Lipoprotein Transport in Borrelia Spirochetes

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显示出多种具有不同生物学功能的丰富脂蛋白。不考虑不同环境中不同脂蛋白表达的调控机制，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转运系统，以及（ii）准确的脂蛋白分选机制。这个建议的总体目标是获得关键的洞察这两个重要的螺旋体发病机制的基本方面使用LB螺旋体伯氏疏螺旋体作为模型。我们的初步研究表明，疏螺旋体脂蛋白分选信号定位于N-末端半胱氨酸后约10个残基的N-末端序列，但与E.杆菌我们也开始描述B。burgdorferiBB 0346是E.杆菌还鉴定了该途径上游分子事件的蛋白质同源物，但未鉴定出BB 0346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E.大肠杆菌，但（ii）在外膜的途径和（iii）脂蛋白分选规则偏离显着从其他diderm细菌中描述的。为了验证这些假设，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定位，进一步确定疏螺旋体脂蛋白分选信号。2.进一步研究B的生物学功能。使用免疫共沉淀、亲和纯化和互补实验，对伯氏Lol蛋白同源物进行纯化。3.使用新的诱变和蛋白质组学方法鉴定和表征疏螺旋体脂蛋白输出机制的其他组分。这些研究将（i）显著增加我们对螺旋体毒力的理解，（ii）揭示更多关于细菌蛋白质输出机制的进化，以及（iii）可能为未来干预策略的设计提供重要线索。

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.