OspC variation THE ROLE OF RECOMBINATION IN OSPC VARIATION IN LYME DISEASE BORRELIA C. P. Gibbs, l. Livey and F. Dorner ABSTRACT The Osp C protein of Lyme disease Borrelia (Borrelia burgdo,feri sensu lato) is highly immunogenic and is a protective antigen, thus an ideal candidate for a subunit vaccine. However, this protein is extremely heterogeneous. This study was undertaken to analyze the variability of the OspC protein at the genetic level. The ospC gene was amplified by the polymerase chain reaction (PCR) from 76 Borrelia burgdo,feri sensu lato strains. PCR products were subjected to restriction fragment length polymorphism (RFLP) analyses and genes from different RFLP types were sequenced. A total of 33 ospC RFLP types were identified, and two additional RFLP types were deduced from published ospC sequences. Genes from different RFLP types were found to be extremely divergent, while within a given RFLP type, no sequence differences were detected. The majority of amino acid changes are localized to the central, highly variable portion of the mature OspC. Pairwise sequence comparisons indicate a mosaic structure of ospC. These results suggest that OspC variation is based on frequent recombination between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences between strains. KEY WORDS Lyme disease, Borrelia, ospC, recombination INTRODUCTION The OspC protein of Lyme disease (LD) Borrelia is highly immunogenic in the natural infection (1-3) and is a protective immunogen in animal models ( 4, 5). This protein is thus a good candidate for a subunit vaccine and may be of value in the serological diagnosis of LD. OspC is a surface-localized lipoprotein with a molecular weight of approximately 22kD. The protein is encoded by a single gene located on acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 a circular plasmid (6,7); expression of OspC correlates inversely with that of OspA and OspB (8), and is regulated by environmental factors (9,10). Comparisons of OspC proteins from different strains of Borrelia burgdo,feri (Bb) indica te this protein is antigenically extremely heterogeneous (11-13). To investigate the nature and extent of this variation at the genetic level, we have analyzed and compared ospC genes from a large collection of LD Borrelia strains. 179 OspC variation MATERIALS AND METHODS The ospC gene was analyzed from a collection of 76 Bb sensu lato strains. The strains were obtained from geographically divergent locations and included isolates from infected humans, rodents and ticks; a list of the strains used and relevant characteristics is given in Livey, et al. (14). Oligo- A. ss C1 V1 40 35 30 25 VARIABILITY 20 15 10 5 nucleotide primers corresponding to the 5' and 3' sequences of the ospC gene from strain Orth were used in the polymerase chain reaction (PCR) to amplify the ospC gene from each of the 76 strains analyzed. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using the enzymes Ddel, Dpnll and Dral. For nucleotide sequence analysis, the ospC gene sv V2 C2 o .j.1.W.U.W.lll,WlliljJllWJ.Wj.U.W 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 AMINO ACID POSITION 8. SPECIES RFLP B.garinii 15 B.garinii 16 8.garinii 14 B.garinii 15 11 1 1 m)// Yn41 /i,m%111 m II 1 m m III 111111 11 O B.garinii 18 11 11 B.garinii 19 111 l 1111 B.burgdorferi 2 111101 group DN127 3 111111 11111 111 m 11111 1111 1 111 1111 m 1111111 10 Fig. l. Variability in the OspC protein and sequence inconsistencies in the ospC gene. A . Variation in OspC proteins. The sequence variability (ratio of the number of different amina acid residues to the frequency of the most common residue) of the 35 RFLP types is plotted for each amina acid position of the mature protein. Constant and variable domains are indicated. B. Nucleotide sequence inconsistencies observed in pai,wise comparisons of ospC RFLP types. Vertical fines indicate residues differing between the two alleles. Significant stretches of sequence identity are indicated by shading and horizontal bars define regions of significant nucleotide divergence. 180 acta dermatovenerologica A.P A. Vol 5, 96, No 3-4 OspC variation was amplified by PCR and the nucleotide sequence of the PCR product determined (14). Amplification of ospC genes, RFLP analyses, and nucleotide sequencing have been described (14). Sequence comparisons were performed using the test of Stephens (15). RESULTS From analysis of the ospC genes of 76 Bb sensu lato isolates, 33 different RFLP types were identified (14), and two additional RFLP types could be deduced from published ospC sequences (16,17). Forty-two isolates, including at least one from each RFLP type, were selected for further analysis; the nucleotide sequence of the ospC gene from each of these strains was determined. Within a given RFLP type, all ospC sequences were identical. However, a comparison of ospC genes from different RFLP types revealed extreme sequence divergence, with sequence homology ranging from 74.4% to 99.0%. A comparison of the deduced amina acid sequences of the mature OspC protein from the different ospC RFLP types allows the protein to be divided into several domains (Fig. lA). The amina terminal one-third and carboxy terminal one-eighth form relatively constant domains (Cl and C2), while the SPECIES RFLP ss C1 V1 8 7 22 B. afzelii 6 11 10 5 17 B. garinii 25 18 central portion of the molecule