INFECTION AND IMMUNITY, Aug. 1988, p. 2047-20530019-9567/88/082047-07$02.00/0Copyright © 1988, American Society for Microbiology

Immunochemical Characterization of and Isolation of the Gene for a

Borrelia burgdorferi Immunodominant 60-Kilodalton AntigenCommon to a Wide Range of Bacteria

KLAUS HANSEN,'* JETTE MARIE BANGSBORG,2 HELLE FJORDVANG,l NILS STRANDBERG PEDERSEN,'AND PETER HINDERSSON'

Borrelia Laboratory, Department of Treponematoses, Statens Seruminstitut,l and Department of Clinical Microbiology,Rigshospitalet,2 Copenhagen, Denmark

Received 6 January 1988/Accepted 6 May 1988

By crossed immunoelectrophoresis and Western blotting (immunoblotting), it was shown that Borreliaburgdorferi expresses the 60-kilodalton Common Antigen (CA) that is cross-reactive with an equivalent antigenin a wide range of remotely related bacteria. B. burgdorferi CA is strongly immunogenic. A B. burgdorferigenomic library was constructed by using a plasmid cloning system. Escherichia coli recombinants werescreened for expression of immunodominant B. burgdorferi antigens. One of the recombinant clones expressedthe 60-kilodalton CA of B. burgdorferi. The DNA region encoding B. burgdorferi CA was localized on a2.3-kilobase fragment of the plasmid pKHl. CA may have pathogenetic implications in Lyme borreliosis, sincethe CA of mycobacteria recently has been shown to play a role in the etiology of experimental autoimmunearthritis. The extensive cross-reactivity of this antigen may account for the low diagnostic specificity of thecurrently used serological tests in Lyme borreliosis.

Borrelia burgdorferi, the etiologic agent of Lyme borreli-osis (25), contains a major immunoreactive protein of 60kilodaltons (kDa) which in Western blot (immunoblot) stud-ies frequently showed unspecific reactivity with control sera

(4, 31; B. Wilske, V. Preac-Mursic, G. Schierz, R. Kuhbeck,A. G. Barbour, and M. Kramer, Ann. N.Y. Acad. Sci., inpress). This study was initiated by our assumption that this60-kDa antigen could be Common Antigen (CA), a widelycross-reacting antigen first and independently described byKaijser (17) for Escherichia coli and by Hiiby (15) forPseudomonas aeruginosa. Since their observation, CA hasnow been shown to cross-react with an equivalent antigenfrom more than 60 different bacteria, including gram-nega-tive and positive bacteria, treponemes, and even archebac-teria (14, 20, 27). The CA of all bacteria investigated so farhas a subunit molecular mass in the range of 58 to 65 kDa.The definite function and localization of CA is still unknown,but it must play an essential role since it is phylogeneticallyso well conserved. The wide distribution of this antigen inbacteria, including those of the normal bacterial flora, ex-

plains the frequent occurrence of antibodies to CA in humanand animal sera and may account for the low diagnosticspecificity of serological tests using whole bacterial antigenpreparations as the test antigen. Recently, the CA of Myco-bacterium bovis has been shown to be involved in thepathogenesis of experimental autoimmune arthritis (29, 29a).

Since all these observations could be of great importanceto the serology and clarification of the pathogenesis in Lymeborreliosis, we decided to look for a B. burgdorferi CA. Inthis study, we have demonstrated that B. burgdorferi indeedexpresses the 60-kDa CA. B. burgdorferi CA was character-ized by immunochemical techniques, and the gene encodingCA was isolated.

* Corresponding author.

MATERIALS AND METHODS

Immunochemical techniques. (i) B. burgdorferi antigen. B.burgdorferi ACA-1 isolated from the skin of a patient withacrodermatitis chronica atrophicans (ACA) by Eva Asbrink(1) was used for all antigen preparations. The spirocheteswere grown in BSK medium (3) at 32°C for 5 to 7 days to a

cell density of 108 cells per ml. The cells were harvested bycentrifugation at 10,000 x g for 30 min and washed threetimes in phosphate-buffered saline (pH 7.4) containing 5 mMMgCl2. The final pellet was stored at -20°C until use.

(ii) Antibodies. Polyspecific rabbit antisera to B. burgdor-feri were raised by subcutaneous inoculation of 5 x 109sonicated borrelia in Freund incomplete adjuvant every thirdweek. The most polyspecific sera obtained 4 to 12 monthsafter immunization were selected by crossed immunoelec-trophoresis (CIE) and pooled; the immunoglobulin fractionwas isolated as described by Harboe and Ingild (11). Thisantibody preparation was designated BN98. Monospecificrabbit antibodies to the CA of P. aeruginosa, Treponemapallidum, and Legionella micdadei were obtained by rabbitimmunization with CA precipitates excised from CIE plates.Furthermore, we used sera from patients with ACA andcystic fibrosis.

