CYTOPATHIC EFFECT OF THE BORRELIA BURGDORFERI EXOPROTEINS OMS28 AND ENOLASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 伯氏疏螺旋体是一种莱姆病螺旋体，它对温度、pH、营养物质的可获得性以及其哺乳动物和壁虱宿主的独特环境的变化做出反应。我们已经使用内源性放射性标记、LC-MS/MS蛋白质测序和免疫化学实验来确定伯氏杆菌选择性地分泌Oms28和BGP/PFS-2，Oms28是以前描述的外膜孔蛋白，BGP/PFS-2是一种双功能酶，与糖胺聚糖结合，可能参与群体感应。 在这个提案中，我们确定了另外11个外源蛋白，并希望进一步鉴定其中的两个，Oms28和烯醇化酶。此外，我们还将确定伯氏杆菌外源蛋白是否能被莱姆病患者抗血清识别。该项目的具体目标是：(1)确定天然Oms28的寡聚体组织，(2)确定出口的Oms28是否对红细胞和角质形成细胞靶标具有细胞毒性，以及(3)检验烯醇化酶是一种双功能蛋白的假设，该蛋白在细胞质中起脱水酶的作用，但当从螺旋体中释放时作为纤溶酶原结合蛋白。一个重要的假设是，伯氏杆菌产生的外源蛋白可能与宿主细胞和组织相互作用，并在发病中起作用。在这里，我们将使用胞外形式的Oms28和烯醇化酶来解决这个问题。 天然和重组Oms28的寡聚结构将通过化学交联法、非变性SDS-PAGE和质谱仪来确定。来自几个来源的天然和重组Oms28和烯醇化酶将被检测对红细胞、角质形成细胞和人类单核细胞白血病细胞系的细胞毒性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Borrelia burgdorferi, the Lyme disease spirochete, responds to changes in temperature, pH, nutrient availability, and the distinct environments of its mammalian and tick hosts. We have used intrinsic radiolabeling, LC-MS/MS protein sequencing and immunochemical experiments to establish that B. burgdorferi selectively secretes Oms28, a previously characterized outer-membrane porin, and Bgp/Pfs-2, a bifunctional enzyme that binds glycosaminoglycan binding and may be involved in quorum-sensing. In this proposal we identify 11 additional exoproteins and wish to further characterize two of these, Oms28 and enolase. In addition, we will determine if B. burgdorferi exoproteins are recognized by Lyme disease patient antisera. The specific aims of this project are to: (1) to determine the oligomeric organization of native Oms28, (2) to establish if exported Oms28 is cytotoxic against erythrocyte and keratinocyte targets, and (3) to test the hypothesis that enolase is a bifunctional protein that functions as a dehydratase when in the cytoplasm, but as a plasminogen-binding protein when released from the spirochete. An overarching hypothesis is that exoproteins produced by B. burgdorferi may interacts with host cells and tissues and contribute to pathogenesis. Here, we will address this question using the extracellular forms of Oms28 and enolase. The oligomeric organization of native and recombinant Oms28 will be determined by chemical-cross linking, nondenaturing SDS-PAGE, and mass spectrometry. Native and recombinant Oms28 and enolase from several sources will be assayed for cytoxicity against erythrocytes, keratinocytes, and a human monoblastic leukemia cell line.