Lipoprotein Transport in Borrelia Spirochetes

疏螺旋体中的脂蛋白运输

• 批准号: 7433306
• 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/7433306
• 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; Event; Evolution; Fluorescence; Future; Genome; Goals; Growth; Homologous Gene; Homologous Protein; In Vitro; Infection; Intervention; Life Cycle Stages; Light; Lipoprotein Binding; Lipoproteins; Localized; 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; in vivo; insight; mutant; novel; periplasm; prevent; promoter; protein aminoacid sequence; 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）：疏螺旋体属螺旋体是媒介传播的莱姆疏螺旋体病（LB）和回归热（RF）的病原体，在其整个生命周期中表现出多种丰富的具有独特生物学功能的脂蛋白。无论不同环境中不同脂蛋白表达的调节机制如何，这些螺旋体毒力因子的成功部署取决于（i）有效的脂蛋白修饰和运输系统，以及（ii）准确的脂蛋白分选机制。该提案的总体目标是使用 LB 螺旋体伯氏疏螺旋体作为模型，获得对螺旋体发病机制这两个重要的基本方面的关键见解。我们的初步研究表明，疏螺旋体脂蛋白分选信号定位于 N 端半胱氨酸后约 10 个残基的 N 端序列，但与大肠杆菌中的特征不同。我们还开始将伯氏疏螺旋体 BB0346 鉴定为大肠杆菌中周质脂蛋白载体 LolA 的功能同源物。还鉴定了该途径上游而非 BB0346 下游分子事件的蛋白质同源物。因此，我们假设（i）疏螺旋体脂蛋白输出机制与大肠杆菌中描述的类似，但（ii）外膜的途径和（iii）脂蛋白分选规则与其他二层细菌中描述的规则显着不同。为了检验这些假设，我们制定了以下三个具体目标： 1. 通过研究荧光报告蛋白和脂蛋白突变体的亚细胞（错误）定位来进一步定义疏螺旋体脂蛋白分选信号。 2.利用免疫共沉淀、亲和纯化和互补实验进一步表征伯氏疏螺旋体Lol蛋白同源物的生物学功能。 3. 使用新的诱变和蛋白质组学方法来鉴定和表征疏螺旋体脂蛋白输出机制的其他成分。这些研究将（i）显着增加我们对螺旋体毒力的理解，（ii）进一步阐明细菌蛋白质输出机制的总体演变，以及（iii）可能为未来干预策略的设计提供重要线索。