(iii) CIE. CIE with an intermediate gel was done accordingto standard methods (2). Thawed spirochetes were sus-pended in phosphate-buffered saline (1011 cells per ml),sonicated on ice by seven 15-s blasts with an MSE 150 Wultrasonic disintegrator (Manor Royal, England), and centri-fuged at 10,000 x g for 10 min. Ten microliters of thesupernatant, equivalent to 109 borrelia, were applied in eachCIE. Electrophoresis was done in 1% agarose (HSA, Litex,Denmark) at 20°C by using Tris-barbital buffer (pH 8.76;ionic strength, 0.02). The first-dimensional electrophoresisof the antigens was run at 10 to 12 V/cm until a bromphenolblue-stained albumin marker had migrated 3.0 cm. Second-dimensional electrophoresis of the antigens was done at 2 to4 V/cm for 16 h. Monospecific rabbit serum or patient serum(200 ,ul) was added to the intermediate gel. The second-

2047

Vol. 56, No. 8

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

2048 HANSEN ET AL.

dimensional gel contained 400 RI of polyspecific rabbitanti-B. burgdorferi immunoglobulin (BN98). Gels werestained with Coomassie blue.

(iv) SDS-polyacrylamide gel electrophoresis and WB. So-dium dodecyl sulfate (SDS)-polyacrylamide gel electropho-resis and Western blotting (WB) were performed essentiallyas described by Laemmli (18) and Towbin et al. (28). Antigenwas suspended in a sample buffer containing (final concen-trations) 8% (vol/vol) glycerol, 0.1 M dithiothreitol, 0.6%SDS, 0.02 M Tris hydrochloride (pH 6.8), and bromphenolblue and heated to 100°C for 3 min before electrophoresis.Antigen lysate (20 ,lI, equivalent to approximately 107 bac-teria) was applied to each lane of the gel. The separating gelcontained 12.5% acrylamide-0.33% bisacrylamide, 0.1%SDS in 0.06 M Tris hydrochloride (pH 8.8). The stacking gelcontained 5% acrylamide-0.13% bisacrylamide, 0.1% SDSin 0.12 M Tris hydrochloride (pH 6.8). Electrophoresis wasdone at 25 mA for 4 to 6 h (vertical electrophoresis systemmodel V16; Bethesda Research Laboratories, Gaithersburg,Md.).The separated antigens were transferred from the gel

overnight at 25 V to a 0.20-,um-pore-size nitrocellulose (NC)filter (BA83; Schleicher & Schuell, Dassel, Federal Republicof Germany) by using a Trans Blot cell (Bio-Rad Laborato-ries, Richmond, Calif.) in a transfer buffer containing 0.025M Tris hydrochloride, 0.2 M glycine, 20% (vol/vol) methanol(pH 8.5).Immunostaining of transferred antigen was performed as

described in detail by Hindersson et al. (13). The NC filterwas blocked by incubation for 30 min at room temperature inimmunostaining (IS) buffer (0.16 M Tris hydrochloride, 0.5M NaCl, and 0.5% Tween 20 [pH 7.4]). The NC filters wereincubated for 1 h with the first antibody diluted in IS bufferand washed three times for 20 min in IS buffer. The bindingof the first antibody was detected by incubating the NC filterfor 1 h with a peroxidase-conjugated swine anti-rabbit im-munoglobulin G (P217; Dako, Copenhagen, Denmark) orrabbit anti-human immunoglobulin G (P214; Dako) diluted 1:2,000 in IS buffer. Bound peroxidase activity was visualizedby incubation of the NC filter for 5 to 15 min at roomtemperature with a substrate solution containing 0.5 ml oftetramethyl benzidin (Merck, Darmstadt, Federal Republicof Germany) dissolved in dimethyl sulfoxide (Merck) at aconcentration of 70 mg/ml, 15 ml of dioctyl sodium sulfosuc-cinate (Merck) dissolved in 96% (vol/vol) ethanol at aconcentration of 8 mg/ml, 45 ml of citrate phosphate buffer(0.08 M sodium hydrogen phosphate [Merck], 0.05 M citricacid [Merck] [pH 5.0]), and 30 Rl of stabilized hydrogenperoxide (Merck product 8597). The enzymatic reactionindicated by the blue color was stopped and stabilized bywashing the NC filter in a solution containing 13 ml of dioctylsodium sulfosuccinate-ethanol in 37 ml of water.

