INFECTION AND IMMUNITY, May 1994, p. 2051-2057 Vol. 62, No. 50019-9567/94/$04.00+0Copyright ©) 1994, American Society for Microbiology

Characterization of a Linear Epitope on Chlamydia trachomatisSerovar L2 DnaK-Like Protein

SVEND BIRKELUND,I* BENTE LARSEN,"2 ARNE HOLM,3 ANKER G. LUNDEMOSE,'4AND GUNNA CHRISTIANSEN'

Institute of Medical Microbiology, University ofAarhus, DK-8000 Aarhus C, Denmark'; Howard Hughes MedicalInstitute, University of Utah, Salt Lake City, Utah2; Department of Chemistry, Research Center for Medical

Biotechnology, Royal Veterinary and Agricultural University, DK-1870 Copenhagen, Denmark3;and Diabetes Research, Novo Nordisk AIS, DK-2880 Bagsvwrd, Denmark4

Received 11 October 1993/Returned for modification 13 December 1993/Accepted 9 February 1994

A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterizedas being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for onemonoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA technique, the epitope waslimited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six ofthese amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The aminoacid sequence homologous to the epitope is located in a linear part of the HSP70 molecule known as connect II.

The genus Chlamydia is divided into three species, Chla-mydia trachomatis, C. pneumoniae, and C. psittaci. C. tracho-matis and C. pneumoniae are human pathogens. The hosts forC. psittaci are nonprimate vertebrates, but occasionally humansare infected with C. psittaci. Chlamydiae have a membranestructure and lipopolysaccharide (LPS) like those of gram-negative bacteria. C. trachomatis is an obligate intracellularparasite with a characteristic biphasic life cycle. The extracel-lular form, the elementary bodies (EBs), are the infectiousparticles, which upon infection enter the eukaryotic host cells.After internalization (2 to 8 h), the EBs transform intoreticulate bodies. Reticulate bodies reorganize to EBs 20 to 36h after infection of the host cell. C. trachomatis causes humandiseases such as trachoma, genital tract infections, lympho-granuloma venereum, inclusion conjunctivitis, and pneumonia.C. trachomatis has also been associated with Reiter's syndromeand with reactive arthritis (20).The major targets of the humoral immune response to a C.

trachomatis infection are the two chlamydial outer membraneproteins MOMP (major outer membrane protein) and Omp2,LPS, and the two cytoplasmic heat shock proteins (HSPs)DnaK and GroEL (8). During the transformation of EBs toreticulate bodies, the genes dnaK and groEL, encoding theHSPs DnaK and GroEL and which are involved in proteinfolding and are also known as chaperones, are highly tran-scribed (23). Antibodies to C. trachomatis DnaK and GroELmay have an impact on other infections because HSPs arehighly conserved in evolution. C. trachomatis L2 DnaK has49% amino acid identity to human HSP70 (22), with theN-terminal two-thirds of HSP70 and DnaK being the mostconserved part. It contains an ATP binding site and ATPaseactivity as well as autophosphorylating activity (37). TheATPase part of bovine HSP70 has been analyzed by X-raycrystallography (13). The C-terminal one-third of DnaK andHSP70 comprises the variable substrate recognition domain;this part of the molecule has a possible three-dimensional

* Corresponding author. Mailing address: Institute of Medical Mi-crobiology, The Bartholin Building, University of Aarhus, DK-8000Aarhus C, Denmark. Phone: (+45) 8613-9711. Fax: (+45) 8619-6128.Electronic mail address: chlam@biobase.aau.dk.

structure similar to that of human major histocompatibilitycomplex class I antigen HLA (28).We have previously cloned the C. trachomatis dnaK-like

gene and shown that patient sera contained antibodies againstC. trachomatis DnaK (6, 7).

