FEMS Microbiology Letters 154 (1997) 117^122

Localization and characterization of a speci¢c linear epitope of the Brucella DnaK protein Nieves Vizca|èno 1 , Michel S. Zygmunt, Jean-Michel Verger, Maggy Grayon, Axel Cloeckaert * Institut National de la Recherche Agronomique, Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France

Received 7 January 1997; revised 17 June 1997; accepted 7 July 1997

Abstract

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the K-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the K-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes. Keywords : Brucella

; DnaK ; Monoclonal antibody; Epitope

1. Introduction

: B. abortus, B. melitensis, B. suis, B. ovis, , and B. neotomae [1]. This classi¢cation is mainly based on the di¡erence in pathogenicity and in host preference [1]. The Brucella species are further subdivided into biovars depending on di¡erential tests based on serotyping, phage typing, dye sensitivity, CO2 requirement, H2 S production, and metabolic properties [2]. A close genetic relationship of brucellae with members of the K-2 subdivision of the class Proteobacteria has been reported [3^5]. Additionally, brucellae share common epitopes, mainly on the smooth lipopolysaccharide, with cross-reactBrucella

B. canis

Brucellae are Gram-negative, facultative intracellular bacteria that can infect many species of animals and man. Six species are recognized within the genus

* Corresponding author. Tel.: +33 2 47 42 78 72; Fax: +33 2 47 42 77 79; E-mail: [email protected] 1

Present address: Dpto. Microbiolog|èa y Geneètica, Edificio Departamental, Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain.

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 3 1 1 - X

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ing strains, of which the most important is Yersinia enterocolitica O:9 [6]. Virulence of brucellae is thought to be essentially due to their capacity to survive and multiply in phagocytic cells such as macrophages. The DnaK protein has been reported to be an essential factor for intracellular multiplication of brucellae in phagocytes [7]. This protein belongs to the hsp70 family which has been highly conserved during evolution [8]. The DnaK protein of Brucella shares 60^78% homology at the amino acid level with that from other bacteria [9]. DnaK induction in Brucella has been shown upon heat stress and acidity [7]. The Brucella DnaK protein has, moreover, been found to be immunogenic in B. melitensis and B. ovis infected sheep [9,10]. We have previously produced and characterized four monoclonal antibodies (MAbs) to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction [11]. In the present study we localized, by a recombinant DNA technique, the DnaK epitopes recognized by the MAbs and further analyzed their speci¢city by reactivity in immunoblotting with all Brucella species and biovars, crossreacting bacteria, and genetically related bacteria of the K-2 subdivision of the class Proteobacteria. 2. Materials and methods

2.1. Bacterial strains and cultivation

The strains used in this study are listed in Table 1. spp., O:9, O:157, , , , , and cultures were grown on tryptic soy agar (Gibco BRL) supplemented with 0.1% (w/v) yeast extract (Difco) (TSAYE) at 37³C. For fastidious strains (B. abortus bv. 2 and B. ovis), sterile horse serum (Gibco BRL) was added to TSAYE to a ¢nal concentration of 5% (v/v) (TSAYES). The Brucella strains were checked for purity and species and biovar characterization by standard procedures [2]. Rhizobium leguminosarum was cultured in tryptone-yeast (TY) medium [12] at 30³C.

Brucella Yersinia enterocolitica Escherichia coli Salmonella urbana Ochrobactrum anthropi Phyllobacterium rubiacearum Agrobacterium radiobacter Agrobacterium tumefaciens

E. coli JM109 carrying plasmid pAC7040 containing the dnaK gene of B. melitensis 16M was cultured on liquid selective LB medium with 50 Wg/ml ampicillin. 2.2. MAbs

Anti-DnaK MAbs A53/01C10/A10 (IgG1), A53/ 09G03/D02 (IgG1), V78/09D04/D08 (IgG2a), and V78/07B01/G11 were produced and characterized as described previously [11]. 2.3. DNase I digestion of plasmid DNA and immunoscreening