is extremely variant. This variable region can be further divided into two hypervariable domains (HVl and HV2) flanking a semi-variable region (SV) centered around a stretch of hydrophobic amina acids. Additionally, close to the carboxy terminus, species-specific motifs (SS) are present. Pairwise sequence comparisons reveal several se- quence inconsistencies, that is, focal regions of extremely high sequence divergence within relatively similar ospC genes, or stretches of sequence identity within the variable domains of otherwise dissimilar ospC alleles. Some examples are presented in Fig. lB. Far instance, RFLP types 15 and 16, both from Bo1Telia garinii strains, differ in only six nucleotides, but five of these differences are localized in the conserved region near the beginning of the gene. On the other hand, RFLP type 2 (Bb sensu stricto) and RFLP type 3 (genogroup DN127) are highly divergent alleles, but the sequence is identical in these two genes in the region spanning the Vl and part of the SV domains. Indeed, short regions of sequence homology are frequently seen, leading to a mosaic nature of ospC (Fig. 2). DISCUSSION The heterogeneity observed in the OspC protein is characterized by the large number of alleles sv V2 C2 Fig. 2. Mosaic nature of ospC genes. The nucleotide sequences of ospC genes from different RFLP types . are compared. Regions of extensive homology between two or more strains within the variable portion of the molecule are indicated by the different shading pattems. Vertical lines indicate nucleotide differences located within homologous stretches. acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 181 OspC variation present in the Bo1Telia population as well as by the extensive sequence divergence between alleles. The amina acid differences are localized primarily in the variable domains in the central portion of the protein, a region containing the most hydrophilic portions of the molecule and thus likely to be immunodominant. The high degree of variability suggests OspC is evolving very rapidly, perhaps subject to intense positive selection. Hypermutability of the ospC gene could account far the sequence heterogeneity observed. However, in all cases when ospC genes from different strains with the same RFLP type were compared, no nucleotide differences were detected, even among strains from diverse geographic locations. These results would imply that ospC genes are relatively stable and that point mutations occur infrequently, a hypothesis that can not be easily reconciled with a mechanism of hypermutability. Any explanation far the molecular mechanism of ospC variation must account far both the high variability observed as well as far the lack of genetic intermediates. A recombination-based mechanism far the generation of OspC variability could account far this dichotomy; major sequence changes would result from a single recombination event between different ospC alleles. Pairwise sequence comparisons support this hypothesis. Sequence inconsistencies reveal a mosaic structure observed among ospC RFLP types (Fig. 2), where scattered, short regions of sequence identity can be faund among divergent genes. The precise mechanism responsible far the recom- bination among ospC genes remains unknown. The ospC gene maps to a single locus on a circular plasmid (6,7), and pseudogenes have not been detected (CPG, unpublished). Thus genetic exchange between resident gene copies within a single cell is unlikely to occur. We propose that OspC variation occurs via lateral transfer of genetic material between strains, fallowed by recombination with the resident ospC gene. The location and extent of recombination probably depends upon the degree of sequence homology between the two genes. Fram analysis of the mosaic structure of the ospC genes, it can be seen that genetic transfer probably occurs frequently between strains within a given species, but may also occur between species. REFERENCES 1. Dressler F, Whalen JA, Reinhardt BN et al. Westem blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993; 167: 392-400. 2. Wilske B, Fingerle V, Herzer P et al. Recombinant immunoblot in the serodiagnosis of Lyme Borreliosis. 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Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi. Infect Immun 1993; 61 : 2182-91. 13. Stevenson B, Barthold SW. Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi. FEMS Microbiol Lett 1994; 124: 367-72. 14. Livey I, Gibbs CP, Schuster R et al. Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia. Mol Microbiol 1995; 18: 257-69. 15. Stephens JC. Statistical methods of DNA sequence analysis: Detection of intragenic recombination or gene conversion. Mol Biol Evol 1985; 2: 539-56. 16. Jauris-Heipke S, Fuchs R, Motz M et al. Genetic heterogeneity of the genes coding for the outer surface protein C (OspC) and the flagellin of Borrelia burgdorferi. Med Microbiol Immunol (Berl) 1993; 182: 37-50. 17. Padula SJ, Sampieri A, Dias F et al. Molecular characterization and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi. Infect Immun 1993; 61: 5097-105. AUTHORS' ADDRESSES Carol P. Gibbs, PhD, Immuno AG, Biomedical Research Center, UferstraBe 15, A-2304 Orth an der Donau, Austria lan Livey, PhD, same address Friedrich Dorner, PhD, professor, same address acta dermatovenerologica A.P A. Vol 5, 96, No 3-4 183