Antigenic determinants, or epitopes, are the specific seg-ments of antigens that are recognized by antibodies or T-cellreceptors. Since bacterial and parasite HSPs are major targetsof the immune system, cross-reacting and autoantibodies maybe formed against these proteins. To characterize the immuneresponse to C. trachomatis DnaK and to search for conservedepitopes on the molecule, we performed epitope mapping of amonoclonal antibody (MAb) against C. trachomatis DnaK. Theepitope mapped to a highly conserved part of the ATPasedomain. The antibody cross-reacted with DnaK from genera inwhich the full epitope was conserved and thus did not react inan autoimmune manner.

MATERIALS AND METHODS

Production and isotyping of MAbs. BALB/c mice wereimmunized according to the method of Birkelund et al. (5); atday 93, the spleen cells (5 x 108) were fused with 2.5 x 108NS-1 azoguanine-resistant cells (American Type Culture Col-lection) in the presence of 50% polyethylene glycol. Fusionand subcloning were done as described by Birkelund et al. (5).

Isotyping of antibodies. Isotyping of MAbs was done byusing the Ouchterlony double-diffusion test. For detection ofsubtypes, rabbit anti-mouse antibodies specific to each of theimmunoglobulins (Ig) (IgGi, IgG2A, IgG2B [Meloy, Spring-field, Va.], and IgG3 [Zymed]) were used.

Microorganisms and cultivation. Campylobacter jejuniNCTC 11168 was cultivated in serum bouillon in a 5%C02-10% 02-85% N2 atmosphere at 37°C. Yersinia enteroco-litica P1089 was cultured in serum bouillon at 37°C. Neisseriagonorrhoeae was cultured on chocolate agar plates at 37°C ina 10% CO2 atmosphere. Salmonella typhimurium U7 wascultured in LB medium at 37°C. Campylobacter jejluni, Yenterocolitica, N. gonorrhoeae, and S. typhimuirium were kindlyprovided by Wilhelm Frederiksen, Statens Seruminstitut

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2052 BIRKELUND ET AL.

Copenhagen, Denmark. Borrelia burgdorferi antigen was ob-tained from H. Ern0, Institute of Medical Microbiology,Aarhus University, Aarhus, Denmark.HeLa cells were cultivated in RPMI 1640 (GIBCO Labora-

tories, Grand Island, N.Y.) containing HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, 10% basalmedium supplements (Bio-Chrom KB, Berlin, Germany), and5 pLg of gentamicin per ml. Escherichia coli K-12 p1248 wascultured in Luria-Bertani medium containing kanamycin (5jg/ml). Legionella pneumophila (kindly provided by BirgitteKorsager, Institute of Clinical Microbiology, Aalborg, Den-mark) was grown on buffered charcoal-yeast extract agar withOL-ketoglutarate (pH 6.9) at 35°C and 90% humidity.An overnight culture of the microorganisms in liquid me-

dium was diluted 1:100 in Luria-Bertani or serum medium andthen incubated for 4 h. Half of the culture was then heatshocked (43°C for 1.5 h).

C. trachomatis LGV-II/434/Bu, C. psittaci (MN), and C.pneumoniae (VR-1310) were cultivated as previously described(5), except that C. pneumoniae was cultivated in HeLa cellsinstead of McCoy cells.Recombinant clones. Recombinant E. coli K-12 pL248 har-

boring plasmid pCtX2-43 was used. pCtX2-43 has a 4-kb insertof C. trachomatis L2 DNA with the grpE and dnaK-like genes.The complete Chlamydia DnaK-like protein is expressed bythe recombinant bacteria (6, 7). An alkaline lysis method wasused for plasmid extraction (30).

Immunoblotting (Western blotting). Antigens suspended insodium dodecyl sulfate (SDS) sample buffer were incubatedfor 30 min at 60°C and separated by SDS-10% polyacrylamidegel electrophoresis (PAGE). Transfer and immunodetection ofantigens were done according to the Bio-Rad immunoblottingprocedure, except that 150 mM NaCl was used instead of 500mM NaCl in all buffers. BA85 nitrocellulose membranes(Schleicher & Schuell, Inc., Keene, N.H.) were used fortransfer.

