Detection of genetic diversity in linear plasmids 28-3 and 36 in Borrelia burgdorferi sensu stricto isolates by subtractive hybridization Yun Xu, John F. Bruno * , Benjamin J. Luft SUNY at Stony Brook, Department of Medicine, State University of New York at Stony Brook, T-15 Room 060, Stony Brook, NY 11794-8154, USA Received 7 April 2003; received in revised form 15 July 2003; accepted 23 July 2003 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. q 2003 Elsevier Ltd. All rights reserved. Keywords: Borrelia; Virulence factors; Subtractive hybridization; Invasive strains 1. Introduction Lyme disease, the most common vector-borne disease in the United States and Europe [1], is a progressive multisystem disorder characterized by an initial cutaneous infection that can spread early in infection to secondary sites that include the nervous system, heart and joints [2,3]. The causative agent is Borrelia burgdorferi, a spirochete first isolated from Shelter Island, NY [4]. B. burgdorferi infection induces a strong humoral response against the endoflagellar protein, p41, and a protein constituent of the protoplasmic cylinder, p93 [5], both of which are enveloped within the outer membrane, and some lipoproteins which are the major components of the membrane [6]. Of these outer surface proteins (Osps), OspA and OspC are the most prominent and have been viewed as likely vaccine candidates. OspA and experimental OspC vaccines, how- ever, have limited utility since they are usually only effective against challenge by homologous genotypes [7–11]. Thus, understanding the genetic variation in the Lyme disease spirochetes will be important in creating a successful vaccine(s). The limitations of the OspA and OspC based vaccines highlight the need to evaluate additional gene products that could afford protection against Borrelia infection. However, the extensive genetic and antigenic diversity of OspC in all three pathogenic genospecies of B. burgdorferi sensu lato has led us to propose that OspC could play a role in determining the potential for a given isolate to lead to chronic infection and, therefore, be used as a marker to discover new protective antigens [12–14]. We have shown that alleles of OspC collected from a single site on Shelter Island, NY could be clustered into 19 major groups or types designated A–S based on DNA sequence homology [14]. Sequence variation within a major group is , 2% but . 8% 0882-4010/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0882-4010(03)00152-9 Microbial Pathogenesis 35 (2003) 269–278 www.elsevier.com/locate/micpath * Corresponding author. Tel.: þ 1-631-444-2054; fax: þ1-631-444-2493. E-mail address: jbruno@notes.cc.sunysb.edu (J.F. Bruno).