Moiecuiar Microbioiogy (1992) 6(20). 3031-3040

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi Patricia A. Rosa,'* Tom Schwan^ and Daniel Hogan^ ^Laboratory of Microbial Structure and Function, and ^Laboratory of Vectors and Pathogens, Rocky fvlountain Laboratories, National Institute of Allergy and Infectious Diseases. National Institutes of Health, Hamilton, Montana 59840, USA.

Summary Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologous genes encoding the major outer surface proteins (Osps) A and B of B. burgdorferi which both deletes osp gene sequences and creates chimaeric gene ^sions. Recombinant osp genes occur in multiple strains and encode unique proteins that lack some characteristic Osp epitopes. Antigenic variation in Osp through recombination may be relevant to the persistence of B. burgdorferi in an infected host, and has important implications for the utility of OspA and OspB as diagnostic or vaccine candidates for Lyme disease. We also describe Osp variation arising from nonsense mutations and sequence divergence, which may also represent significant sources of Osp polymorphism.

et ai, 1985; 1984; Schwan and Burgdorfer 1987) and clonaiiy within a strain (Bundoc and Barbour, 1989), but the genetic basis for this phenomenon is not compieteiy understood. In some cases sequence divergence accounts for altered mobility and antigenicity of Osp proteins (Fikrig efa/., 1992b; Jonsson efa/., 1992). iJniike the vmp genes of B. hermsii, osp gene sequences are present at oniy a singie locus, an operon on a iarge iinear plasmid of S. burgdorferi, with the ospA gene proximai to the promoter (Barbour and Garon, 1987; Howe ef a/., 1986; 1985). Sequenceanaiyses have identified homoiogy betvi/een the ospA and ospB genes (Bergstrom ef ai, 1989). in this study we examined the osp ioci from several North American and European strains expressing characteristic Osp phenotypes in an attempt to identify mechanisms of ceii surface change in B. burgdorferi. Poiymerase chain reaction (PCR) amplification (Muilis ef ai, 1986; Saiki ef ai, 1985) of the osp ioci from both cioned and uncioned organisms, and sequence anaiyses of the osp fragments, demonstrated recombination between ospA and ospB genes. The molecular characteristics of these osp recombinants and their potential impiications are discussed.

Results and Discussion Introduction In many patients Lyme disease is a chronic infection with diverse manifestations (Steere, 1989; Steere etai, 1977 a.b). The means by which the causative agent. Borrelia burgdorferi (Barbour etai, 1983a; Burgdorferefa/., 1982), evades the immune response and establishes chronic infection is unknov^fn; one possibility is through changes in surface-exposed proteins. The related spirochaete Borretia hermsii, the agent of relapsing fever, varies its major outer membrane protein (Vmp) (Barbour et ai, 1982; Stoenner et ai, 1982) by a gene conversion-like mechanism vtfhereby piasmid-encoded sequences from muitipie siient ioci are introduced into an expression locus (Meier ef ai, 1985; Piasterk ef a/., 1985). The expression and mobiiities of the major outer surface proteins (Osps) of B. burgdorferi, OspA and OspB, vary among strains (Barbour

Received 24 April, 1992; revised 8 July, 1992; accepted 15 July, 1992. *For correspondence. Tel. (406) 363 3211; Fax (406) 363 6406,

Amplification of the osp loci from B. burgdorferi strains with distinct Osp phenotypes Whoie-ceil iysates of severai North American and European e. burgdorferi strains (B31, HB19, Sh.2.82 and G1, G2, IP2, respectiveiy) were separated by SDS-poiyacrylamide gel eiectrophoresis (PAGE) (Fig. 1). Strain Sh.2.82 (high passage) expresses oniy OspA, and strains GI and G2 express a singie major protein, intermediate in size between OspA and OspB. The OspB molecules of strains i-IB19 and B31 have slightly different mobiiities and are antigenicaily distinguishabie (Barbour et ai, 1984), Strain IP2 expresses Osp proteins simiiar in size to those of strain B31. The osp ioci from North American strains B31, HB19 and Sh.2.82 were amplified by PGR (Muilis ef a/., 1986; Saiki ef ai. 1985) (Fig. 2A) with primers derived from sequences flanking the B31 osp operon (Bergstrom etai, 1989). A POR product of the predicted size (1.9 kb) was ampiified from ail three strains; strain Sh.2.82 aiso

3032

P. A. Rosa, T. Schwan and D. Hogan Total Protein

CM . CO

m m

CD X ^ X 05 O

CM

O

_ H

I l l l l - 45 kDa

OspB OspA

- 31 kDa

(1.4kb) is comparabie to that of the prototype versus variant Sh.2.82 osp fragments (1.9 kb versus 0.9 kb) and is approximateiy the size of the ospB gene. Hybridization with an internai osp probe demonstrated that ail of these ampiified fragments contained osp sequences and were not non-specific PCR products (Fig. 2A), The G2 osp fragments do not hybridize as weli to the B31 DNA probe because of sequence divergence between the strains (Rosa et ai, 1992). Under the conditions used here, PCR amplification of the osp locus was not quantitative, and thus the ratio of osp PCR products does not reflect the ratio of deieted and undeleted osp loci in the unamplified genomic DNA. By genomic Southem analysis the undeieted osp iocus is the predominant form in the uncioned G2 and Sh.2.82 populations (data not shown).

