Molecular Analysis fo Borrelia P66-Integrin Binding

描述（由申请人提供）：莱姆病的病原体伯氏疏螺旋体能够从蜱虫叮咬的接种部位传播，并在宿主免疫反应的情况下持续存在，这表明在感染过程中，与哺乳动物细胞的相互作用是重要的。伯氏疏螺旋体与整合素alphaIIbbeta3和alphavbeta3结合，而蜱传回归热的一种病原体hermsii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。我们鉴定了伯氏疏螺旋体外膜蛋白P66作为整合素alphaIIbbeta3和alphavbeta3的配体。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疏螺旋体中得到验证。我们还生成了不表达P66的伯氏疏螺旋体突变体，并表明这些突变体在β a3整合素附着方面存在缺陷。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疏螺旋体菌株和不同伯氏疏螺旋体菌株的P66氨基酸多态性的意义，将为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Aim 1中，将通过将不同的P66等位基因引入P66缺陷突变体来测试特定P66氨基酸在β - 3链整合素识别中的作用。在Aim 2中，我们将验证B. hermsii P66也与β - 3链整合素结合的前提，并探讨B. burgdorferi和B. hermsii P66之间氨基酸差异的重要性。在Aim 3中，我们将研究B. burgdorferi P66附着在整合素alphavbeta3上是如何影响哺乳动物细胞生物学的。免疫荧光显微镜、免疫沉淀、免疫印迹和微阵列将被用于鉴定伯氏疏螺旋体P66结合alphavbeta3特异性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疏螺旋体的反应。通过这项工作，我们期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常规配体。

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.