CYTOPATHIC EFFECT OF THE BORRELIA BURGDORFERI EXOPROTEINS OMS28 AND ENOLASE

伯氏疏螺旋体外蛋白 OMS28 和烯醇酶的细胞病变作用

• 批准号: 7610036
• 负责人: Robert Gregory Cluss
• 金额: $ 9.62万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7610036
• 关键词: Address; Amino Acid Sequence; Binding; Binding Proteins; Biological Assay; Borrelia burgdorferi; Cell Line; Cells; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Environment; Enzymes; Erythrocytes; Funding; Glycosaminoglycans; Grant; Human; Hydro-Lyases; Immune Sera; Institution; Lyme Disease; Mass Spectrum Analysis; Membrane; Nutrient; Order Spirochaetales; Pathogenesis; Patients; Peptide Sequence Determination; Plasminogen; Radiolabeled; Recombinants; Research; Research Personnel; Resources; Source; Temperature; Testing; Ticks; Tissues; United States National Institutes of Health; crosslink; cytotoxic; enolase; extracellular; keratinocyte; leukemia; porin; protein function; quorum sensing; radiotracer; research study

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 莱姆病螺旋体伯氏疏螺旋体对温度、pH值、营养物质可用性以及其哺乳动物和蜱宿主的不同环境的变化有反应。我们使用内在放射性标记、LC-MS/MS蛋白质测序和免疫化学实验来确定B。burgdorferi选择性分泌Oms 28（一种先前表征的外膜孔蛋白）和Bgp/Pfs-2（一种结合糖胺聚糖结合的双功能酶，可能参与群体感应）。 在这个建议中，我们确定了11个额外的外蛋白，并希望进一步表征其中的两个，Oms 28和烯醇化酶。此外，我们将确定是否B。莱姆病患者抗血清可识别伯氏菌外蛋白。该项目的具体目的是：（1）确定天然Oms 28的寡聚体组织，（2）确定输出的Oms 28是否对红细胞和角质形成细胞靶标具有细胞毒性，以及（3）检验以下假设：烯醇化酶是双功能蛋白质，其在细胞质中时作为脂肪酶发挥作用，但在从螺旋体释放时作为纤溶酶原结合蛋白发挥作用。总体假设是由B产生的外蛋白。Burgdorferi可能与宿主细胞和组织相互作用并导致发病。在这里，我们将使用Oms 28和烯醇化酶的细胞外形式来解决这个问题。 通过化学交联、非变性SDS-PAGE和质谱法测定天然和重组Oms 28的寡聚组织。将测定天然和重组Oms 28和来自几种来源的烯醇化酶对红细胞、角质形成细胞和人单核细胞白血病细胞系的细胞毒性。