Fig. 1. SDS-PAGE profile of Borrelia whole-celt lysates. B. burgdorferi strains B31. HB19, and Sh.2.82 are North American isolates and strains G I , G2, and IP-2 are European isolates. Arrows indicate the mobilities of OspA and OspB from the prototype strain B31. Protein size standards are indicated to the right of the figure.

Amplification ofthe osp loci from B. burgdorferi clones

generated a smalier fragment (0.9 kb) and faint bands larger than the prototype 1.9 kb fragment (Fig. 2A). We have previousiy demonstrated that strain G2 represents a distinct European subtype of B. burgdorferi that is not ampiified with these B31 osp primers (Rosa et ai, 1991). Primers derived from sequences flanking the G2 osp operon (Rosa ef ai, 1992) ampiify 2.3kb and 1.4kb fragments from G2 DNA (Fig. 2A); the G2 primers are siightiy more distal to the osp operon than are the B31 primers. The difference in size between the anticipated G2 osp PCR fragment (2.3 kb) and the smalier fragment

The tempiate DNAs used in the PCR anaiysis discussed above vi/ere extracted from uncioned strains. Therefore it was unclear whether both forms of the osp locus were present In a single bacterium or if they were components of separate bacteria. In order to address this issue, approximateiy 100 B. burgdorferi ciones from strain Sh.2.82 were analysed by PCR ampiification of the osp iocus. Ali of the clones contained either the 1.9 kb or the 0,9 kb osp fragment but none contained both, osp-related fragments bigger than the prototype osp locus were not ampiified from any clones. Figure 2B shows the amplification products of DNA from a cione that contains only the smalier osp locus (SH#14), a cione that contains only the fuil-length osp iocus (SH#1), and, for comparison.

A.

B.

Fig. 2. A. PCR amplification of the osp locus from uncioned B. burgdorferi strains. DNAs from strains B31, HB19, and Sh,2.82 were amplified with primers derived from sequences flanking the osp operon of strain B31: the predicted size ot the B31 amplified fragment is 1.9kb. G2 DNA was amplified with primers flanking the G2 osp operon; the predicted size of the G2 amplified fragment is 2.3kb. The left side ot the figure represents total PCR products separated on an agarose gel and visualized with ethidium bromide. The right half of the Figure represents a Southern blot of the gel hybridized with a *^P-labelled DNA probe encompassing the B31 ospoperon (nucleotides 1-1915) (Bergstrom et a/., 1989). B. A comparison of the osp loci from cloned and unctoned 8. burgdorferi. DNAs from uncioned strain Sh.2,82 and clones SH#1 and SH#14 were amplified with the B31 osp primers and analysed as described in (A),

osp gene recombination uncioned strain Sh.2.82, which contains both forms of the osp iocus. Hence the muitipie osp PCR fragments from uncioned strain Sh.2.82 probably represent the amplification products from separate subpopuiations of bacteria rather than from more than one form of the osp iocus within a singie bacterium, Cionai derivatives of strain G2 aiso contained either the fuli-iength or the deleted form of the osp iocus (data not shown).

3033

osp loci encode OspA/B chimaeras, but the junction of the fusion is different for each strain. In aii cases the deletion has occurred between homoiogous sequence in ospA and ospB. such that the OspB open reading frame is maintained, isolates CA.20 and CA.17 were made from ticks and had been grown for fewer than five passages in vitro when these variants were detected and cloned.

Nucleotide sequence analysis of variant osp loci

Generation of chimaeric csp genes by homologous recombination

We sequenced the osp locus from cione SH#14 to define the genetic basis of its variation, A deietion of approximately 1 kb extends from the 3' end of the ospA gene to the homologous position in the ospB gene (Fig. 3A), The resulting Osp moiecuie expressed by variant SH#14 is an OspA/B fusion protein; the 3' end of the ospB gene, encoding 16 amino acids, has replaced the corresponding segment of the ospA gene. The fusion junction occurs as shown between residues 919 of ospA and 1819 of ospB. Genomic Southern analyses demonstrated that the variant osp locus of cione SH#14 resides on the 49kb linear piasmid at approximateiy the same site as the fuii-length osp ioci of B31 and Sh.2.82, and that its fianking sequences were unaitered (Rosa et ai. 1992). Therefore an internal deletion is the oniy gross structurai difference that we have detected between the osp iocus cf SH#14 and a prototypic twc-gene osp locus.

Bergstrom etai pointed out that OspA and OspB are the products of homoiogous genes that presumabiy diverged foliowing dupiication of an ancestrai osp gene (Bergstrom et ai, 1989). This homcicgy is readiiy apparent when comparing both the nucieotide and amino acid sequences (Fig. 3, bottom). The chimaeric osp ioci that we have described ccuid have arisen by homologous recombination between the ospA and ospS genes on separate copies of the linear piasmid (inter-piasmid recombination, diagrammed in Fig. 5A, product #1). The reciprocai product of such an unequai crossover would be an osp locus with three genes: ospA, an ospB/A chimaera and osps (Fig. 5A, product #2). Although such an osp iocus has not been identified in clonal derivatives of these strains, PCR fragments larger than the wiid-type osp iocus are aisc amplified from the uncioned strains Sh.2.82 and G2 (Fig. 2).