Antibodies were diluted 1:10 in antibody buffer (20 mM Tris,150 mM NaCl, 0.05% Tween 20, 0.2% gelatin [pH 7.5]) andused for immunodetection. Goat anti-mouse IgG (heavy andlight chain specific)-horseradish peroxidase conjugates diluted1:3,000 (Bio-Rad Laboratories, Richmond, Calif.) were used assecondary antibodies.

Immunofluorescence. C. trachomatis serotypes L2, D, E, F,G, H, and K and C. psittaci MN were cultivated in McCoy cells.C. pneumoniae VR-1310 was cultivated in HeLa cells. Immu-nofluorescence analysis was done as described by Birkelund etal. (6).

Subcloning of digested pCtX2-43 DNA into vector pEX2.The pCtX2-43 DNA was digested with DNase I in a buffercontaining 20 mM Tris-HCl (pH 7.5), 4 mM MnCl2, and 100,ug of bovine serum albumin per ml according to the method ofMehra et al. (25). The DNA was digested at room temperature(24°C) for 10 to 60 min to produce short random fragments.The DNA was fractionated in a 1.2% agarose gel, and

fragments of 100 to 500 bp were isolated in a Biotrap (Schlei-cher & Schuell, Dassel, Germany).The isolated DNA fragments were end repaired by treat-

ment with bacteriophage T4 DNA polymerase (BoehringerGmbH, Mannheim, Germany) in the presence of deoxynucleo-side triphosphates. A blunt-end ligation was performed withthe expression vector pEX2 (Boehringer) (30), which wasopened with SmaI (Boehringer), and the hybrid DNA mole-cules were transformed into E. coli pL248. Recombinantbacteria were screened by colony blotting with MAb 35.2, aspreviously described (6, 32).DNA sequence analysis of recombinant clones. The plasmid

DNA was sequenced by the method described by Hattori andSakaki (16), with [at-32P]dATP (Amersham) and T7 DNApolymerase (Pharmacia).

Peptide synthesis. Solid-phase peptide synthesis was per-formed by the fluorenylmethyloxycarbonyl (Fmoc) strategy (1),using Fmoc amino acid pentafluorophenol esters (Milligen)and a Pepsyn KA resin (Milligen) as the solid support with thefirst amino acid attached (substitution = 0.1 mmol/g). Synthe-sis of the overlapping eight-residue peptides (amino acids 357to 362) was performed on a parallel multiple-column peptidesynthesizer developed in our laboratory (18, 19, 26). The otherpeptides were synthesized by the continuous-flow version ofthe polyamide solid-phase method (12) on a fully automatedpeptide synthesizer, as described previously (27). Solvent N,N-dimethylformamide for peptide synthesis was distilled at re-duced pressure and analyzed for free amines by the addition of3-hydroxy-1,2,3-benzotriazin-4(3H)-one (Aldrich) prior to use.The peptides assembled on the resins were cleaved from theresin with trifluoroacetic acid (TFA-H20; 95:5, vol/vol) atroom temperature for 2 h, which was followed by a wash withTFA-H20 (95:5, vol/vol). The combined TFA washing solu-tions were concentrated in vacuo, and the peptide was precip-itated and washed with ether, dried, purified by gel filtration ona Sephadex G-15 column (Pharmacia, Uppsala, Sweden), andlyophilized. Amino acid analyses were performed by the Pl-COTAG method (Waters). High-performance liquid chroma-tography (HPLC) was performed on a Waters HPLC system,using a C18 reversed-phase column (Hamilton PRP-3; flowrate, 1.5 ml/min for analytical preparations), with a bufferconsisting of 0.1% TFA and a buffer consisting of 0.1%TFA-10% water in acetonitrile. Amino acid analysis wassatisfactory for all peptides.

Inhibitory enzyme-linked immunosorbent assay (ELISA).The recombinant clone pCtX2-43 expressing C. trachomatisDnaK was lysed in 2% SDS and diluted 1:500 in carbonatebuffer (pH 9.6) to a final concentration of 2 pLg of protein perml. Microtiter plates (Nunc) were coated for 1 h at 37°C withthe antigen suspension. Excess binding capacity was blockedwith 1% gelatin in phosphate-buffered saline. MAb (1.5 pig/ml)and peptides (0.5 to 2,280 nM) were preincubated for I h in amicrotiter plate before the mixtures were transferred to theantigen-coated microtiter plate. The antibody binding wasmeasured with goat anti-mouse IgG horseradish peroxidaseconjugate and OPD (15).