(v) Indirect immunofluorescence assay. B. burgdorferi wasfixed to glass slides in methanol. The monospecific rabbitantibody to P. aeruginosa CA was used as the first antibody,diluted 1:20 in phosphate-buffered saline, and fluorescein-conjugated anti-rabbit immunoglobulin (F205; Dako) wasused as the second antibody, diluted 1:200 in phosphate-buffered saline.Recombinant DNA techniques. (i) Isolation of B. burdorferi

DNA. B. burgdorferi ACA-1 (10" cells) was suspended in 10ml of SET buffer (25% sucrose, 5mM EDTA, 50 mM Trishydrochloride [pH 7.5]). The bacteria were lysed by addingSDS (final concentration, 0.5% [wt/vol]) and further treatedwith DNase-free RNase (0.1 mg/ml) and proteinase K (0.1mg/ml). The cells were incubated at 37°C for 45 min with

gentle shaking every 15 min. DNA was extracted with threesuccessive phenol extractions and a chloroform-isoamylalcohol extraction and finally dialyzed overnight against TEbuffer (10 mM Tris hydrochloride, 1 mM EDTA [pH 7.0]).The NaCl concentration was adjusted to 0.2 M, and DNAwas precipitated with 2 volumes of 99% (vol/vol) ethanol for3 h at -20°C and then pelleted by centrifugation at 15,000rpm for 1 h. The DNA pellet was washed in cold 70% (vol/vol) ethanol, air dried for 30 min, and resuspended in 200 ,ulof TE buffer. Agarose gel electrophoresis revealed that themolecular size of the DNA in the preparation was approxi-mately 50 kilobases (kb). The DNA was partially digestedwith the restriction enzyme Sau3A, titrated to yield frag-ments of 6 to 10 kb. The fragments were concentrate(d byethanol precipitation and resuspended in TE buffer at a DNAconcentration of 0.1 ig/jIl.

(ii) Construction of B. burgdorferi DNA library. We usedthe plasmid vector pPLc236, a derivative of the vectorpBR322 containing the PL promoter of the phage lambda(21). The PL promoter was controlled by a thermolabile cIrepressor encoded by a helper plasmid, pcI857. pPLc236carries an ampicillin resistance gene, and pcI857 carries akanamycin resistance gene.pPLc236 was opened with BamHI and dephosphorylated

to prevent self-ligation. Approximately equal amounts ofBamHI-cut, dephosphorylated pPLc236 and partiallySau3A-digested B. burgdorferi DNA were ligated with T4ligase. The total DNA concentration in the ligation mixturewas 0.2 jig/p,l. E. coli DH5, containing the helper plasmidpcI857, was transformed as described by Hanahan (9; pro-tocol 1). Recombinant clones were selected and grown at28°C on LB plates (19) containing 200 jig of ampicillin and 50jig of kanamycin per ml. Replica from the masterplate wereproduced on NC filters. The colonies on the replica filterwere first grown at 28°C for 6 h before the temperature wasraised to 42°C for 4 h for induction of the PL promoter. Therecombinant E. coli colonies were lysed in saturated chloro-form vapor for 1 min. After lysis, the NC filters were washedextensively in IS buffer, followed by immunostaining asoutlined in the description of the WB technique (see above).E. coli recombinants expressing an immunodominant B.burgdorferi antigen were identified by immunoscreening ofthis plasmid library with a high-titer serum from an ACApatient. To avoid a high background staining of E. coliantigens, the serum used was selected for a low level ofanti-E. coli antibodies by WB. Residual anti-E. coli activitywas removed by negative affinity chromatography on aCNBr-activated sepharose column (Pharmacia, Uppsala,Sweden), coupled with E. coli. The absorbed serum wasdesignated BN102.

Colonies reactive with BN102 were isolated from themaster plate, amplified on liquid LB medium (16 h at 28°C, 4h at 42°C), and subjected to WB to confirm the expression ofB. burgdorferi antigens.

Plasmid purification was performed according to themethod of Birnboim and Doly (5) and, if necessary, furtherpurified on a CsCl density gradient. Restriction sites withinthe plasmid pKH1 were mapped with a panel of restrictionenzymes (Boehringer GmbH, Mannheim, Federal Republicof Germany; New England BioLabs, Inc., Beverly, Mass.)by standard procedures (19). The CA-encoding region waslocalized by deletion of DNA between mapped restrictionsites and by opening the plasmid with BamHI and SphI tocreate unidirectional deletions with exonuclease III, as de-scribed by Henikoff (12). A commercially available kit wasused for this (Erase-a-Base; Promega, Madison, Wis.).

INFECT. IMMUN.

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

B. BURGDORFERI 60-kDa COMMON ANTIGEN 2049

C97 -

66 -_A _

43 -

31-X

22-A

14-

1 2FIG. 1. (A) CIE of B. burgdorferi (Bb) against polyspecific rabbit antiserum to B. burgdorferi (a-Bb; BN98). Blank intermediate gel. (B)

Same as A, except that 200 ,u1 of monospecific rabbit antiserum to P. aeruginosa CA (f-PaCA) was included in the intermediate gel. Theretained precipitate is designated B. burgdorferi CA (BbCA). (C) WB analysis of an excised B. burgdorferi CA-anti-P. aeruginosa CAprecipitate (lane 2) and B. burgdorferi whole-cell lysate (lane 1). Immunostaining was done with polyspecific rabbit antiserum to B.burgdorferi (BN98). B. burgdorferi CA has a subunit molecular weight of 60 kDa. Numbers on the left indicate molecular weights inkilodaltons.

