FEMS Microbiology Letters 124 (1994) 367-372 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 06303

Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi

Brian S tevenson a and S t e p h e n W. B a r t h o l d *

Section of Comparative Medicine, Yale University School of Medicine, PO Box 208016, New Haven, CT 06520-8016, USA

(Received 9 September 1994; revision received and accepted 10 October 1994)

Abstract: The expression of outer surface protein C (OspC) was determined for North American Borrelia burgdorferi isolates HB19, DN127c19-2, 25015 and both low and high culture passage B31. A monoclonal antibody detected the presence of OspC protein in only two isolates, while polyclonal antiserum identified this protein in all five isolates. The ospC gene was cloned and sequenced for isolates HB19, DN127cl9-2 and 25015, and compared with the published ospC sequences of other Lyme disease spirochetes. Both the nucleotide and amino acid sequences were found to vary as much among isolates from the same geographic area as between isolates of different species.

Key words." Borrelia burgdorferi; Lyme disease; OspC

Introduction

Lyme disease is a disease of humans and other mammals which affects the skin and muscu- loskeletal systems, as well as the cardiac, neuro- logical and other organ systems. It is caused by the spirochete Borrelia burgdorferi, which has been recently reclassified into at least three

* Corresponding author. Tel.: (203) 785 2525; Fax: (203) 785 7499.

i Present address: Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA.

species, including B. burgdorferi sensu stricto, B. garinii, and B. afzelii [1,2].

The outer surface protein C (OspC), a 22-kDa lipoprotein, is an important immunogen in Lyme disease, with anti-OspC antibodies being pro- duced quite early in the infection of both humans and laboratory animals ([3-5]; our unpublished results). The ospC gene has been found present in all isolates examined [6-8]. It has been re- ported that North American isolates of B. burgdorferi, such as the type strain B31 (ATCC 35210), are unable to express OspC when grown in culture, based upon studies which failed to detect this protein with polyclonal serum and monoclonal antibodies generated against the

SSDI 0378-1097(94)00456-0

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OspC protein of European isolates [3,5,7,8]. To further explore the expression (or lack thereof) of OspC in North American isolates, we performed immunoblotting studies using both a monoclonal antibody and polyclonal serum directed against the recombinant OspC of a North American B. burgdorferi sensu stricto isolate.

A wide variability in OspC sequences has been reported between isolates of B. burgdorferi [3,7,8]. We have determined the ospC gene sequences of three additional North American isolates of B. burgdorferi, and compared both the nucleotide and amino acid sequences with those of other B. burgdorferi sensu stricto isolates, as well as B. garinii and B. afzelii. Alignment analysis of both ospC genes and proteins indicated that these sequences suggest a different phylogenetic tree than those based on other sequences of Lyme disease spirochetes, raising the possibility of ge- netic exchange of ospC between these bacteria.

Materials and Methods

Bacterial strains All Borrelia burgdorferi used in this work were

originally isolated in North America: HB19 from Connecticut [9] (provided by A. Barbour), DN127c19-2 from California [10] (provided by M. 13issett), and 25015 from New York [11]. Two

cultures of isolate B31 were utilized: low-passage B31, an uncloned isolate which was passaged in culture medium no more than five times before storage at -70°C (provided by A. Barbour); and high-passage B31 (ATCC 35210), a derived iso- late which has been passaged continuously in culture medium for several years (provided by D. Persing). Isolate N40, which produces OspC when grown in culture [6], was used as a positive con- trol for OspC expression. Recent evidence [1] has suggested that DN127c19-2 and 25015 may be members of groups which are genetically distinct from B. burgdorferi sensu stricto, the species which includes HB19, B31 and N40.

Analysis of OspC protein expression Bacteria were grown to late log phase in BSK

II medium [6] at 33°C. The bacteria were har- vested by centrifugation, washed twice with phos- phate-buffered saline containing 5 mmol MgC12 and lysed in distilled water. 15 ng of total protein was separated by polyacrylamide electrophoresis, transferred to nitrocellulose membranes and blocked as previously described [6]. Membranes were incubated at room temperature with either a 1:100 dilution of monoclonal antibody L221F8, which is directed against the OspC of 13. afzelii isolate PKo [3] (provided by B. Wilske), or with a 1:5000 dilution of a mouse hyperimmune poly- clonal serum raised against purified recombinant

A B C D E F A B C D E F

Fig. 1. (A) Immunoblot analysis of OspC expression by different B. burgdorferi sensu stricto isolates, using anti-OspC monoclonal antibody L221F8 [3]. A, high-passage B31; B, low-passage B31; C, N40; D, HB19; E, DN127c19-2; F, 25015. (B) Immunoblot analysis of OspC expression by different B. burgdorferi sensu stricto isolates using OspC polyclonal antiserum [6]. A, high-passage

B31; B, low-passage B31; C, N40; D, HB19; E, DN127c19-2; F, 25015.