Sequencing of the 1,4 kb G2 osp fragment demonstrated that it also represents a chimaeric ospA/B gene, but that in this case the deletion between ospA and ospB occurred ciose to the 5' ends of the genes. Figure 3B shows the structure cf the osp operon of variant clone G2.22 in which the carboxy-terminai 225 amino acids cf OspB have replaced the corresponding segment of OspA. Again, the recombinant sequence is aligned with the homologous sequences of the donor ospA and ospB genes.

Aiternativeiy, homologous recombination between ospA and ospB genes on the same piasmid copy (intraplasmid recombination, diagrammed in Fig, 5B) would generate a singie chimaeric ospA/B gene on the iinear piasmid (product #1) in addition to a circuiar DNA containing the intervening sequences (product #2), which presumabiy would not be repiicated. Aithough we cannot distinguish between intermoiecuiar and intramolecuiar recombination in B. burgdorferi at this time, an analogous event in S. hermsii, non-reciprocai recombination between homoiogous vmp sequences, occurs between genes encoded on separate iinear piasmids (Meier et ai, 1985; Piasterk efa/., 1985).

It is possibie that bacteria bearing deleted or undeieted osp loci in a given strain are progeny of a mixed popuiation present in the original isoiate and therefore are not cicnaiiy reiated. Aithcugh we cannot eliminate this possibility with the current data, the foliowing points argue against it for strains Sh.2.82 and G2. (i) The nucieotide sequences of the deieted and undeleted forms of the osp iocus from a given strain are identicai to each other (except for the deletion) but distinct from the osp sequences cf other strains, (ii) Deleted osp ioci are not detectable by PCR in DNA from very eariy passages of strains G2 and Sh.2.82 (data not shown). We have detected deieted osp loci in additional 6, burgdorferi isolates; the reiative structures of the ioci that we have anaiysed to date, including Sh#14 and G2,22, are diagrammed in Fig. 4. Where characterized, the variant

The homologous nature of the osp gene sequences could foster chimaeric ospAJB artefacts from a prototypic two-gene osp operon by inappropriate priming during PCR amplification. The foilowing points demonstrate that this has not occurred in these experiments, (i) Genomic Southern anaiyses of unampiified B. burgdorfen DNA ccnfirm the presence of vanant osp ioci whose structures were predicted by PCR ampiification (Rosa et ai, 1992; our unpublished data), (ii) Recentiy cloned cultures of 6. burgdorferi give rise to only a singie osp PCR fragment, fuii-iength or deleted, but not both (Fig. 2B). (iii) The osp amplification products differ among uncioned strains, but are reproducible from a given strain (Fig. 2).

3034

P. A. Rosa, T. Schwan and D. Hogan

A

SH.2.82 OspA 1

256

o«pX/B 871 aapA

871

o«pB

B

OspB

1

260 296

AACACAATTACAGTACAACAATACGACTCAAAIGGCACCAAATTAGAGGGATCAGCflAGTGAAATTAAAAATCTTTCAGAGCTTAAAAArnfTTTft BiS/1819

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297

976 991

1200

296

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252

ospB

GATTTACCTGGTGGAATGACAGTTCTTGTAAGTAAAGAAAAAGACAAAJI,fcCMTjAATATGTGrTCAGAGCAArAGTTGATACAGTTGAGCTTAAAGGGGTT tc....ttcaa..dt.gtqa.a..t,

297

igtc

us

.aac, 353 1256

Fig. 3. A. Diagram of the chimaeric osp gene of SH#14. The top of the diagram represents the undeleted ospA (solid bar) and ospB (hatched bar) genes of the 1.9kb Sh.2.82 osp locus. The 0.9kb ospAlB locus of vanant SH#14 is schematically represented in the centre of the diagram. The bottom of the diagram represents the sequence encompassing the endpoints of the deletion in SH# 14, relative to the homologous sequences in undeleted ospA and ospB genes. Shading beneath the ospAlB nucleotide sequence designates the osp gene from which it originated. Dots represent nucleotide identity of ospA or ospB with the chimaeric ospAIB sequence. The nucleotides that define the transition from ospA to ospB are underlined. Boxed amino acids represent residues that differ between OspA and OspB. The numbering of the ammo acid residues (above the line) and nucleotides (below the line) is analogous to the cioned B31 osp sequence (Bergstrom etal.. 1989). B. Diagram of the chimaeric osp gene of G2. The undeleted and deleted forms of the G2 osp tocus are depicted as described for SH.2.82 in (A),

osp gene recombination ospB

ospA

B31 prototype

1719

819

CA-20.2A

780/1676

Sh.2.82 #14

766/1666

3035

Fig. 4. Diagram of chimaeric osp loci from differer^t B. Owrgdorfen strains. The ntjcleotide sequences of recombinant osp loci from various strains have been determined, and the structures of these loci are schematically represented below the prototype osp locus of strain B31. ospA gene sequences are indicated with a solid bar and ospB gene sequences with a hatched bar. In addition to the deleted osp ioci depicted here, clones with prototypic loci were also isolated from these same strains. The numbering of the nucleotides (underneath the diagrams) starts with the first base of ospA. With the recombinant loci, the endpoints of the deletions are numbered relative to the prototype ospA and ospS genes of the undeleted loci. Deleted forms of the osp locus have been detected in strains CA-11 and CA-19, but the positions of the crossover in these strains have not been determined.

