Lipoprotein Transport in Borrelia Spirochetes

疏螺旋体中的脂蛋白运输

• 批准号: 7624331
• 负责人: WOLFRAM R ZUECKERT
• 金额: $ 35.01万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624331
• 关键词: ATP phosphohydrolase; Affinity; Affinity Chromatography; Animal Model; Antibodies; Bacteria; Bacterial Proteins; Binding Proteins; Biological Assay; Biological Process; Borrelia; Borrelia burgdorferi; Co-Immunoprecipitations; Complement; Complex; Cysteine; Cytoplasm; Environment; Enzymes; Escherichia coli; Essential Genes; Event; Evolution; Fluorescence; Future; Genome; Goals; Growth; Homologous Gene; Homologous Protein; In Vitro; Infection; Intervention; Life Cycle Stages; Light; Lipoprotein Binding; Lipoproteins; Lyme Disease; Membrane; Modeling; Modification; Molecular; Molecular Chaperones; Mutagenesis; N-terminal; Order Spirochaetales; Pathogenesis; Pathway interactions; Phenotype; Protein Export Pathway; Proteins; Proteomics; Recombinant Proteins; Relapsing Fever; Reporter; Research Personnel; Signal Transduction; Sorting - Cell Movement; Surface; System; Techniques; Technology; Testing; Tetanus Helper Peptide; Transmembrane Transport; Tungsten; Virulence; Virulence Factors; Yeasts; base; cellular targeting; design; genome-wide; in vivo; insight; mutant; novel; periplasm; prevent; promoter; protein aminoacid sequence; protein complex; relapsing fever borrelia; research study; sortase; translocase; transmission process; vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Spirochetes of the genus Borrelia, the causative agents of vector-borne Lyme borreliosis (LB) and relapsing fever (RF), display throughout their lifecycle a variety of abundant lipoproteins with distinct biological functions. Irrespective of the regulatory mechanisms governing the expression of different lipoproteins in different environments, the successful deployment of these spirochetal virulence factors hinges on (i) an efficient lipoprotein modification and transport system, and (ii) an accurate lipoprotein sorting machinery. The overall objective of this proposal is to gain key insights into these two important underlying aspects of spirochetal pathogenesis using the LB spirochete Borrelia burgdorferi as a model. Our preliminary studies indicate that Borrelia lipoproteins sorting signals localize to an N-terminal sequence of about ten residues after the N-terminal cysteine, yet differ from the ones characterized in E. coli. We have also begun to characterize B. burgdorferi BB0346 as a functional homolog of the periplasmic lipoprotein carrier LolA in E. coli. Protein homologs for molecular events in this pathway upstream, but not downstream of BB0346 were identified as well. We therefore hypothesize that (i) the Borrelia lipoprotein export machinery is similar to the one described in E. coli, but (ii) pathways at the outer membrane and (iii) the lipoprotein sorting rules diverge significantly from the ones described in other diderm bacteria. To test these hypotheses, we have formulated the following three specific aims: 1. To further define borrelial lipoprotein sorting signals by studying subcellular (mis)localization of fluorescent reporter proteins and lipoprotein mutants. 2. To further characterize the biological function of B. burgdorferi Lol protein homologs using co- immunoprecipitation, affinity purification, and complementation experiments. 3. To identify and characterize other components of the borrelial lipoprotein export machinery using novel mutagenic and proteomic approaches. These studies will (i) significantly increase our understanding of spirochetal virulence, (ii) shed more light on the evolution of bacterial protein export mechanisms in general, and (iii) may yield important clues for the design of future intervention strategies.

描述（由申请人提供）：Borrelia属的螺旋体，载体 - 传播的莱姆毛毛虫病（LB）的病因和复发性发烧（RF），在其整个生命周期中都显示出各种具有不同生物学功能的丰富脂蛋白。不论管理不同环境中不同脂蛋白表达的调控机制如何成功地部署了这些螺旋体性毒力因子（i）有效的脂蛋白修饰和运输系统，以及（ii）精确的脂蛋白分类机器。该提案的总体目的是使用LB Spirochete Borrelia Burgdorferi作为模型，以对这两个重要的脊柱发病机理的基本潜在方面获得关键见解。我们的初步研究表明，在N末端半胱氨酸后，伯罗利脂蛋白分类信号定位于大约十个残基的N末端序列，但与大肠杆菌中特征的bor骨序列不同。我们还开始将B. burgdorferi BB0346表征为大肠杆菌中周质脂蛋白载体LOLA的功能同源物。在该途径上游的该途径中的分子事件的蛋白质同源物也被鉴定出BB0346的下游。因此，我们假设（i）（i）伯罗利脂蛋白的导出机械与大肠杆菌中描述的机械相似，但是（ii）外膜处的途径和（iii）脂蛋白分选规则与其他DIDERM细菌中描述的脂蛋白分选规则显着差异。为了检验这些假设，我们提出了以下三个具体目的：1。通过研究荧光报告基因蛋白和脂蛋白突变体的亚细胞（MIS）定位，进一步定义骨脂蛋白分类信号。 2。进一步表征B. burgdorferi LoL蛋白同源物的生物学功能，使用共沉淀，亲和力纯化和补体实验。 3。使用新型的诱变和蛋白质组学方法来识别和表征骨脂蛋白导出机械的其他成分。这些研究将（i）显着提高我们对螺旋毒力的理解，（ii）一般来说，（ii）更多地了解了细菌蛋白出口机制的演变，并且（iii）可能会为未来的干预策略设计而产生重要的线索。