Purification of C. trachomatis DnaK. Plasmid DNA from therecombinant clone pCtX2-43 (6, 7), containing the completednaK gene from C. trachomatis serovar L2, was digested withrestriction endonuclease StyI (Boehringer). A 2,175-bp frag-ment starting at bp 45 in the open reading frame of the dnaKgene was produced. The sticky ends of the fragment were filledin with T4 DNA polymerase (Boehringer) and deoxynucleo-side triphosphate. pGEX-3X DNA (31) was opened with SmaI(Boehringer), treated with calf intestinal alkaline phosphatase(Boehringer), and ligated with the 2,175-bp fragment, using T4ligase (Boehringer). The mixture was transformed into E. coliXL1-blue (Stratagene, La Jolla, Calif.). The recombinantclones were induced for 3 h with 0.4 mM IPTG (isopropyl-3-D-thiogalactopyranoside) and tested with MAb 18.1 specific forChlamydia DnaK (5, 31). The produced fusion protein con-tained glutathione S-transferase (GST) and DnaK from aminoacids 16 to 660. The GST-DnaK fusion protein was purified onglutathione beads as described before (31) and was used in theinhibitory ELISA in concentrations of 1.25 to 50 nM.

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TABLE 1. Reaction of MAb 35.2 to different microorganisms

Microorganism Reaction" by:or cells Immunoblotting Immunofluorescence

C. trachomatis L2 + +C. trachomatis A NDh +C. trachomatis B ND +C. trachomatis C ND +C. pneumoniae ND +C. psittaci ND +B. burgdorferi + NDM. tuberculosis + NDE. coli - NDY enterocolitica - NDN. gonorrhoeae - NDS. typhimurium - NDL. pneumophila - NDBacillus subtilis - NDHeLaMcCoy ND

" +, positive reaction; -, negative reaction.h ND, not determined.

RESULTS

Characterization of MAb 35.2. MAb 35.2 was obtained afterimmunization of a mouse with purified EB of C. trachomatis L2and fusion of spleen cells with NS-1 myeloma cells, as previ-ously described by Birkelund et al. (6). ELISA-positive cloneswere tested by immunoblotting, and one MAb (MAb 35.2)reacted with a 75-kDa C. trachomatis protein. The MAb alsoreacted with the recombinant E. coli pCtX2-43 that expresses

C. trachomatis L2 DnaK (6, 7). The antibody was of the IgGlimmunoglobulin subtype.To characterize the possible cross-reaction of MAb 35.2 with

other microorganisms and eukaryotic HSP70, indirect immu-nofluorescence was used for Chlamydia species and C. tracho-matis serotypes. MAb 35.2 reacted with all tested serotypes ofC. trachomatis, with C. pneumoniae, and with C. psittaci.

CoCo cJ .0N m

00)r 00r N a cs e N C'h C'J

c'x x x x x x

KDa V

.

67

43.

FIG. 1. Coomassie brilliant blue-stained SDS-7.5% PAGE of re-

combinant E. co/i strains containing insets of different sizes. Arrow

indicates the location of the Cro-JI-galactosidase DnaK hybrid proteins

containing the epitope for MAb 35.2.

TABLE 2. Alignment of recombinant clones, expressing the epitopefor MAb 35.2, to C. trachomatis DnaKa

Clone Amino acids

43X79a .............................................. 359-43443X23a .............................................. 337-61943X34a .............................................. 357-42343X73a .............................................. 352-38843X62a .............................................. 334-38943X3b .............................................. 353-37243X87a .............................................. 352-384

a Limitations of the epitope shared with DnaK were amino acids 359 to 372.