CA-17.13C

667/1587

G2.22

1 144/1047

s

CA-11 CA-19

1719

Reactivity of chimaeric Osp proteins with anti-Osp monoclonal antibodies The chimaeric OspA/B protein expressed by SH#14 migrates with a mobiiity on SDS-PAGE simiiar to that of wiid-type OspA. it is recognized on immunoblots by the OspA monoclonal antibody H5332 (Fig. 6A) but not by the OspB monocionai antibody H4610 (Fig. 6B). Escherichia coli 4.28 containing this variant osp operon exhibits the same pattern of antibody reactivity {Fig. 6). However, with different recombinants or antisera, the extent to which a chimaeric protein resembles OspA versus OspB reflects the particular moiecuiar event that took piace. For exampie, only the first 48 amino acids (18%) of the G2.22 chimaeric Osp protein are encoded by ospA sequences (Fig. 3); the subsequent 226 residues are from ospB. Although this OspA/B protein is expressed at a level comparabie to that of the non-recombinant G2 Osp protein, it is no ionger recognized by the monocionai antibody H5332 (Fig. 7). This is consistent with moiecuiar mapping studies which demonstrate that H5332 recognizes a non-linear epitope within amino acids 66-273 of OspA (Sears et ai, 1991; Shanafeit ef ai, 1992).

ospB nonsense mutation Uncioned high-passage strains Sh.2.82 and G2 do not appear to express fuil-iength OspB (Fig. 1), yet within the popuiation are bacteria with intact as weli as deleted osp ioci (Fig. 2). Similarly, Bundoc and Barbour have previously described 6. burgdorferi strain HB19 clonal variants that iacked fuli-length OspB, for which there was no evidence of iarge DNA deletions or rearrangements (Bundoc and Barbour, 1989), Therefore, generation of osp deietions by recombination cannot be the only mechanism by which OspB expression is apparently iost. Sequence anaiysis of the undeieted 1.9kb Sh.2.82 osp operon (P. A. Rosa, unpublished) has demonstrated a premature stop codon at nucieotide 716 in the ospS gene (TTG-»TAG) that terminates OspB 58 amino acids short of the fulllength protein. Consistent with this, a smaller OspB fragment can be detected with the OspB monoclonal antibody H4610 in both high-passage Sh.2.82 (SH"^ and the corresponding E. coli recombinant, 3.39 (Fig. 6B, OspB^'). Only full-length OspB is detected in iow-passage Sh,2.82 (SH^), As described above, neither full-length OspB nor an OspB fragment is detected in variant SH#14

3036

P. A. Rosa, T. Schwan and D. Hogan

A. Inter-ptasmid recombination

and E. co//recombinant 4.28 bearing the recombinant osp locus. Bundoc and Barbour aiso reported smailer proteins recognized by OspB antibodies in their OspB-deficient variants from strain HB19 (Bundoc and Barbour, 1989); a point mutation in ospB could aiso account for what they observed. Co-migrating Osp proteins? A different explanation is necessary to account for the presence of only a singie Osp protein in G2 spirochaetes that contain an undeleted osp operon. Sequence analysis of the undeleted G2 osp operon has identified homologues of both ospA and ospB with intact open reading frames for both genes (Rosa etai, 1992). The chimaeric Osp protein expressed by variant G2.22 is mostly OspB in sequence, yet migrates with a mobility similar to that of the prototype G2 Osp protein (Fig. 7A). It is possible that the G2 ospA and ospS gene products co-migrate, despite differences in predicted molecular weights. This is not improbable as there is a general lack of correspondence between the predicted sizes of the Osp proteins and their mobilities on SDS-PAGE. Thus, the single major band detected by SDS-PAGE in uncioned G2 could be composed of three separate species of proteins: OspA, OspB and chimaeric OspA/B.

B. Intra-plasmid recombination

Fig. 5. A. Intemioleciilar recQmbtr>ation. An unequal crossover between ospA (solid bar) and ospB (hatched baO sequences on separate copies of the 49kb linear plasmid would generate (1) a single chimaeric ospAIB gene on one plasmid and (2) an osp locus with three genes on the other plasmid. The crossover could occur anywhere along the ospA gene. B. Intramolecular recombination. Homologous recombination between ospA {solid bar) and ospB (hatched bar) genes on the same copy of the 49 kb linear plasmid would generate (1) a single chimaeric ospAJB gene on the plasmid and (2) a 1 kb circular fragment of the osp operon containing the deleted intervening sequences.