RESULTSCIE of sonified B. burgdorferi against polyspecific rabbit

antibody to the same organism (BN98) revealed at least 20different precipitates (Fig. 1A). When a monospecific anti-body to the CA of P. aeruginosa was added to the interme-diate gel, a well-defined precipitate was retained (Fig. 1B).This indicated a strong cross-reactivity between the CA ofP.aeruginosa and the antigen of the retained precipitate, whichwas therefore designated the CA of B. burgdorferi.The retained B. burgdorferi CA precipitate was excised,

homogenized, and subjected to SDS-polyacrylamide gelelectrophoresis and WB. A single 60-kDa band was detectedby a polyspecific rabbit serum (BN98) (Fig. 1C) and by themonospecific antibody to the CA of P. aeruginosa (data notshown).

Figure 2 shows a WB of B. burgdorferi whole-cell lysateimmunostained with a polyspecific serum from a patient withACA and monospecific antibodies against the CA of threedifferent bacteria: P. aeruginosa, T. pallidum, and L. mic-dadei. The 60-kDa subunit was recognized by each of thesera, indicating the broad antigenic cross-reactivity of thisprotein.To evaluate the human immune response in Lyme borre-

liosis against B. burgdorferi CA, we used CIE, including serafrom patients with ACA in the intermediate gel. Ten serafrom different patients were investigated. All sera showed astrong reaction with B. burgdorferi CA (Fig. 3A). To dem-onstrate the unspecificity of this reaction, we investigatedten sera from patients with cystic fibrosis known to havefrequent P. aeruginosa infections. Four of these showed asimilar retention of B. burgdorferi CA (Fig. 3B). All the seraof patients with cystic fibrosis were negative when subjectedto the B. burgdorferi-flagellum enzyme-linked immunosor-bent assay indicating (10), that they were not infected withB. burgdorferi.

Indirect immunofluorescence showed no fluorescence onwhole B. burgdorferi cells with the monospecific antibody toP. aeruginosa CA.

To study antigens of B. burgdorferi, we constructed a B.burgdorferi DNA gene library. Approximately 8,000 recom-binant E. coli clones were generated. Of the randomlyselected colonies, 80% housed a recombinant plasmid withmore than 0.5 kb of B. burgdorferi DNA. Because weprimarily were interested in clones expressing immunodomi-nant B. burgdorferi antigens, the library was screened with ahigh-titered E. coli-absorbed serum from an ACA patient.Several positive colonies were identified. Their expressionof a B. burgdorferi antigen was confirmed by WB. One ofthese clones, designated DH5(pKH1, pcI857), expressed a60-kDa antigen. The molecular mass of this antigen sug-

97 - IN66- -

43

w

221 -

22- *

lisi: | -

14 -

1 2 3 4FIG. 2. Reactivity of a WB of B. burgdorferi antigens with a

polyspecific high-titered serum from a patient with ACA (lane 1) andmonospecific rabbit antisera against the CA of P. aeruginosa (lane2), T. pallidum (lane 3), and L. micdadei (lane 4).

Ba-Bb

(NBbCA

I

..^ ~ ~ ~ ~ ~.iot. I.

o _ ~~iBbCA

5-PaCA

Bb Bb

VOL. 56, 1988

r

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

2050 HANSEN ET AL.

a-Bb

INFECT. IMMUN.

a-Bb BA

BbCA

BbCA ....._.

ACA

Bb

CF

3Bb

FIG. 3. (A) CIE of B. burgdorferi (Bb) antigen against polyspecific rabbit antiserum to B. burgdorferi (a-Bb; BN98), with 200 ,ul of serumfrom a patient with ACA included in the intermediate gel. B. burgdorferi CA (BbCA) precipitate is retained, demonstrating that B. burgdorferiCA belongs to the immunodominant antigens of B. burgdorferi. (B) Same as A, but the intermediate gel contained 200 [±l of serum from apatient with cystic fibrosis (CF). The reactivity of the B. burgdorferi CA precipitate with this serum demonstrates the unspecificity of B.burgdorferi CA.

gested that it could be the CA of B. burgdorferi. The identityof the cloned 60-kDa antigen with the CA of B. burgdorferiwas established by reactivity with the monospecific antibodyto P. aeruginosa CA (Fig. 4).The expression of the cloned B. burgdorferi CA was not

controlled by the PL promoter of pPLc236, since equalamounts of the 60-kDa antigen were produced at 28 and42°C. The redundant helper plasmid pcI857 was removed byexcision of the plasmid pKH1 encoding CA from an agarosegel electrophoresis, followed by retransformation of DH5.