N40 OspC [6]. Specifically bound proteins were identified as previously described [6].

Cloning and sequencing of ospC genes B. burgdorferi isolates were grown to late log

phase and 1 ml of each culture was pelleted in a microcentrifuge. The cells were washed twice with PBS, and lysed by resuspension in 30 /x l distilled water and heating to 94°C for 5 min. 2 / z l of each lysate was subjected to PCR amplification of the ospC gene using ol igonucleotide primers based on the sequence of the ospC gene of B. afzelii isolate PKo [12]: 5 ' - C G C G G A T C C T G C A A A G A - AAAATTGT-FGGAC-3' and 5'-CGCGGATCC- C C A G T T A C T T T T T T A A A A C A A - 3 ' , compli- mentary to regions approximately 30 bp upstream and downstream of the gene, respectively. PCR conditions consisted of 30 cycles of 1 min at 94°C, 1:30 min at 45°C and 1:30 min at 74°C. In each case, a single band of approximately 700 bp was observed after agarose electrophoresis of the completed reactions, corresponding with the known size of the PKo ospC gene [12]. The PCR product for each isolate was cloned using the T A Cloning System (Invitrogen, San Diego, CA). The cloned D N A s were purified using a Qiagen plas- mid kit (Qiagen, Chatsworth, CA) and both strands were sequenced in entirety using a 373A D N A sequencer (Applied Biosystems, Foster City, CA).

Nucleotide sequence accession numbers The ospC sequence data for isolates HB19,

DN127c19-2 and 25015 have been submitted to the E M B L / G e n B a n k nucleotide sequence data library and given the accession numbers U04281, U04280 and U04282, respectively.

Results and Discussion

Analysis of OspC protein expression Based upon reactivity with OspC monoclonal

antibodies, others [3,5,7,8] have surmised that many isolates of B. burgdorferi, including B31, fail to synthesize the OspC protein when grown in culture. As illustrated in Fig. 1A, one of these same monoclonal antibodies, L221F8, likewise

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failed to detect OspC production in the high-pas- sage isolate of B31, which is the most commonly available B31 isolate. This monoclonal antibody did, however, bind to OspC in the low-passage isolate of B31. It is possible that these observa- tions stem from the inadvertant selection of a variant sub-population within the uncloned low- passage B31 which fails to produce OspC when grown in culture medium.

OspC monoclonal antibody L221F8 also recog- nized OspC production by isolate N40, as we have reported elsewhere [6] (Fig. 1A). Binding of the monoclonal antibody could not be detected

HBI9

25015

DNI27

B31 N40 CAll

2591

297

DK7

PKO

PBI

HBI9

25015

DNI27

B31 N40 CAll

2591

297

DK7

PKo

PBi

HBI9

25015

DNI27

B31 N40

CAll

2591

297

DK7

PKo

PBi

HBI9

25015

DNI27

B31 N40 CAll

2591

297

DK7

FKo

PBi

55

MKKNTLSAI LMTLFLF I SCNNSGKDGNT- SANSADESVKGPNLTEI SKKI TESNA

........................... AA.T.P .................. D...

........................... SA.ToP .... A ............. D...

............................ - ...................... D.•.

........................... -A ..........................

............................ - ...................... D...

.... G...- ..........................

............................ - ...................... D...

........................ G.DSA.T.P .... A ............. D...

....................... --°DSA.T.P-...A ...... V ...... D...

ii0

VVLAVKEVETLLT S I DELA- KAI GKK I KNDVS LDNEADHNGS L I SGAY L I S TL I T

........ GA ......... T ....... HQNNG. .T.NN ..... LA. o °A ......

I ........... L ...... - ....... N.NG-..VLQNF.A..LG..HT° .K...

.L ....... A..S .... I .A ....... HQNNG..T.NN ..... LA...A ..... K

............ A ...... T ....... G.N-G.EANQSK°T..L .... A..D..A

.L ...... V..S ...... K ....... DQNNA.GTLDN ..... LA. o .A..A...