A.

monoclonal anti - osp A CO

CM

CO CO

X X tn tn

X tn

m

When the sequence of the G2 osp operon is compared with that of G02, another European B. burgdorferi strain that expresses only a single major protein (Eiffert et ai, 1992), a surprising alignment results: the osp sequences of these two strains are identical except for a 79 bp deletion in the 5' end of the G02 ospSgene relative to G2. This deletion alters the reading frame in G02 such that OspB coding sequences are no longer preceded by an

B.

monoclonal antI - osp B 09

kDa

^'

-47 osp B-33

n

n

en

z

X

Ln

CQ

X

m

X Q

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•33

osp -24 osp B%. Fig. 6. A. Immunoblot analysis of Sn.2.82 Osp proteins expressed in S. burgdorferi and E. coli with an anti-OspA monoclonal antibody, B. burgdorferi lysates are as follows: SH# 14, cloned variant of high-passage Sh.2.82; SH^ uncloned, high passage Sh,2,82; SH^ uncioned, low-passage Sh,2.82; HB19, high-passage, uncioned strain; B31, high-passage, uncioned strain, E. co//recombinants 4.28 and 3.39 contain the 0.9kb and 1.9kb osp fragments, respectively, of high-passage strain Sh,2.82, cloned into pUC18; DH5i*, E. cali containing the pUC18 plasmid vector without an insert. The sizes of molecular mass standards are indicated at the right-hand side of the figure. The immunoblot was reacted with the OspA monoclonal antibody H5332 (Barbour efa/., 1983b). B. Immunoblot analysis of Osp proteins expressed in B. burgdorieri and E. coii with an anti-OspB monoclonal antibody. Bacterial lysates are as described in (A). OspB* designates the truncated fragment of OspB, The immunoblot was reacted with the OspB monoclonal H4610.

osp gene recombination A. Total Protein

B. OspA Immunoblot CM CM

OJ CO

CM

CQ

o

O

initiator methionine codon (Eiffert etai, 1992) and cannot be translated. The 100% nucleotide sequence identity of the G2 and G 6 2 ospA genes indicates that these two strains are very closely related, but that G 6 2 has undergone a discrete change in ospB that alters its expression. Conclusions We have found what appears to be homologous unequal recombination between the genes encoding OspA and OspB, the major outer surface proteins of B. burgdorferi. Recombination between osp genes theoretically could give rise to a large repertoire of chimaeric Osp proteins that have lost or altered pertinent epitopes. The frequency with which this occurs in the tick vector or mammalian host, and the effect that it has on the spirochaete's ability to establish a chronic infection are unknown. Although it is not possible to assess the frequency of recombination from our present data, we believe that this is not a rare event in B. burgdorferi because we have identified recombinant osp loci in multiple strains of high and low in vitro passage histories without imposed selective pressure. The fraction of bacteria with deleted osp loci in cloning experiments varied from 2-100%, but the plating efficiencies in these experiments were generally quite low, and thus the ratio of deleted to undeleted clones is not necessarily representative of the original cultures. It is also possible that deleted and prototype osp loci confer different selective advantages during in vitro growth and passage, and therefore the ratio of osp forms in a culture would not reflect the frequency with which recombinant variants arise. In addition, the method that we have used to screen for recombination would not identify any doublecrossover or gene conversion events in which segments of

CM

o

OJ O

3037

Fig. 7. A. S D S - P A G E profile of S. burgdorfen with prototypic and chimaeric osp loci. Wholecell S. burgdorferi lysates were separated on a potyacrylamide gel. Uncioned strains B31 and G2 consist primarily of bacteria with prototypic, t w o gene osp operons. Clone G2.22 contains a single, chimaeric osp gene; the molecular nature of this gene is described in Figs 3 and 4. M o l e c u lar mass standards are included at the left-hand side of the figure, and their sizes are indicated. B. Immunoblot analysis of G2 Osp proteins with an OspA monoclonal antibody. Bacterial iysates are as described in (A). The immunoblot was reacted with OspA monoclonal antibody H5332 ( B a r t j o u r e f a / . , 1983b).

ospA and ospB were exchanged, but where the resulting operon still contained two genes. We have also found Osp variation that is accounted for by point mutations and sequence divergence; this may also represent a significant means of altering the expression or function of the Osp proteins. OspA and OspB have been proposed as potential vaccine candidates for Lyme disease (Fikrig ef ai, 1990; 1992a; Schaible et ai. 1990; Simon er ai, 1991a,b). Protective polyclonal and monoclonal murine antibodies, as well as antibodies in the sera of patients with Lyme disease, bind a conformationally dependent epitope in the carboxy-terminal half of OspA (Sears ef ai. 1991; Shanafeit et ai, 1992), Similarly, the 60 carboxy-terminal amino acids of OspA contain human T-cell epitopes (Shanafeit etai, 1992). Thus the carboxy terminus of OspA is important to its antigenicity, and deletion of part of it through recombiriation could generate B. burgdorferi that would not be recognized by immunized individuals. Consistent with this, we have demonstrated the loss of an OspA epitope in an OspA/B chimaera. It is not known if additional epitopes are involved in protective immunity and, if so, whether they are eliminated in OspA/B chimaeras. Recombination of the osp gene sequences could also limit their usefulness as potential diagnostic targets(Malloy etai, 1990; Nielsen etai, 1990; Persing et ai. 1990). On the positive side, B. burgdorferi clones with defined Osp alterations provide an opportunity to assess the role of these major surface proteins in the pathogenesis of Lyme disease and their function in the spirochaete. The osp genes also represent a potentially interesting system in which to investigate genetic rearrangements among linear plasmids in B. burgdorferi.