97 -

66 -

After purification of pKH1 on a CsCl density gradient, arestriction enzyme analysis was performed to map restric-tion sites for deletion of irrelevant DNA in pKH1. pPLc236contained 4.5-kb B. burgdorferi DNA. The CA-encodingDNA region was isolated by construction of plasmid deletion

+

-gl

31 -

*0.Xz

Ori Amp oLPL

020 - - KL K.YUJ (a X e uiUJ coen CA 2en o ,

COenOC.X

r-r-1 12 I AS+

+

*1-

+

+

-0.

+

22 -

14 - +

1 2 3 4 5FIG. 4. WB analysis of B. burgdorferi and E. coli DH5(pKH1,

pcI857) expressing B. burgdorferi CA with polyspecific E. coli-absorbed serum from a patient with ACA (BN102) and monospecificrabbit antiserum to P. aeruginosa CA. B. burgdorferi (lane 1), E.coli DH5(pKH1, pcI857) (lane 2), and E. coli without B. burgdorferiDNA inserted DH5(pPLc236, pcI857) (lane 3) immunostained withthe E. coli absorbed serum from the patient with ACA (BN102). B.burgdorferi (lane 4) and E. coli DH5(pKH1, pcI857) (lane 5) reactedwith monospecific antiserum to P. aeruginosa CA that was notabsorbed with E. coli.

pKHI

pKH3089pKH3091p KH3092

pKH3093pKH3094pKH3095pKH3096pKH3097pKH3098p KH3099pKH 3100

p KH3101

pKH3113

1000b CA GENE

FIG. 5. Restriction map of plasmid pKH1 encoding B. burgdor-feri CA. Symbols: Ef and _, pPLc236 DNA; =, B. burgdor-feri DNA. The plasmid pKH1 was opened with BamHI and SphI tocreate unidirectional deletions with exonuclease III, resulting inplasmid pKH3089 to pKH3101. The expression of B. burgdorferiCA was lost abruptly (-) when the deletion exceeded the deletion ofpKH3096, indicating the localization of the B. burgdorferi CA gene.A further deletion between the EcoRV and MluI sites of the plasmidpKH3096 resulted in plasmid pKH3113, containing a 2.3-kb B.burgdorferi DNA fragment still encoding the authentic 60-kDa B.burgdorferi CA (+). Abbreviations: Ori, ori sequence; Amp, ampi-cillin resistance gene; OLPL, operator-promotor region; b, bases.

43 -

ir

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

B. BURGDORFERI 60-kDa COMMON ANTIGEN 2051

mutants (Fig. 5). The resulting plasmid pKH3113 contained2.3-kb B. burgdorferi DNA and was still able to code for theauthentic 60-kDa subunit of CA.

DISCUSSION

CIE of B. burgdorferi antigens, including a monospecificantibody to the CA of P. aeruginosa in the intermediate gel,unambiguously showed that B. burgdorferi expresses CA(Fig. 1A and B). The position and morphology of the B.burgdorferi CA precipitate corresponds to the equivalent CAprecipitate of two other spirochetes, T. pallidum and Trepo-nema phagedenis (14). Excision of the CA precipitate,application to SDS-polyacrylamide gel electrophoresis andWB, and subsequent immunostaining with a polyspecificrabbit antiserum to B. burgdorferi revealed a single 60-kDaband (Fig. 1C). This subunit molecular mass is in accordancewith the findings for other bacteria. The CA of P. aeruginosahas been purified and characterized as a protein with a nativemolecular mass of 695 to 900 kDa, consisting of 59- to62-kDa subunits (24). The subunit molecular mass of T.pallidum and L. micdadei is 60 kDa (13, 20; J. M. Bangs-borg, M. T. Collins, N. Hoiby, and P. Hindersson, manu-script submitted), whereas the CA of mycobacteria is 65 kDa(23, 27, 32). The 60-kDa protein of B. burgdorferi, as well asthe 41-kDa protein, is a major protein of all B. burgdorferistrains (Wilske et al., in press). The cross-reactivity of thenative and cloned B. burgdorferi CA is illustrated by thereactivities with monospecific sera against the CA fromdifferent bacterial species (Fig. 2 and 4).The extent of amino acid homology between the CA of

different bacteria is a matter of great interest. Using CIE,HsSiby (16) demonstrated that the immunological cross-reactivities ranged from 25 to 100%. He proposed that CAhad highly conserved and thus cross-reactive epitopes, aswell as species-specific epitopes. This concept is supportedby several recent studies using different panels of monoclo-nal antibodies to the mycobacterial 65-kDa antigen (23, 27,32). Some of these monoclonal antibodies reacted broadlywith a protein in the range of 58 to 65 kDa in many differentbacteria, whereas the pattern of reactivity of others wasgenus or species restricted. This also explains why a widelycross-reactive monospecific antiserum to purified LegionellaCA could be made species specific by sequentiel absorptionwith different non-Legionella bacteria (20).The amount of comparative data on CA from different

bacteria is increasing, especially since the genes encodingthe CA of Mycobacterium tuberculosis (33), M. bovis (26),Mycobacterium leprae (34), T. pallidum (13), Coxiella bur-netii (30), L. micdadei (Bangsborg et al., submitted), andnow also B. burgdorferi have been cloned and expressed inE. coli. The DNA sequence data and the deduced amino acidsequences are now available for all three mycobacteria andC. burnetii. The amino acid sequence for the CA of myco-bacteria showed more than 95% homology (22), a remark-able finding in light of the only 20 to 30% homology shown intotal genomic DNA hybridization studies. The amino acidsequence of the CA of C. burnetii was 55% identical withthat of the CA of M. leprae (30).

