Detection of genetic diversity in linear plasmids 28-3 and 36 in Borrelia burgdorferi sensu stricto isolates by subtractive hybridization

Abstract

Recent studies based on sequence divergence in the ospC gene have identified limited subpopulations of B. burgdorferi associated with invasive human disease. Spirochetes with certain OspC types never cause human disease, while some others cause local infection at the primary skin site but do not hematogenously disseminate. Only four OspC genotypes (A, B, I and K) are responsible for disseminated disease and are found in the blood and cerebrospinal fluid, and hence are termed invasive strains. Subtractive hybridization was carried out between a prototype of a low passage invasive type, strain B31, and a strain associated only with local infection, group E, to identify genes associated with hematogenous dissemination. Two clones isolated from the subtraction library were unique to the B31 genome and mapped to locus BBH26 located on linear plasmid 28-3 (lp28-3) and to locus BBK48 located on linear plasmid 36 (lp36). Sequence analysis of the BBH26 locus revealed an amino acid repeat motif in the group E DNA that was absent in the B31 genome. This in-frame repeat motif was present yet variable in DNA isolated from several major OspC groups. However, no consistent sequence diversity was noted when other invasive and non-invasive strains were compared. In contrast, analysis of the BBK48 locus revealed a striking distinction between invasive and non-invasive spirochetes. PCR and Southern blot analysis indicated this locus was only present in invasive groups A, B, I, and K. BBK48 is a member of a gene family clustered on lp36. Therefore, these findings indicate that this genetic loci may participate in differentiating pathogens from non-pathogens and that its presence, which is correlated with ospC type, may play a role determining infectivity in humans.