3038

P. A. Rosa, T. Schwan and D. Hogan

Experimental procedures

For immunoblots, electrophoretically separated proteins were transferred to nitrocellulose and incubated with antisera and ^^^I-Protein A as described (Caldweil and Hitchcock, 1984),

Enzymes and chemicals Recombinant Taq polymerase was obtained from Perkin Elmer Cetus, nucleotides were from Pharmacia, Sequenase sequencing reagents were from USB, restriction endonucieases were from New England Biolabs, rabbit anti-mouse immunoglobulin was from Cappel-Organon Teknika Corp, and radiolabel was from NEN-DuPont. All chemicals were reagent grade. Oligonucleotides were synthesized with a SAM ONE DNA Synthesizer (Biosearch).

Culture conditions and strains Spirochaetes were grown in static liquid cultures under microaerophilic conditions in BSKII medium (Barbour, 1984) at 35°C. The biological and geographical origins of B. burgdorferi strains B31 (tick, US), HB19 (human, US), Sh.2.82 (tick, US), GI (human, Germany), G2 (human, Germany) and IP2 (human, France) and 6, hermsii strain HS1 (tick, US) were previousiy described in greater detail (Rosa ef a/., 1991). B. burgdorferi isolates CA-11 (Sacramento County, 2/3-7-90, passage 4), CA-17 (Amador County, 6/3/90, passage 3), CA-19 (Alameda County, 4/10/88, passage 7), and CA-20 (Butte County, 2/10/88, passage 5) were isolated from pools of Ixodes pacificus ticks and were obtained from Jane Wong and Michael Janda at the California State Health Department.

B. burgdorferi clones Sh.2.82, G2. CA-20 and Ca-17 clones were obtained from early log-phase cultures with a modification of a previously described protocol (Kurtti ef ai, 1987); bacteria were plated within a layer of top agarose (0.6%) rather than on the surface ofthe solid medium, and plates were placed in a 5% C02 incubator at SS^C instead of in a candle jar (Rosa and Hogan, 1992). Colonies were usually visible within a week. With strain G2, the efficiency of plating was usually less than 1%, and it took up to a month for colonies to appear. In order to obtain larger numbers of clones, G2 was subjected to limiting dilution plating in liquid BSKII medium in 96-well plates in a 5% C02 incubator. Under these conditions, the efficiency of plating was approximately 100%. Cultures were considered clonal when the mean number of spirochaetes per well was approximately 0.1 and the probability of more than one spirochaete per well was less than 1 % , as determined by the Poisson Distribution. One out of 50 G2 clones screened contained a deleted osp iocus. With strains Sh.2.82, CA-17, and CA-20, a larger fraction of the screened clones contained deleted osp loci; however, the efficiency of plating with these strains was not 100% so it is not possible to estimate the relative frequency of osp genotypes in the original populations.

Electrophoretic separation Late-log-phase bacteria were pelleted, washed twice in phosphate-buffered saline, lysed in reducing sample buffer, and separated on 12 % polyacrylamide gels (Laemmli, 1970). Approximately 10' bacteria were run per lane. Gels were stained with Coomassie brilliant blue R-250 in order to visualize total protein.

Antibodies The monoclonal antibody H5332 (Barbour et at.. 1983b) recognizes an epitope on OspA that is expressed by most B. burgdorferi isolates (Barbour ef al.. 1985). The monoclonal antibody H4610 recognizes an OspB epitope, and was the product of a fusion of a BALB/c mouse spleen with NSl myeloma cells as previously described (Barstad etai, 1985). On day 1, the mouse was inoculated intraperitoneally (i.p.) with approximately 10^ live spirochaetes of B. burgdorferi Sh.2.82 (passage 5). On day 24, the mouse was boosted I.p. with an identical inoculum, and on day 35 the mouse was boosted again intravenously. The spleen was harvested on day 38 for fusion and subsequent cloning in 96-well microtitre tissue culture plates. The isotype of H4610 was determined to be lgG2A by immunoblot analysis with whole cell lysates of S. burgdorferi Sh.2.82 and rabbit anti-mouse immunoglobulin (Ig) M, IgGI, lgG2A, and lgG2B and by '^^l-protein A radiography,

PCR amplification and primers The B31 primers represent nucleotides 1-20 and 1896-1915 of the published osp sequence (Bergstrom ef ai, 1989) and are as follows: 5' primer AAGCTTAATTAGAACCAAAC; 3' primer CTTCCTACACTAGCTGATGC. The G2 primer sequences are located slightly more distally to the osp locus and are as follows: 5' primer ATGAAGCGCATCTTTCAAGC; 3' primer GGCCTGATTTGTGTATTGTT (Rosa ef al.. 1992). One-hundred nanograms of totai e. burgdorferi DNA was amplified (Muilis ef ai. 1986; Saiki ef ai, 1985) for 20 cycles under the following conditions: 94°C for 1 min, 50°C for 0.5 min, 70°C for 2 min. Thirty picomoles of each primer was used per 100-^.1 reaction. Buffer conditions were as recommended by Perkin Elmer-Cetus. PCR amplification products were separated on 0.8% agarose gels and visualized with ethidium bromide (Maniatis etai, 1982). Genomic DNA isolation and hybridization conditions with ^^P-labelled probes were as previously described (Rosa and Schwan, 1989).