Further comparative data will soon be added, since thesequencing of the DNA encoding the CAs of T. pallidum, L.micdadei, and B. burgdorferi is in progress.The subcellular localization of CA has not been finally

clarified. Gillis et al. (8) found that the 65-kDa protein of M.leprae was associated with the cell wall, since the antigenwas found in the insoluble fraction after disruption of the

cells. De Bruyn et al. (7) showed that M. bovis excreted largeamounts of the 65-kDa antigen into the culture fluid but onlyunder zinc deficiency. When cultured under normal condi-tions, the 65-kDa antigen was only present in the solublecellular extract. Hitherto, only Thole et al. (27) tried tolocalize CA by immunoelectron microscopy. Gold-labeledmonoclonal antibody to the 65-kDa mycobacterial antigenwas used. In a recombinant E. coli hyperexpressing myco-bacterial CA, gold-stained antigen was found exclusively inthe cytoplasm. In M. bovis, labeled antigen was foundmainly in the cytoplasm and only occasionally in the peri-plasm. Plikaytis et al. (20), investigating the 60-kDa CA ofLegionella spp., found no evidence of surface exposure byindirect immunofluorescence, using a monospecific antibodyto purified Legionella CA. These observations are in accor-dance with our findings regarding B. burgdorferi CA. Indi-rect immunofluorescence with a monospecific antiserum toP. aeruginosa CA was negative, and differential centrifuga-tion of sonicated B. burgdorferi and sonicated E. coliDH5(pKH1, pcI857) expressing B. burgdorferi CA showedthat the CA was found in the soluble fraction.The phylogenetic stability of this antigen is puzzling and

suggests a vital but so far unknown physiological function.Recently, several investigators found a striking amino acidsequence homology (65%) of the carboxy-terminal third ofmycobacterial CA (22) and C. burnetii CA (30) with an aminoacid sequence deduced from the DNA sequence data of anE. coli gene cloned by Chanda et al. (6). This gene wasbelieved to be the ams gene, which is thought to influencemRNA stability in E. coli. However, plasmids encoding theCA of C. burnetii (30) and T. pallidum (Peter Hindersson,unpublished observation) could not complement the ams-deficient mutant.

Shinnick et al. (23) showed that the amount of M. tuber-culosis CA and of the cross-reacting E. coli 60-kDa antigenincreased considerably in cells grown at a higher tempera-ture. Furthermore, they presented evidence for the corre-spondence of mycobacterial CA with an E. coli 60-kDa heatshock protein known as the GroEL protein. The GroELprotein is involved in phage lambda morphogenesis. Theamino acid sequence of the GroEL protein and the CA ofM.tuberculosis showed about 54% homology. Similar data wererecently presented for the cloned CA of C. burnetii (30),indicating that CA indeed is a heat shock protein. The CAmay thus be essential for the cells in various stress situa-tions, for instance, exposure to environmental hazards andhost defence mechanisms.The CA of M. bovis has been implicated in the pathogen-

esis of experimental adjuvant arthritis in Lewis rats (29).Van Eden et al. (29) were able to isolate a T-cell clonedesignated A2b, which induced arthritis in these rats. ThisT-cell clone was shown to be specifically stimulated by theCA of M. bovis and components of cartilage. A correspond-ing suppressor T-cell clone, A2c, able to protect againstarthritis was also stimulated by M. bovis CA. Using thearthritogenic T-cell clone and truncated peptides of clonedM. bovis CA, the arthritogenic epitope was localized (29a).The presence of this or a cross-reactive epitope in the CA ofdifferent bacteria remains to be elucidated and may explainwhy only some of the many bacteria expressing CA areassociated with arthritis. Bacterial species, including Kleb-siella, Shigella, Salmonella, Yersinia, and Campylobacterspp., have all been suspected to be involved in humanarthritis. Therefore, a possible role of CA in autoimmunearthritis is of particular interest in the context of the unre-solved pathogenesis of Lyme arthritis. The cloning and