........... A .... V.K .... NL.AQN-G.NAG.NQ .... LA° . .V ..... A

...... I .... A ...... T ....... QQNGG.AV. .G. . .T.LA. . .T° .K. . °

.L ...... A..S ...... - .......... G..GD..N..E..LA...T ......

F ......... VL ...... K .... Q.. D o NNG.AALNNQ .... LAA° .A ......

FL ...... A..S ..... S-° ......... GT ..... NR°E...A...E..K...

165

KK I SAI KDSGELKAE I EKAKKCS EEFTAKLKGEHTDLGKE -G -VTDDNAKKA I LK

Q. LGGL.N-E...EK.AAV ........ N...SS..E...QD-A-Q..D ...... R

E.L.KLNG.E...EK..A ..... DD..K..QSS .2rE..VA..AT..E ........

Q. LDGL.NE. -..EK.DA ...... T. oN...EK ........ --o . .AD° .E ....

E.LNVL.N-E...EK.DT..Q. °T...N...S..AV..LDN--L ..... QR ....

E.L.S ................... S. .K. . SDNQAE. .I.N-A-. ..........

E.LDGL.N.E...EK..D .... NKA..D...SS.AE.. IAN.AAS .A...A ....

Q.LDGL.N.EK. .EK. .N ...... D..K. .E. . .AQ. .I.N--. . .E ....... I

Q.L.KLNG.EG. .EKoAA ........ ST. . .DN.AQ. .IQ-.-. . .E ........

E.L.KL.NLE.. .T . .A .......... N. . .SG.A .... QD-A- . . .H. .A ....

Q.L.VLN-.E . . .EK.KE. .D. .QK. .T. . .DS .AE. . IQ--S.Q ..........

TNND -KTKGADELEKLFESVKNL S KAAKEMLTNSVKELT S PWAESPKKP

.H.T-.D. , .E..D...KA.E .......... S ..................

S.A.- ........ G ..... VES.A ....... A ............. T ....

• .GT-. .... E..G ...... EV ......... A ..................

KHAN- ° D...A ...... KA.E ...... QDT.K.A ....... I ........

oH.A-.D...E..V..S...AG.L...QAI .A ..................

• .GT-.D. • .Q ................ QET.N ............. N ....

.DAA-.D.. °A ...... KA.E..A ....... A ......... I ........

A . A A G . D . . V E . . . . . S G . L E S . . . . . . . . . A . . . . S . Q . V .

• H A T - T D . . . K . F K D . . . . . E G . L . . . Q V A . . . . . . . . . . . . . . . . . . . .

. H G T - . D . . . K . . . E . . K . L E S . . . . . Q A A . . . . . . . . . N . . . . . . . . . .

Fig. 2. Comparison of the OspC proteins of North American B. burgdorferi isolates HB19, DN127c19-2 and 25015 with B. burgdorferi sensu stricto isolates B31 [3], N40 [6], CAll [19], 2591 [20], 297 [13] and DK7 [7], B. garinii isolate PBi [3] and B• afzelii isolate PKo [12]. A period indicates an amino acid identical to that of isolate HB19. Dashes indicate spaces introduced to allow for maximum alignment of the amino acid sequences• The complete sequences of OspC from isolates

297 and DK7 are not presently available [7,13].

370

for isolates HB19, DN127c19-2 or 25015. In con- trast, immunoblotting with polyclonal serum against the recombinant OspC protein of N40 indicated synthesis of OspC by all of the isolates (Fig. 1B). Interestingly, high-passage B31 gave a weak signal in response to immunoblotting with the polyclonal antiserum, indicating that OspC protein was indeed present in this isolate, but was either produced in low quantities or was antigeni- cally different from the low-pasage isolate, which may explain why the monoclonal antibody failed to detect the protein. These studies indicate that OspC is in fact produced in culture by a number of North American isolates of B. burgdor/eri, including the commonly used laboratory high- passage isolate B31.

Cloning and sequence analysis of OspC genes The ospC gene of isolates HB19, DN127c19-2

and 25015 were all efficiently amplified via PCR using these oligonucleotide primers. In spite of the fact that the coding regions vary widely, it

appears that the surrounding regions are greatly conserved between different isolates. The oligo- nucleotide primers used in this work should therefore be useful for PCR amplification of the entire ospC gene from other Lyme disease spiro- chetes, rather than the partial sequences which have often been reported [7,8,13].