DNA sequencing The sequences of the deleted (SH#14) and undeleted Sh.2.82 osp loci were determined by dideoxy sequencing of plasmid DNA (Bartlett et ai. 1986; Sanger et at.. 1977) from fragments cloned into pUC18. The nucleotide sequence of the Sh.2.82 osp locus differs from the B31 sequence (Bergstrom ef ai, 1989) at six positions: a G to A transition at nucleotide 89, a G to A transition at nucleotide 596, a T to G transversion at nucleotide 1574, a G to A transition at nucleotide 1697, an A to C transversion at nucleotides 1739, and the insertion of a C after nucleotide 1890. Two of these changes are in non-coding regions, three of them would change the amino acid sequence, and one of them introduces a nonsense mutation (termination signal). Other than the deletion, the sequence of the SH#14 osp locus was identical to Sh.2.82, including three positions that differed from B31. The variant SH#14 sequence was confirmed by dideoxy sequencing of single-stranded POR-generated DNA (Rosa ef a/., 1991). The nucleotide sequence of the undeleted (2.3 kb) G2 osp locus was

osp gene recombination

3039

obtained by dideoxy sequencing of overlapping BgtW and Hindlll genomic fragments cloned into pUC18 (Rosa ef a/., 1992). The nucleotide sequence of the deleted G2 osp operon was determined by direct sequencing of the 1.4 kb osp PCR fragment, following amplification of SamHI-digested G2 DNA. The 2.3kb G2 osp fragment contains a SamHI site in a region of the ospA gene that is deleted in the variant 1.4 kb locus and thus is not amplified from the restricted DNA. This sequence was confirmed by directly sequencing the amplified osp operon of the deleted variant G2.22. The nucleotide sequences of the deleted osp loci of clones CA-20.2A and CA-17,13C were obtained by dideoxy sequencing of single-strand PCR-generated DNA as described (Rosa efa/., 1991).

molecular analysis of North American and European isolates, J /n^D/s 152: 478-484. Barstad, P.A., Coligan, J.E., Raum, M.G., and Barbour, A.G. (1985) Variable major proteins of Borretia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides. J Exp Med 161: 1302-1314. Bartlett, J.A., Gaillard, R.K., and Joklik, W.K. (1986) Sequencing of supercoiled plasmid DNA. BioTechniques 4: 208-210. Bergstrom, S,, Bundoc, V.G., and Barbour, A.G. (1989) Moiecuiar analysis of linear piasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mot Microbiot 3: 47&-486. Bundoc, V.G., and Barbour, A.G. (1989) Clonal polymorphisms of outer membrane protein OspB of Borretia burgdorferi. Infect

DNA cloning

Burgdorfer, W,, Barbour, A.G., Hayes, S,F., Benach, J.L., Grunwaldt, E., and Davis, J.P. (1982) Lyme disease — a tick-borne spirochetosis? Sc/ence 216:1317-1319. Caidwell, H.D., and Hitchcock, P.J. (1984) Monoclonal antibody against a genus-specific antigen of Chlamydia species; location of the epitope on chlamydial lipopolysaccharide. Infect /mmun 44: 306-314. Eiffert, H., Ohienbusch, A., Fehling, W., Lotter, H., andThomssen, R. (1992) Nucieotide sequence of the ospAB operon of a Borretia burgdorferi strain expressing OspA but not OspB. infect Immun 60: 1864-1868, Fikrig, E., Barthold, S.W., Kantor, F.S., and Flavell, R.A. (1990) Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 250: 553-556. Fikrig, E., Barthold, S.W., Marcantonio, N,, Deponte, K,. Kantor, F.S., and Flavell, R.A. (1992a) Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice. Infect tmmun 60: 657-661, Fikrig, E., Barthold, S.W., Persing, D.H., Sun, X,, Kantor, F.S., and Flavell, R.A. (1992b) Borretia burgdorfer/strain 25015: characterization of outer surface protein A and vaccination against infection. J Immunol 148: 2256-2260. Howe, T.R., Mayer, L,W., and Barbour, A.G. (1985) A single recombinant plasmid expressing two major outer surface proteins of the Lyme disease spirochete. Science 227: 645646, Howe, T.R., LaQuier, F.W.. and Barbour, A.G. (1986) Organization of genes encoding two outer membrane proteins of the Lyme disease agent Borretia burgdorferi within a single transcriptionai unit. Infect tmmun 54: 207-212. Jonsson, M., Noppa, L., Barbour, A.G., and Bergstrom, S. (1992) Heterogeneity of outer membrane proteins in Borretia burgdorferi: comparison of osp operons of three isolates of different geographic origins, tnfect Immun 60: 1845-1853. Kurtti, T.J., Munderloh, U.G., Johnson, R.C, and Ahlstrand, G.G, (1987) Colony formation and morphology in Borretia burgdorfert. J Ctin Microbiot 25: 2054-2058. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Malloy, D.C,, Nauman, R.K., and Paxton, H, (1990) Detection of Borretia burgdorferi using the poiymerase chain reaction. J Clin Microbiol 2a: 1089-1093. Maniatis, T., Frltsch, E.F., and Sambrook, J. (1982) Molecutar Ctoning. A Laboratory Manuat. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Meier, J.T., Simon, M.I., and Barbour, A.G. (1985) Antigenic variation is asociated with DNA rearrangements in a relapsing fever Borrelia. Ce//41: 403-409.

