrook et al. 1989). The consensus multiplesequence alignments were performed with the use of CLUSTAL

W program (Thompson et al. 1994).Figure 1 shows the recognition of OMVs from N. me-

ningitidis B385, B16B6, and M978 (TbpB from the strainM978 belongs to the high molecular mass family (Mazarinet al. 1995)) by hTf and two specific rabbit sera directedagainst the TbpA–TbpB complex of N. meningitidis M982and B16B6. The TbpB from strains B385 and M978 exhib-ited a similar molecular mass (Fig. 1A). When the antigenicproperties were analysed, we found that the TbpB fromN. meningitidis B385 was well recognized by the sera di-rected against the Tbps of N. meningitidis M982 (Fig. 1B).

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Fig. 1. Western blotting analysis of OMV from N. meningitidis M978 (lane 1), B16B6 (lane 2), and B385 (lane 3), grown under ironrestriction conditions. Membranes were incubated with (A) human transferrin, (B) rabbit sera directed against the TbpA–TbpB complexof strain M982, or (C) rabbit sera directed against the TbpA–TbpB complex of strain B16B6. The arrow indicates the TbpB protein.

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Surprisingly, the TbpB was also recognized, although withless intensity, by the sera directed against the Tbps ofN. meningitidis B16B6 (Fig. 1C), while the TbpB fromstrain M978 was not recognized by the latter sera. This find-ing prompted us to study the TbpB coding gene at the mo-lecular level.

We designed specific primers for PCR amplification of theDNA fragment containing the gene encoding the matureTbpB (i.e., without the signal peptide) of N. meningitidisB385. The primers were designed based on the previouslyreported sequences of TbpB (Legrain et al. 1993; Mazarin etal. 1995). The tbpB gene was amplified and completely se-quenced. This sequence has been submitted to the EMBLNucleotide Sequence Data Library under the accession No.Y16102. The nucleotide and deduced amino acid (aa) se-quences were compared with the tbpB gene and TbpB pro-tein sequences of N. meningitidis B16B6 (EMBL accessionNo. Z15129), M982 (accession No. Z15130) (Legrain et al.1993), 6940 (accession No. X78939), S3032 (accession No.X78940), M978 (accession No. X78941) (Mazarin et al.1995), BZ163 (accession No. Z50731), and BZ83 (accessionNo. Z50732) (Legrain et al. 1996).

The aa sequence deduced from the 2088-bp sequencedgene was 40% identical to TbpB sequence from strainB16B6, and showed 61–74% identity to the sequences fromM982-like strains. These results indicated that TbpB fromN. meningitidis B385 belonged to the M982-like family, ac-cording to the previously reported homology for tbpB allelesbelonging to different groups (Legrain et al. 1993) or thesame group (Mazarin et al. 1995). A detailed analysis of thededuced aa sequence showed that TbpB from strain B385 in-cludes the sequence PVSDMAARTEANAK in the regioncorresponding to aa 538–551. In the rest of TbpBs belongingto the high molecular mass family, the peptide DEKEIP orDENKIP is highly conserved in this region, followed by amore variable stretch. Surprisingly, a very similar peptide(PVSDVAARTEANAK) is present in TbpB from strainB16B6 (Fig. 2).

This finding could reflect the occurrence of horizontal ge-netic exchange in the tbpB locus between the strain B385and a low molecular mass, TbpB-containing strain such asB16B6. There are data which suggest that this mechanismhas an important influence on the population genetics of thenaturally competent microorganism N. meningitidis (re-viewed by Maiden 1993). Rokbi et al. (1995) have suggestedthat genetic exchange is responsible for the mosaic-like or-ganization observed in the tbpB genes belonging to the highmolecular mass family of TbpBs. The sequence analysis ofthe present study suggests that genetic exchange has also oc-curred among the tbpB genes belonging to different families.

The existence of the common peptide identified in thisstudy may explain the recognition of native TbpB fromstrain B385 by the sera that are raised against the TbpA–

TbpB complex from strain B16B6, as noted in the antigenicanalysis shown in Fig. 1. With the exception of this commonpeptide, the homologous regions between TbpBs fromstrains B16B6 and B385 are basically the same regionsshared by TbpBs from strains B16B6 and M982, and thus itis possible to speculate that the peptide PVSDM/VAARTEANAK may be highly immunogenic. This possi-bility is now under investigation.

The classification of TbpBs from N. meningitidis into highand low molecular mass families is relevant, because as ithas been shown (Rokbi et al. 1993), the antibodies directedagainst a TbpB from one family do not cross-react with theTbpBs belonging to the other one. Therefore, it has beensuggested that a recombinant antimeningococcal vaccineshould include at least one TbpB from each family (Johnsonet al. 1997). The discovery of a cross-reactive, hybrid TbpBin strain B385 raises the possibility that this protein is capa-ble of conferring protection against strains belonging to bothfamilies. Further studies are required to elucidate this issue.

Acknowledgements

The authors want to thank the colleagues from the FinlayInstitute (Havana, Cuba) for providing us the meningococcalstrain B385. We are also grateful to Mark Achtman fromMax Planck Institute for Molecular Genetics, Berlin, Ger-many, for providing us the Neisseria meningitidis B16B6and M978, and Dr. Quentin-Millet from Pasteur-Mérieux-Connaught, Marcy l’Étoile, France for providing us the spe-cific rabbit sera.

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Fig. 2. Deduced amino acids sequence alignment of TbpBs from strains B385 and B16B6, and the previously reported sequences ofTbpBs from the M982-like family of N. meningitidis (i.e., M982, 6940, S3032, M978, BZ163, and BZ83). At the top of each block(line 1) is shown the result of the comparison of TbpB from strain B385 and the TbpBs from the high molecular mass family. Theresult of aligning the TbpBs from strains B385 and B16B6 is displayed at the bottom (line 2). Asterisks indicate identical amino acids.Dots indicate homologous amino acids. The conserved sequence stretch of the high molecular mass TbpB family, which is replaced bya different sequence in the TbpBs from strains B385 and B16B6, is boxed.

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