Upon DNA sequencing, the ospC clones of isolates HB19, DN127c19-2 and 25015 were each found to contain a single open reading frame of 630, 636 and 633 nucleotides, respectively, capa- ble of encoding a protein with an approximate molecular mass of 22.5 kDa. Using the alignment program CLUSTAL V, the amino acid sequences of the corresponding proteins were compared with the published sequences of a number of B. burgdorferi sensu stricto isolates, as well as B. garinii isolate PBi [3] and B afzelii isolate PKo [12] (Fig. 2). Conservation of amino acid se- quence was found in the first 22 amino acids of the amino termini (which includes the putative leader polypeptide [12]), but varied greatly within

A HBI9 (Bb ss)

DK7 (Bb ss)

CAll (Bb ss)

25015 (Bx)

B31 (Bb ss) B

N40 (Bb ss) ~ HBI9 (Bb ss)

I B31 (Bb ss)

2591 (Bb ss) N40 (Bb ss)

297 (Bb ss)

i 25015 (Bx) DNI27 (Bx)

PBi (Bg) PBi (Bg)

PKo (Ba) PKo (Ba)

Fig. 3. Dendrograms based upon the ospC genes (A) and ospA genes (B) of isolates of Lyme disease Borrelia. The species of each isolate is indicated as B. burgdorferi sensu stricto (Bb ss), B. garinii (Bg), B. afzelii (Ba) or ambiguous (Bx). The length of each horizontal branch is to scale and is indicative of the degree of dissimilarity between isolates. Available ospA gene sequences were obtained from GenBank, with accession numbers L23136, X14407, M57248, $88693, $48323 and X62161 for isolates HB19, B31,

N40, 25015, PBi and PKo, respectively.

371

the remainder of the proteins. The low degrees of similarity observed between the isolates studied herein (HB19, DN127c19-2, 25015, and B31) and that of B. afzelii PKo strongly suggests why mon- oclonal antibody L221F8 did not react all isolates. It is apparent that neither the identification of the species of a Lyme disease organism nor the geographic location of its isolation can be used to predict the sequence of, and therefore the im- munoblotting response to its OspC protein.

Recent reports indicate that vaccination of laboratory animals with OspC protein may effec- tively protect against B. burgdorferi challenge [4,14]. In both of these experiments, however, the protective OspC protein used was derived from the same isolate as used in the challenge. The high degree of variability of the OspC proteins and the variability of their recognition by antibod- ies casts significant doubt on whether OspC would prove to be effective in protecting against chal- lenge by heterologous isolates. Such experiments need to be pursued in the development of effec- tive Lyme disease vaccines.

Based upon the sequence conservation of a number of chromosome- and plasmid-encoded genes, it has been speculated that there is no exchange of genetic material between Lyme dis- ease-associated Borrelia [15]. A bootstrap analysis of the ospC gene sequences produced the relat- edness tree shown in Fig. 3A. What was espe- cially surprising about the ospC genes was the lack of correlation between ospC sequence simi- larities and cladistic trees of Lyme disease Borre- lia based upon ospA sequences, ribosomal RNA sequences or restriction fragment length poly- morphisms [15-17], such as that shown in Fig. 3B. In such trees, B. burgdorferi sensu stricto isolates consistently fall into a closely related, distinct group, accompanied with a great degree of dis- similarity between the three species. The dendro- gram in Fig. 3A, based upon ospC sequences, indicates a great dissimilarity between individual isolates with very short branch lengths between groupings, with the ospC genes of isolates B31, HB19 and N40 placed as distant from each other as from members of B. garinii or B. afzelii. It was further indicated that the ospC gene of isolate 25015 is most closely related to that of isolate

B31, whereas all other trees have placed 25015 at a considerable distance from B31, enough to sug- gest that 25015 may be a member of a different genomic group [1,15]. These observations raise the intriguing possibility that the ospC gene, which is located on a circular plasmid [18], may be exchanged laterally and become modified through intragenic recombination.

Acknowledgements

Our thanks to B. Wilske for providing mono- clonal antibody L221F8; to L. Bockenstedt, F. Kantor, K. DePonte and T. Zhu for production of the anti-N40 OspC polyclonal serum; to D. Persing, A. Barbour and M. Bissett for providing bacterial isolates; to P. Rosa for comments on the manuscript: to J. Hoch for the CLUSTAL V program; to D. Beck, F. Coyle, J. Butler and G. Terwilliger for technical assistance. Supported by National Institute of Allergy and Infectious Dis- eases, NIH, Grants AI26815 and AI30548.

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