Immunoblotting of heat-shocked cells was used to analyze thereactivity to other microbial genera and to eukaryotic cells.MAb 35.2 reacted with a 75-kDa antigen in B. burgdorferi andMycobacterium tuberculosis (Table 1). No reaction was seen toheat-shocked E. coli or Salmonella, Yersinia, Neisseria, Campy-lobacter, or Legionella species or to HeLa and McCoy cells ofhuman and murine origins, respectively (Table 1).

Epitope mapping by recombinant fusion protein analysis.Plasmid pCtX2-43, containing a 4.0-kb C. trachomatis L2 DNAfragment encoding the grpE and dnaK genes, was randomlycleaved to fragments of 100 to 400 bp and subcloned intoexpression vector pEX2. The recombinant clones were thenscreened with MAb 35.2 specific for DnaK. Seven clones were

recognized by the MAb (Fig. 1).The insert DNAs of the seven clones were sequenced. The

nucleotide sequences were translated to amino acids andcompared with the DnaK sequence (Table 2). The minimalamino acid sequence shared by the seven clones consisted ofthe 14 amino acids 359PNKGVNPDEVVAIG372.

Epitope mapping with synthetic peptides. To further analyzethe epitope found by recombinant fusion protein analysis, a

peptide (360NKGVNPDEVVAIG372) identical to 13 of the 14amino acids in the epitope and the shorter peptides365PDEVVAIG372 and 63 VNPDEVVAIG372 were synthe-sized. The binding of the oligopeptides to MAb 35.2 was donein solution. The unbound MAb 35.2 was measured by trans-ferring the peptide antibody mixture to microtiter platescoated with recombinant bacteria expressing C. trachomatis L2DnaK. Only the 13'mer oligopeptide could prevent the bindingof MAb 35.2 to DnaK (Table 3). Thus, the N-terminal aminoacids were part of the epitope for MAb 35.2. To further limitthe epitope, six 8'mer peptides displaced by one amino acidwere synthesized with start points from amino acids 357 to 362.The inhibitory effects of the peptides on the binding of MAb35.2 to DnaK were measured by an ELISA at peptide concen-

trations of 0.5 to 2,200 nM (Fig. 2). The 8'mer 360NKGVNPDE367 showed 50% inhibition at 20 nM peptide. The 8'mer359PNKGVNPD366 showed 50% inhibition at 1,000 nM,

TABLE 3. Reaction of MAb 35.2 with synthetic peptidesReaction with MAb 35.2 Peptide

Positive.....36"NKGVNPDEVVAIG372Negative.... 363'VNPDEVVAIG372Negative.....3"5PDEVVAIG372Negative....357KEPNKGVN364Negative....358EPNKGVNPD366Negative.... 359PNKGVNPD 366

Positive...... 36"NKGVNPDE367Negative.... 36'1KGVNPDEV365Negative.... 362GVNPDEVV369

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100 °

80

c

.2 60

c~ ~ ~

40

20

0

2 nM 9 nM 35 nM 140 nM 570 nM 2280 nMPeptide concentration nM

FIG. 2. Graph displaying the percent inhibition by 8'mer peptides(one 9'mer was used) of the binding of MAb 35.2 to C. trachomatisDnaK measured with an ELISA. The peptides cover the sequencefrom amino acids 357 to 369 in C. trachomatis DnaK. A concentrationof approximately 20 nM peptide 360NKGVNPDE367 shows 50% inhi-bition. Symbols: U, KEPNKGVN; *, EPNKGVNPD; A,PNKGVNPD; O, NKGVNPDE; O, KGVNPDEV; A, GVNPDEVV.

whereas the 8'mer 36'KGVNPDEV368 showed no inhibition ofthe binding of MAb 35.2 to DnaK. To analyze whether the8'mer 360NKGVNPDE367 was the full-length epitope, thepeptides were extended to 12'mers. The 12'mer peptides wereobtained by extending the 8'mer peptides. The 12'mer356GKEPNKGVNPDE367 extended four amino acids in theN-terminal end had a decreased inhibitory effect on thebinding of MAb 35.2 to DnaK (Fig. 3). Peptides 357KEPNKGVNPDEV368 and 358EPNKGVNPDEVV369 both showed50% inhibition at 20 nM (Fig. 3). This indicates that extensionwith four amino acids at the N-terminal end of the epitopechanges its conformation, but when one and two amino acidswere added at the C-terminal end of the epitope, 50%inhibition was obtained with 20 nM peptide, as with the 8'mer360NKGVNPDE367. None of the 12'mers had a higher affinityfor the epitope than the 8'mer 360NKGVNPDE36 , indicatingthat it represents the full-length epitope for MAb 35.2.To analyze whether the linear epitope specified by the

peptides had the same conformation as the epitope in thenative protein, we compared the inhibitory effect of purified

