Molecular Analysis fo Borrelia P66-Integrin Binding

描述(由申请人提供)：莱姆病病原体伯氏疏螺旋体能够从扁虱叮咬处的接种部位传播，并在宿主免疫反应的情况下持续存在，这表明在感染期间与哺乳动物细胞的相互作用是重要的。Burgdorferi与整合素αIIbbeta3和Alphavbeta3结合，Hermsii，一种硬蜱传播的复发热剂，也与AlphaIIbbeta3结合，这表明整合素结合对节肢动物传播的螺旋体很重要。整合素是调节细胞生长、迁移和炎症反应的关键信号受体。我们发现伯氏杆菌外膜蛋白p66是整合素αⅡbbeta3和alphavbeta3的配基。两个天冬氨酸残基似乎是整合素与p66结合的关键，但它们的作用尚未在完整的伯氏杆菌中得到测试。我们还产生了不表达p66的伯氏杆菌突变体，并表明这些突变体在Beta3整合素结合方面存在缺陷。由于p66不包含任何经典的整合素结合基序，不同整合素结合特性的不同伯氏杆菌菌株和不同疏螺旋体的p66氨基酸多态性的意义将为研究整合素生物学和螺旋体如何影响宿主细胞生物学提供独特的见解。在目标1中，将通过将不同的p66等位基因引入我们的p66缺失突变体来测试特定的p66氨基酸在β3链整合素识别中的作用。在目标2中，我们将验证Hermsii p66也与β3链整合素结合的前提，并探讨Burgdorferi和B.hermsii p66之间氨基酸差异的重要性。在目标3中，我们将研究Burgdorferi p66与整合素αvbeta3的结合对哺乳动物细胞生物学的影响。免疫荧光显微镜、免疫沉淀、免疫印迹和微阵列将被用来识别与Alphavbeta3结合的伯氏杆菌p66特异激活的信号通路和基因表达变化。这些实验将确定p66-整合素相互作用如何影响宿主对伯氏杆菌的反应。通过这项工作，我们希望深入了解致病螺旋体如何操纵其哺乳动物宿主的生物学，以及整合素如何识别非传统配体。

DESCRIPTION (provided by applicant): The abilities of Borrelia burgdorferi, the agent of Lyme disease, to disseminate from the inoculation site at the tick bite and to persist despite the host immune response indicate that interactions with mammalian cells are important continually during infection. B. burgdorferi binds to integrins alphaIIbbeta3 and alphavbeta3, and B. hermsii, an agent of tick-borne relapsing fever, also binds to alphaIIbbeta3, suggesting that integrin binding is important to the arthropod-borne spirochetes. Integrins are key signaling receptors that regulate cell growth, migration, and the inflammatory response. We identified P66, an outer membrane protein of B. burgdorferi, as the ligand for integrins alphaIIbbeta3 and alphavbeta3. Two aspartic acid residues appear to be key to integrin binding by P66, but their roles have not been tested in intact B. burgdorferi. We have also generated B. burgdorferi mutants that do not express P66 and showed that these mutants are defective for beta3 integrin attachment. Since P66 does not contain any classical integrin-binding motifs, the significance of amino acid polymorphisms in P66 from different B. burgdorferi strains with different integrin binding properties, and from different Borrelia species, will provide unique insights into integrin biology and into how the spirochetes affect host cell biology. In Aim 1, the roles of particular P66 amino acids in beta3-chain integrin recognition will be tested by introducing different p66 alleles into our p66-deficient mutant. In Aim 2 we will verify the premise that B. hermsii P66 also binds to beta3-chain integrins, and the importance of amino acid differences between B. burgdorferi and B. hermsii P66 will be explored. In Aim 3 we will investigate how mammalian cell biology is affected by B. burgdorferi P66 attachment to integrin alphavbeta3. Immunofluorescence microscopy, immunoprecipitation, immunoblotting, and microarrays will be used to identify signaling pathways and gene expression changes that are specifically activated by B. burgdorferi P66 binding to alphavbeta3. These experiments will determine how P66-integrin interaction influences the host response to B. burgdorferi. Through this work, we expect to gain insight into how pathogenic spirochetes manipulate the biology of their mammalian hosts, and into how integrins recognize unconventional ligands.