The XbaI-SacI insert of plasmid pAC7033 [11] containing the dnaK gene of B. melitensis 16M was ¢rst subcloned into plasmid pGEM-7Zf‡ (Promega) using standard methods as described previously [11,13]. Transformation of E. coli JM109 cells (Promega), plasmid isolation, and insert puri¢cation were done by standard procedures [13]. The puri¢ed XbaI-SacI insert (2300 bp) of the resulting plasmid pAC7040 was digested with DNase I (Boehringer) as described by Mehra et al. [14]. 1 ng of DNase I and 10 Wg of the insert DNA were mixed in 1 ml of 20 mM Tris-HCl (pH 7.5)-4 mM MnCl2 -100 Wg of bovine serum albumin per ml. After 20 min of incubation at 24³C, DNA fragments were separated in a 1% agarose gel. The gel region containing fragments of 100^500 bp was excised, and DNA was puri¢ed with a Geneclean II kit (Bio 101, La Jolla, CA) as instructed by the manufacturer. Fragments were end repaired by treatment with T4 DNA polymerase (Boehringer) in the presence of deoxynucleoside triphosphates and then ligated, into the SmaI site of the lac K-peptide, to pGEM-7Zf‡ . E. coli JM109 cells were then transformed with the recombinant plasmid DNA, and bacterial colonies were screened by colony blotting with the anti-DnaK MAbs. Brie£y, plates were overlaid with a 0.45 Wm pore size nitrocellulose membrane (Millipore) and kept for 2 h at 37³C. Next, the membranes were placed for 10 min over a ¢lter paper soaked with 10% w/v SDS and then washed three times in Tris-bu¡ered saline (TBS; 0.15% w/v NaCl, 10 mM Tris-HCl [pH 7.5]), saturated for 30 min at room temperature with TBS-1% w/v skim milk, and incubated over-

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night at room temperature with the anti-DnaK MAbs (culture supernatants) diluted 1/3 in TBS0.33% w/v skim milk. Binding of the MAbs was detected using, as previously described [11], rabbit antimouse immunoglobulin antiserum (Nordic Immunology, Tilburg, The Netherlands) and protein A-peroxidase conjugate (Sigma, St. Louis, MO) followed by incubation with TBS containing 0.06% w/v 4-chloro-1-naphthol (Sigma) and 5 mM H2 O2 . 2.4. DNA sequencing

119

kit (Epicentre Technologies, Madison, WI) and the pGEM-7Zf SP6 and T7 promoter primers (Promega). 2.5. SDS-PAGE and immunoblotting

SDS-PAGE and immunoblotting were performed as described previously [11]. 3. Results and discussion

The DNA of positive immunoscreened recombinant plasmids containing DNase I-digested dnaK inserts was sequenced by primer-directed dideoxy sequencing [15] with a SequiTherm cycle sequencing

3.1. Localization of a linear epitope on the Brucella DnaK protein

Recombinant plasmids coding for fusion proteins

Table 1 Reaction of the anti-DnaK MAbs to di¡erent bacteria in immunoblotting after SDS-PAGE Bacteria Strain (biovar) Sourcea Reactivity in immunoblotting, after SDS-PAGE, with anti-DnaK MAbs: V78/07B01/G11 V78/09D04/D08 A53/09G03/D03 A53/01C10/A10 B. abortus 544 (biovar 1) ATCC + + + + 86/8/59 (biovar 2) ATCC + + + + Tulya (biovar 3) ATCC + + + + 292 (biovar 4) ATCC + + + + B3196 (biovar 5) ATCC + + + + 870 (biovar 6) ATCC + + + + C68 (biovar 9) ATCC + + + + B. melitensis 16M (biovar 1) ATCC + + + + 63/9 (biovar 2) ATCC + + + + Ether (biovar 3) ATCC + + + + B. suis 1330 (biovar 1) ATCC + + + + Thomsen (biovar 2) ATCC + + + + 686 (biovar 3) ATCC + + + + 40 (biovar 4) ATCC + + + + 513 (biovar 5) BCCN + + + + B. ovis 63/290 ATCC + + + + B. canis RM6/66 ATCC + + + + B. neotomae 5K33 ATCC + + + + Ye8 INRA 3 3 3 3 Ec2 INRA 3 3 3 3 Su1 INRA 3 3 3 3 Oa1 INRA 3 3 + + Pr1 INRA 3 3 + 3 Ar1 INRA 3 3 3 3 At1 INRA 3 3 3 3 Rl1 INRA 3 3 3 3 a ATCC: American Type Culture Collection, Rockville, MD, USA; BCCN: Brucella Culture Collection Nouzilly, France; INRA: Institut National de la Recherche Agronomique, Nouzilly, France. b Antigen cross-reacting bacteria. c Bacteria of the K-2 subdivision of the class Proteobacteria. O:9