100

80

nM 2 nM 9 nM 35 nM 140 nM 570 nM 2280 nMPeptide concentration nM

FIG. 3. The 8'mer peptides shown in Fig. 2 were extended withfour amino acids of the DnaK sequence. The histogram shows theinhibitory effect of the peptides, measured with an ELISA, on thebinding of MAb 35.2 to C. trachomatis DnaK. Symbols: E1, EIFGKEPNKGVN; *, IFGKEPNKGVNPD; A, FGKEPNKGVNPD;C]1, GKEPNKGVNPDE; O, KEPNKGVNPDEV; A, EPNKGVNPDEVV.

100-_

80

.2 60

.0

aR 40

20

0 1

O,: 10 100Peptide concentration nM

1000 10000

FIG. 4. Comparison of the inhibition of binding of MAb 35.2 toDnaK by purified DnaK (A) and peptide 357KEPNKGVNPDEV369(O) measured with an ELISA. Fifty percent inhibition of the bindingis seen at approximately 20 nM for both the peptide and purifiedDnaK.

DnaK with that of the peptide 357KEPNKGVNPDEV368. TheC. trachomatis dnaK gene was cloned into the expression vectorpGEX-3X downstream of the GST gene so that a fusionprotein of GST and C. trachomatis DnaK from amino acid 16to the C-terminal end was obtained. The GST-DnaK fusionprotein was purified on glutathione beads, and the binding toMAb 35.2 was measured in an inhibitory ELISA (Fig. 4). TheGST-DnaK fusion protein and peptide 357KEPNKGVNPDEV368 both inhibited 50% of the binding between MAb 35.2and DnaK at approximately 20 nM peptide or protein, indi-cating that the peptide and the native protein had similarconformations.

Alanine scan of the epitope for MAb 35.2. To determine thecontribution of each of the amino acids to the epitope, analanine scan of the epitope was performed. In Table 4 is listedthe inhibitory effect of each of the eight peptides (at aconcentration of 140 nM) on the binding of MAb 35.2 to C.trachomatis DnaK. It is seen that alanine exchange of any of

TABLE 4. Inhibitory effects of peptides on binding of MAb 35.2 toC. trachomatis DnaK

Determination by: Peptide (140 nM)a % Inhibition

Alanine scan 360NKGVNPDE367 >9536OAKGVNPDE367 036ONAGVNPDE367 036ONKAVNPDE367 036ONKGANPDE367 7036ONKGVAPDE367 6036oNKGVNADE367 7036ONKGVNPAE367 >9536ONKGVNPDA367 >95

Homology to MAb 35.2epitopes in other DnaK-HSP70 sequences

Bacillus subtilis HKGVNPDE 0Clostridium perfringens SKGVNPDE 0Escherichia coli RKDVNPDE 0Synechocystis sp. NQGVNPDE 0Erysipelothrix rhusiopathiae NKSVNPDE 0Trypanosoma cruzi mito- FRGVNPDE 0

chondriaEukaryote HSP70 NKSINPDE 0a Boldfaced letters indicate amino acid substitutions.

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FIG. 5. Three-dimensional structure of the ATPase part of bovineHSP70. The amino acid sequence homologous to the epitope for MAb35.2 is localized in connect II of the molecule. Connect II is surfaceexposed and connects two ot-helices in the HSP70 molecule. The areacontaining the epitope for MAb 35.2 is colored purple.

the first three amino acids (360NKG362) completely destroyedthe epitope. Exchange of each of the next three amino acids(363VNP365) with alanine reduced the specificity of the pep-tides (60 to 70% inhibition). Exchange of each of the two lastamino acids (366DE367) in the epitope with alanine did notaffect the binding affinity of the peptides to MAb 35.2.