VOL. 56, 1988

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

2052 HANSEN ET AL.

expression of B. burgdorferi CA may become helpful toclarify these questions.The wide cross-reactivity of CA has implications for the

interpretation of serological tests using whole-cell antigenpreparations. Hyiby (16) showed by CIE that 54% of 151healthy controls had precipitating antibodies against the CAof P. aeruginosa. These antibodies are probably a result offrequent exposure to common bacteria, including the normalbacterial flora. CIE with sera from ACA patients demon-strates that the human immune response to B. burgdorferiincludes a strong reaction to CA (Fig. 3A). A comparablereactivity of sera from patients with cystic fibrosis known tohave frequent infections with P. aeruginosa illustrates theunspecificity of the B. burgdorferi CA (Fig. 313). Unspecificantibody reactions to the 60-kDa band of B. burgdorferi inWB studies have been reported by others (4, 31). Thus,inclusion of this and similar unspecific antigens in sero-diagnostic tests for Lyme borreliosis may account for thelow diagnostic performance of the currently used sonicextract enzyme-linked immunosorbent assay. Since CA is amajor immunoreactive protein, the species-specific epitopesof B. burgdorferi CA should theoretically be a suitable testantigen. However, a complete separation from the con-served epitopes is necessary, since the diagnostic specificityotherwise would be invalidated. It is probably easier toimprove serological tests for Lyme borreliosis by eliminatingwidely cross-reacting antigens such as CA by the use ofpurified immunodominant and more specific antigens. Theefficiency of this strategy has recently been demonstrated bythe use of B. burgdorferi flagellum as the test antigen in anenzyme-linked immunosorbent assay (10).

ACKNOWLEDGMENTS

Klaus Hansen and Jette Marie Bangsborg were supported by agrant from the University of Copenhagen. Det Danske PasteurSelskab and the Novo Foundation supported Peter Hindersson. Thestudy was further supported by Direktir Jacob Madsen og hustruOlga Madsens fond and the Center of Medical Biotechnology.We thank Dorte Soeborg Petersen for perfect technical assis-

tance, Eva Asbrink, Department of Dermatology, Sodersjukhuset,Stockholm, for supplying B. burgdorferi ACA-1, and Niels Hoiby,Department of Clinical Microbiology, Rigshospitalet, Copenhagen,for the monospecific antibody to the CA of P. aeruginosa.

LITERATURE CITED1. Asbrink, E, B. Hederstedt, and A. Hovmark. 1984. The spiro-

chetal etiology of acrodermatitis chronica atrophicans Herx-heimer. Acta Dermatol.-Venerol. 64:506-512.

2. Axelsen, N. H. 1983. Intermediate gel immunoelectrophoresis.Scand. J. Immunol. Suppl. 10:141-149.

3. Barbour, A. G. 1984. Isolation and cultivation of Lyme diseasespirochetes. Yale J. Biol. Med. 57:521-525.

4. Barbour, A. G., W. Burgdorfer, E. Grunwaldt, and A. C. Steere.1984. Antibodies of patients with Lyme disease to componentsof the Ixodes damini spirochete. J. Clin. Invest. 72:504-515.

5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

6. Chanda, P. K., M. Ono, M. Kuwano, and H.-F. Kung. 1985.Cloning, sequence analysis, and expression of alteration of themRNA stability gene (ams+) of Escherichia coli. J. Bacteriol.161:446-449.

7. De Bruyn, J., R. Bosmans, M. Turneer, M. Weckx, J. Nyabenda,J.-P. van Vooren, P. Falmagne, H. G. Wiker, and M. Harboe.1987. Purification, partial characterization, and identification ofa skin-reactive protein antigen of Mycobacterium bovis BCG.Infect. Immun. 55:245-252.

8. Gillis, T. P., R. A. Miller, D. B. Young, S. R. Khanolkar, andT. M. Buchanan. 1985. Immunochemical characterization of a

protein associated with Mycobacterium leprae cell wall. Infect.Immun. 49:371-377.

9. Hanahan, D. 1985. Techniques of transformation of Escherichiacoli, p. 109-135. In D. M. Glover (ed.), DNA cloning, vol. 1. Apractical approach. IRL Press, Oxford.

10. Hansen, K., P. Hindersson, and N. S. Pedersen. 1988. Measure-ment of antibodies to the Borrelia burgdorferi flagellum im-proves serodiagnosis in Lyme borreliosis. J. Clin. Microbiol. 26:338-346.

11. Harboe, N., and A. Ingild. 1983. Immunisation, isolation ofimmunoglobulins and antibody titer determination. Scand. J.Immunol. Suppl. 10:345-351.

12. Henikoff, S. 1984. Unidirectional digestion with exonuclease IIIcreates targeted breakpoints for DNA sequencing. Gene 28:351-359.

13. Hindersson, P., J. D. Knudsen, and N. H. Axelsen. 1987. Cloningand expression of Treponema pallidum common antigen (Tp-4)in Escherichia coli K12. J. Gen. Microbiol. 133:587-596.

14. Hindersson, P., C. S. Petersen, N. S. Pedersen, N. H$iby, andN. H. Axelsen. 1984. Immunological crossreaction between an-tigen Tp-4 of Treponema pallidum and an antigen common to awide range of bacteria. Acta Path. Microbiol. Immunol. Scand.Sect. B 92:183-188.

