burgdorferistrain B31 MI-16 using oligonucleotides5-TGTAAAGCATATGTAGAAGAAAAG-3and5-TTAATTAGTGCCCTCTTCGAGGAA-3

burgdorferistrain B31 MI-16 using oligonucleotides5-TGTAAAGCATATGTAGAAGAAAAG-3and5-TTAATTAGTGCCCTCTTCGAGGAA-3. comparable levels ofB. burgdorferiDNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect againstBorrelia burgdorferiinfection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible power of RevA-based immunotherapeutics or vaccine. == INTRODUCTION == Borrelia burgdorferiis the causative agent of Lyme disease, the most common arthropod-borne infection in the United States (1). Early diagnosis and treatment are key to preventing the debilitating long-term sequelae such as musculoskeletal, cardiovascular, and neurological damage (2). A preventative vaccine was approved for human use in 1998, but production was discontinued in early 2002 (3). The incidence of this disease has been continuously increasing since it was first Regorafenib (BAY 73-4506) explained in the late 1970s, and all evidence indicates that Lyme disease will continue to be a common public health problem. B. burgdorferican infect immunocompetent humans and other vertebrates for considerable periods of time, even for the animal’s lifetime (4,5,6). The Lyme disease spirochete is an extracellular organism, but a complete picture of how it manages to avoid clearance from its hosts is usually lacking. Antigenic variance at thevlslocus, which occurs onlyin vivo, is usually continuous throughout contamination (7,8). The outer surface protein VslE appears to be crucial for persistence in the mammalian host, as bacteria lacking VslE are completely cleared (9,10). Antibody appears to be important for clearance ofB. burgdorferi, as the variable regions of VslE are accessible to antibodies (11). Other aspects ofB. burgdorferi, including its tropism for immunologically isolated sites, may also contribute to its persistencein vivo. Extracellular matrix (ECM) has been suggested to provide a Rabbit Polyclonal to SUCNR1 protective market for the spirochete (12).B. burgdorferiis frequently found associated with connective tissues (12,13,14,15) and is often detected in and isolated from infected cartilaginous or membranous tissues, such as skin and joints. This suggests specific interactions between the pathogen and host skin tissues (5,16,17,18).In vitro,B. burgdorferishows affinity for host extracellular matrix components, such as fibronectin (12,19,20,21). Bacteria deficient in one of the fibronectin-binding proteins, BBK32, exhibit reduced virulencein vivo(22,23). Together, these data indicate thatB. burgdorferiinteracts with its host’s ECM and suggest that those interactions are crucial in bothB. burgdorferipathogenesis and persistence in mammals. Recently, we discovered that an antigenic 17-kDa outer surface lipoprotein, RevA, binds to fibronectin (19). We hypothesize that borrelia-ECM interactions, especially those mediated by RevA fibronectin-binding protein, are crucial for mammalian Regorafenib (BAY 73-4506) contamination and persistence in the host. The gene encoding RevA (so named because it is usually transcribed in the reverse direction from its neighboring genes) is located on a circular prophage (cp32). RevA has no significant homology to any proteins outsideBorreliaspecies, yet it is highly conserved within the Lyme disease borrelial genospecies. TherevAgenes are widely distributed among Lyme disease spirochetes, and the predicted amino acid sequences of RevA proteins are highly conserved (19). Many strains ofB. burgdorfericarry two copies of therevAgene; for example, the type strain B31 has two copies, and the well-characterized isolate 297 also has two copies ofrevA. In contrast,B. burgdorferistrain N40 andBorrelia gariniistrain PBi each carry only onerevAlocus (19). Serological studies show that humans and laboratory animals are frequently exposed to RevA duringB. burgdorferiinfection (24,25). Using quantitative real-time PCR, it was confirmed thatrevAis indeed transcribed during mammalian contamination, but not during colonization of vector ticks (19). Sera from patients in the initial stages of Lyme disease contained antibodies against RevA, demonstrating that this protein is usually expressed early in human infection (26). In the current study, we propose that RevA is the target of protective antibodies and that RevA expression remains elevated throughout mammalian contamination. To test our hypotheses, we examined mammalian response to RevA expression throughout the natural course of infection. In addition, we vaccinated mice with recombinant RevA Regorafenib (BAY 73-4506) antigen and challenged them withB. burgdorferi. == MATERIALS AND METHODS == == Bacteria. == B. burgdorferistrain B31 MI-16 is an infectious clone of the sequenced type strain (27,28) which contains all parental plasmids (29). Bacteria were produced at 34C to cell densities of approximately 1 107bacteria/ml in altered Barbour-Stoenner-Kelly (BSK-II) medium supplemented with 6% rabbit serum (30). Total DNA (genomic and plasmids) was isolated using a DNeasy blood and tissue kit (Qiagen, Valencia, CA). Plasmid content was monitored by multiplex PCR by the method of Bunikis et al. (31). == Recombinant proteins. == Recombinant proteins contained amino-terminal polyhistidine tags, with the RevA segment beginning with that protein’s first amino acid following the cysteine lipidation site. TherevAgene was PCR amplified from total genomic DNA ofB. burgdorferistrain B31 MI-16 using oligonucleotides5-TGTAAAGCATATGTAGAAGAAAAG-3and5-TTAATTAGTGCCCTCTTCGAGGAA-3. Amplicons were.