Substitution of amino acids in the epitope with amino acidsfrom other DnaK sequences. The epitope sequence wassearched against the PIR and GenEMBL data bases, usingFASTA and TFASTA. The last four amino acids (364NPDE367)were conserved in all known DnaK and HSP70 sequences. Theepitope was conserved in some bacteria, among which weanalyzed B. burgdorferi and M. tuberculosis to test for reactivitywith MAb 35.2. The antibody recognized an antigen of the sizeof DnaK in both bacteria (Table 1). To verify the negativereactions shown in Table 1, we synthesized the amino acidsequences homologous to the MAb 35.2 epitope from E. coli,Bacillus subtilis, DnaK, and HSP70. None of these peptidescould inhibit the binding of MAb 35.2 to C. trachomatis DnaK(Table 4). Four other peptides from bacterial and mitochon-drial DnaK that had one or two amino acid changes from the

C.traL2 1 S VVKEIFC.pneumol S * ETVKELFE.coli 1 T KKVAEFFB.subtill ST EAIKKETM.leprael DLVKELTHuman 2iEVX^3Mouse 2 1HOM__3 LVGG R P

MAb 35.2 epitope were synthesized. None of these peptidesshowed any inhibitory effect on the binding of MAb 35.2 to C.trachomatis DnaK (Table 4).

Localization of the epitope on DnaK and HSP70. Thethree-dimensional structure of the ATPase part of bovineHSP70 has been determined (13). Five of the eight amino acidsin the epitope for MAb 35.2 are conserved in bovine HSP70.The amino acid sequence is localized in a part of the moleculenamed connect II that connects two cx-helices. In Fig. 5 thethree-dimensional structure of a bovine HSP70 ATPase frag-ment (13) is shown; ot-helices are indicated in red and 1-sheetsare indicated with green arrows. The amino acids homologousto the epitope sequence are indicated in purple. The epitope islocalized in the longest stretch of the molecule which is surfaceexposed without being either an a-helix or a 1-sheet. Thelinear form of the epitope is thus accessible for the antibody.The sequence of the epitope is highly conserved between

DnaK and HSP70 molecules (Fig. 6). In Bacillus subtilis, oneamino acid is replaced (N--H), (17, 35), and in E. coli, threeamino acids are different (3). No reaction of MAb 35.2 toeither Bacillus subtilis or E. coli DnaK (Table 1) was seen. Thesequences of human and mouse HSP70s had two amino acidchanges at the epitope, and the antibody did not react withthese HSP70s (Table 1 and Fig. 6). The epitope sequence wasconserved in C. pneumoniae (21) and B. burgdorferi (34), whichboth reacted with MAb 35.2 (Table 1). It is thus likely that anyamino acid changes within the epitope prevent binding ofMAb35.2.

DISCUSSION

The epitope for MAb 35.2 was mapped to the amino acidsequence 360NKGVNPDE367 in connect II of the C. trachoma-tis L2 DnaK-like protein. The epitope is likely to be linearbecause the affinity of the antibody to the 12'mer syntheticpeptide and that to the purified DnaK-GST protein wereidentical. The antibody reacted with C. pneumoniae DnaK, inwhich the amino acid sequence of the mapped epitope isconserved (21). One amino acid change in the epitope, as seenin Bacillus subtilis DnaK (17), abolished the reaction of MAb35.2, and a synthetic peptide of the Bacillus subtilis DnaKepitope homolog did not inhibit the binding of MAb 35.2 to C.trachomatis DnaK. These data confirm the localization of theepitope. The epitope was not present in DnaK from othergram-negative bacteria, e.g., E. coli (59.7% identity to C.trachomatis DnaK [3, 4]), Campylobacter jejuni, Y enteroco-litica, N. gonorrhoeae, S. typhimurium, and L. pneumophila.However, as revealed by a data base search using TFASTA(11), the amino acid sequence of the epitope was present inMycobacterium leprae (24), M. paratuberculosis (33), M. tuber-

IIFIG. 6. Multiple sequence alignment of DnaK and HSP70 amino acid sequences. The epitope for MAb 35.2 is indicated by the bar. The epitope

is localized in a highly conserved sequence of DnaK and HSP70 molecules. The Chlamydia sequences are identical to the M. leprae sequence. InBacillus subtilis, one amino acid is replaced. MAb 35.2 did not react with Bacillus subtilis DnaK or with mouse or human HSP70.