b Yersinia enterocolitica b Escherichia coli Salmonella urbanab Ochrobactrum anthropic Phyllobacterium rubiacearumc Agrobacterium radiobacterc Agrobacterium tumefaciensc Rhizobium leguminosarumc

O:157

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Fig. 1. Recombinant plasmids, harboring small fragments of B. melitensis 16M dnaK, expressing the epitope(s) recognized by anti-DnaK MAbs V78/07B01/G11 and V78/09D04/D08. Insert DNA was sequenced and the amino acid sequence deduced. The minimal amino acid sequence shared by the inserts of the ¢ve plasmids extended from amino acid 617 to 637 of the B. melitensis DnaK protein.

containing the epitopes recognized by the anti-DnaK MAbs were identi¢ed by a colony blot technique as described in Section 2. A number of recombinant plasmids gave a positive reaction with MAbs V78/ 07B01/G11 and V78/09D04/D08 but not with MAbs A53/09G03/D02 and A53/01C10/A10. Both MAbs V78/07B01/G11 and V78/09D04/D08 reacted with the same fusion proteins suggesting that these MAbs recognize the same epitope or closely located epitopes. The nucleotide sequences of the insert DNAs from ¢ve recombinant plasmids were determined and translated to amino acids and compared with the B. ovis DnaK sequence [9] (Fig. 1). The determination of the minimal amino acid sequence of B. melitensis 16M DnaK shared by the ¢ve inserts has allowed the localization of the epitope(s) recognized by MAbs V78/07B01/G11 and V78/09D04/D08 within the region of DnaK corresponding to the last

Fig. 2. Reactivity in immunoblotting of anti-DnaK MAbs A53/ 09G03/D02 (lanes 1^3) and A53/01C10/A10 (lanes 4 and 5) after SDS-PAGE of B. melitensis 16M (lanes 1 and 4), Ochrobactrum anthropi (lanes 2 and 5), and Phyllobacterium rubiacearum (lane 3).

21 amino acids of the C-terminal end (amino acids 617^637): SSKDDVVDADYEEIDDNKKSS. This B. melitensis 16M DnaK sequence was identical to that previously reported for the B. ovis DnaK protein [9]. A search for amino acid sequence homologies using the BLAST program [16] revealed that this C-terminal amino acid sequence had the greatest similarity to that of Agrobacterium tumefaciens (84%) and Rhizobium meliloti (80%) DnaK proteins. The C-terminal region represents the peptide-binding domain and the most variable region among DnaK proteins and has been shown in other intracellular pathogens such as Chlamydia trachomatis [17] and Mycobacterium leprae [18,19] to constitute an interesting region regarding speci¢c humoral immune responses. No recombinant fusion proteins were detected with MAbs A53/09G03/D02 and A53/01C10/A10. Therefore, these MAbs could possibly recognize discontinuous epitopes of the DnaK protein. Both MAbs nevertheless showed good antibody reactivities in immunoblotting after SDS-PAGE of whole Brucella cell extracts or E. coli producing the B. melitensis DnaK protein [11]. Simply removal of SDS after transfer of the denatured DnaK protein to nitrocellulose probably allowed its renaturation. Such behavior has also previously been shown with MAbs recognizing discontinuous epitopes of the Chlamydia trachomatis DnaK protein [17]. Although these MAbs also reacted in immunoblot assay, it was not possible to map the epitopes completely by use of synthetic peptides [17]. Interestingly, the discontinuous epitopes recognized by these MAbs were also

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mydia trachomatis

Chla-

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Acknowledgments

DnaK protein [17]. èno was supported by an EEC fellowship. N. Vizca|

3.2. Occurrence of the DnaK epitopes in Brucella spp. and related bacteria

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