tmmun 57:

Recombinant plasmids 3,39 and 4.28 were constructed from PCR amplification products ofthe undeleted and deleted osp loci, respectively, of strain Sh.2,82. Primers flanking the osp locus (see PCR amplification and primers) v/ere modified to include SamHI sites, and PCR fragments were ligated into the BamHI site of pUC19 and transformed into E. coti DH5u. The consensus sequence from several clones was determined to assure that incorrect bases had not been introduced during PCR amplification.

Acknowledgements We thank Dr John Swanson for originally suggesting the possibility of homologous recombination between osp genes as a mechanism for Osp variation. We are grateful to Jane Wong and Michael Janda at the California State Health Department for their generosity in providing numerous low-passage B. burgdorferit\ck isolates. We thank Merry Schrumpf for assistance in monoclonal antibody production, Drs J, Swanson, R. Belland, S. Hill and K. Tilly for critical review ofthe manuscript, S. Smaus for manuscript preparation, and R. Evans and G. Hettrick for artwork and photography. Supported in part by a Biomedical Science Grant from the Arthritis Foundation (P.A.R.).

References Barbour, A.G. (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biot Med 57: 521-525, Barbour, A.G,, and Garon, C.F. (1987) Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends. Science 237: 409-411. Barbour, A.G.. Tessier, S.L., and Stoenner, H.G. (1982) Variable major proteins of So/Te/za/jerms/V, J Exp Med 156:1312-1324, Barbour, A.G., Burgdorfer, W., Grunwaldt, E., and Steere, A.C, (1983a) Antibodies of patients with Lyme disease to components of the Ixodes dammini spirochete. J Ctin tnvest72: 504-515. Barbour. A,G,. Tessier, S.L.. and Todd, W.J. (1983b) Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect tmmun 41: 795-804. Barbour, A.G., Tessier. S.L., and Hayes, S.F. (1984) Variation in a major surface protein ol Lyme disease spirochetes. Infect /mmun 45: 94-100. Barbour, A.G,, Heiland, R.A., and Howe, T.R. (1985) Heterogeneity of major proteins in Lyme disease borreliae: a

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Schaible, U.E,, Kramer, M.D., Eichmann, K., Modotell, M., Museteanu, C , and Simon, M.M. (1990) Monoclonal antibodies specific for the outer surface protein A (OspA) of Borretia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency {scid) mice. Proc Natt Acad Sci USA 87: 3768-3772. Schwan, T.G., and Burgdorfer, W. (1987) Antlgenic changes of Borretia burgdorferi as a result of in vitro cultivation. J tnf Dis 156: 852-853. Sears, J.E., Fikrig, E., Nakagawa, T.Y., Deponte, K., Marcantonio, N., Kantor, F.S., and Flavell, R.A. (1991) Molecular mapping of Osp-A mediated immunity against Borrelia burgdorferi. the agent of Lyme disease. J tmmunot 147: 1995-2000. Shanafeit, M.-C, Anzola, J., Soderberg, C, Yssel, H., Turck, C.W., and Peltz, G, (1992) Epitopes on the outer surface protein A of Borrelia burgdorferi recognized by antibodies and T cells of patients with Lyme disease. J Immunol 148: 218-224. Simon, M.M., Schaible, U.E., Kramer, M.D., Eckerson, C , Museteanu, C , Muller-Hermelink, H.K., and Wallich, R. (1991a) Recombinant outer surface protein A from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mce.JInfDis 164: 123-132. Simon, M.M., Schaible, U.E., Wallich, R,, and Kramer, M.D. (1991b) A mouse model for Borretia burgdorferi infection: approach to a vaccine against Lyme disease. Immunot Today 12: 11-16. Steere, A.C. (1989) Lyme disease. N Engt J Med 321: 586-596, Steere, A.C, Malawista, S.E., Hardin, J,A., Ruddy, S,, Askenase, P.W., and Andrman, W.A, (1977a) Erythemachronicum migrans and Lyme arthritis. The enlarging clinical spectrum. Ann tntern Med a6: 665-698. Steere, A.C, Malawista, S.E., Snydman, D,R,, Shope, R.E,, Andiman, W.A., Ross. M.R., and Steele, F.M. (1977b) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis Rheum 20: 7-17. Stoenner, H.G., Dodd, T., and Larsen, C. (1982) Antigenic variation in B. hermsii. J Exp Med 156: 1297-1311.

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi.

Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologou...
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