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culosis (X5806), Streptomyces coelicolor (L08201), Streptomycesgriseus (D14499), and B. burgdorferi (34) DnaK. The predictedreactions of MAb 35.2 with B. burgdorferi and M. tuberculosisDnaK were confirmed by immunoblotting (Table 1). Theamino acid identity between C. trachomatis DnaK and M.leprae DnaK is 56.9% and that between C. trachomatis DnaKand B. burgdorferi DnaK is 63.3%. Thus, the presence of theepitope sequence did not correlate with the overall identity ofthe genes (4).The epitope recognized by MAb 35.2 is localized in the

ATPase part of DnaK. This part of the molecule was not foundimmunogenic in M. leprae DnaK, in which three MAbs used forepitope mapping all map to the C-terminal variable domain ofM. leprae DnaK (28). However, Zhong and Brunham (36) didnot find the C-terminal part of C. trachomatis DnaK to beparticularly immunogenic, using 10'mers of synthetic peptidesand six rabbit hyperimmune serum samples. One of the rabbitsin their assay reacted with the epitope for MAb 35.2. Thedifference in the detection of immunogenic regions of DnaKmay be due to differences in the methods used. Fusion proteinsthat allow detection of discontinuous epitopes were used forthe M. leprae investigation. Discontinuous epitopes were notdetected by the 10'mer peptides used for analysis of the C.trachomatis DnaK.

In C. trachomatis research, epitope mapping has mainlyfocused on the MOMP because of its potential as a vaccinecomponent. With the method of Geysen et al. (14), severallinear epitopes located in the surface-exposed variable do-mains have been determined (9, 17, 30). However, Conlan etal. (10) have shown that even though a MAb reacts with a shortlinear MOMP sequence, the binding is affected by the sur-rounding amino acids. MAb B3/B9 paratope recognized bothvariable sequences II and IV, indicating that the native epitopewas located on two loops of MOMP. This is in agreement withthe model proposed by Baehr et al. (2) of the topology ofMOMP in the outer membrane. MOMP is a membraneprotein that interacts with LPS to obtain its tertiary structure(5). It can therefore not be purified for use in an inhibitoryELISA. In the present investigation, we performed the reac-tion between the peptides and antibody in solution. It wastherefore possible to measure the concentrations of bothpeptide and native DnaK, which gave 50% inhibition of thebinding of MAb 35.2.The present epitope mapping shows how specific the hu-

moral immune response is to a member of the HSP family.MAb 35.2 did not react with mouse HSP70 even though six ofthe eight amino acids in the epitope were conserved. DnaKcannot be detected on the host cell or chlamydial surface byusing a MAb (6), so the biological significance of antibodies toDnaK is not known. Specific antibodies to DnaK may have afunction in eliminating Chlamydia DnaK quickly from thecirculation to prevent inexpedient T-cell stimulation.

ACKNOWLEDGMENTS

We are grateful to Karin Skovgaard, Charlotte Holm, and IngerAndersen for skillful technical assistance, Henning Ern0 for cultivatingB. burgdorferi, and Ase B. Andersen (Statens Seruminstitut, Copenha-gen, Denmark) for testing MAb 35.2 reactivity to M. tuberculosis.

This work was supported by the Danish Health Research Council(grants 12-0850-1 and 20-3503-1), Aarhus University Research Foun-dation, Novo's Foundation, Nationalforeningen til Bekempelse afLungesygdomme, and Fonden til Lwgevidenskabens Fremme.

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