Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic illness that can affect the joints, heart, skin, and central nervous system. The spirochete is able to establish persistent infection in one or more of the affective tissues, despite the host immune response mounted. B. burgdorferi binds to several members of the integrin family of cell surface receptors, including alphaIIb beta/3, alpha/vbeta/3, and alpha/5 beta/1. The applicant's hypothesis is that binding to integrins contributes to the virulence of B. burgdorferi. As a step toward understanding the relevance of integrin binding to the pathogenesis of Lyme disease, the investigators have identified a strong candidate ligand for beta/3-chain integrins. To analyze the pathogenic significance of this candidate, the 66 kDa B. burgdorferi surface antigen, the following Aims will be pursued:
Aim 1. Generation of recombinant p66 and of specific antibodies directed against p66. Recombinant p66 will be expressed in E. coli and purified. Rabbit antisera and mouse monoclonal antibodies will be generated against p66 and used in the experiments described in Aim 2 to assess the relevance of p66 in attachment to mammalian cells.
Aim 2. Evaluation of the role of p66 in the attachment of live B. burgdorferi to alphaIIB beta/3 and other integrins in vitro. A series of independent experiments will be performed to determine whether the putative integrin binding domain of p66 is exposed on the surface of the spirochete, and whether p66 is relevant in the attachment of live spirochetes to purified integrins, platelets, and cultured epithelial cells.
Aim 3. Evaluation of the importance of p66 in a mouse model of Lyme disease. Mutant B. burgdorferi that do not express p66, or that express altered forms of the protein, will be selected using the antibodies generated in Aim 1. These mutants will be tested for binding to integrins in vitro, and for infectivity in a mouse model of Lyme disease. Together, the in vitro investigation of the interaction between this novel B. burgdorferi ligand and the integrins that it recognizes, are likely to contribute to our understanding of the pathogenesis of Lyme disease.