15. Hoiby, N. 1975. Cross-reactions between Pseudomonas aerugi-nosa and thirty six other bacterial species. Scand. J. Immunol.Suppl. 2:187-196.

16. Hiiby, N. 1975. Normally occurring precipitating antibodiesagainst Pseudomonas aeruginosa. Prevalence, specificity andtitres. Scand. J. Immunol. Suppl. 2:197-202.

17. Kaijser, B. 1975. Immunological studies of an antigen commonto many gram negative bacteria with special reference to E. coli.Int. Arch. Allergy Appl. Immunol. 48:72-81.

18. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

20. Plikaytis, B. B., G. M. Carlone, C.-P. Pau, and H. W. Wilkin-son. 1987. Purified 60-kilodalton Legionella protein antigen withLegionella-specific and nonspecific epitopes. J. Clin. Microbiol.25:2080-2084.

21. Remaut, E., P. Stansseus, and W. Fiers. 1981. Plasmid vectorsfor high-efficiency expression controlled by the pL promoter ofcoliphage lambda. Gene 15:81-93.

22. Shinnick, T. M., D. Sweetser, J. Thole, J. van Embden, andR. A. Young. 1987. The etiologic agents of leprosy and tuber-culosis share an immunoreactive protein antigen with the vac-cine strain Mycobacterium bovis BCG. Infect. Immun. 55:1932-1935.

23. Shinnick, T. M., M. H. Vodkin, and J. C. Williams. 1988. TheMycobacterium tuberculosis 65-kilodalton antigen is a heatshock protein which corresponds to common antigen and to theEscherichia coli GroEL protein. Infect. Immun. 56:446-451.

24. Sompolinsky, D., J. B. Hertz, N. Hoiby, K. Jensen, B. Mansa, V.Barkholt-Pedersen, and Z. Samura. 1980. An antigen common toa wide range of bacteria. 2. A biochemical study of a "commonantigen" from Pseudomonas aeruginosa. Acta Pathol. Micro-biol. Scand. Sect. B 88:253-260.

25. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft,A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, andS. E. Malawista. 1983. The spirochetal etiology of Lyme dis-ease. N. Engl. J. Med. 308:733-740.

26. Thole, J. E. R., H. G. Dauwerse, P. K. Das, D. G. Groothuis,L. M. Schouls, and J. D. A. van Embden. 1985. Cloning ofMycobacterium bovis BCG DNA and expression of antigens inEscherichia coli. Infect. Immun. 50:800-806.

27. Thole, J. E. R., P. Hindersson, J. de Bryun, F. Cremers, J. vander Zee, H. de Cock, J. Tommassen, W. van Eden, and J. D. A.van Embden. 1985. Antigenic relatedness of a strongly immuno-genic 65 kD mycobacterial protein antigen with a similarly sizedubiquitous bacterial common antigen. Microbiol. Pathogenesis4:71-83.

INFECT. IMMUN.

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

B. BURGDORFERI 60-kDa COMMON ANTIGEN 2053

28. Towbin, H., J. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

29. Van Eden, W., W. J. Holoshitz, Z. Nevo, A. Frenkel, and A.KIa man. 1985. Arthritis induced by a T-lymphocyte clone thatresponds to Mycobacterium tuberculosis and to cartilage pro-teoglycans. Proc. Natl. Acad. Sci. USA 82:5117-5120.

29a.Van Eden, W., J. E. R. Thole, R. van der Zee, A. Noordzi,J. D. A. van Embden, E. J. Hensen, and I. Cohen. 1988. Cloningof the mycobacterial epitope recognized by T-lymphocytes inadjuvant arthritis. Nature (London) 331:171-173.

30. Vodkin, M. H., and J. C. Williams. 1988. A heat shock operon

in Coxiella burnetii produces a major antigen homologous to a

protein in both mycobacteria and Escherichia coli. J. Bacteriol.170:1227-1234.

31. Wilske, B., V. Preac-Mursic, G. Schierz, and K. V. Busch. 1986.Immunochemical and immunological analysis of European Bor-relia burgdorferi strains. Zentralbl. Bakteriol. Hyg. A 263:92-102.

32. Young, D. B., J. Ivanyi, J. H. Cox, and J. R. Lamb. 1987. The65 kDa antigen of mycobacteria-a common bacterial protein?Immunology Today 8:215-219.

33. Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivanyi, D.Thomas, and R. W. Davis. 1985. Dissection of Mycobacteriumtuberculosis antigens using recombinant DNA. Proc. NatI.Acad. Sci. USA 82:2583-2587.

34. Young, R. A., V. Mehra, D. Sweetser, T. M. Buchanan, J.Clark-Curtiss, R. W. Davis, and B. R. Bloom. 1985. Genes forthe major protein antigens of Mycobacterium leprae. Nature(London) 316:450-452.

VOL. 56, 1988

on August 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from