Lipoprotein Transport in Borrelia Spirochetes

描述(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)和复发性热病(RF)的病原体，在其生命周期中显示出各种丰富的脂蛋白，具有不同的生物学功能。不管不同环境中不同脂蛋白表达的调控机制如何，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。这项建议的总体目标是以伯氏疏螺旋体为模型，获得对螺旋体致病的这两个重要潜在方面的关键见解。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，但与大肠杆菌中的不同。我们还开始将伯氏杆菌BB0346鉴定为大肠杆菌中周质载脂蛋白载体LOLA的功能同源物。该途径上游的分子事件的蛋白质同源物也被鉴定出来，但不是BB0346的下游。因此，我们假设(I)疏螺旋体脂蛋白输出机制类似于在大肠杆菌中描述的机制，但(Ii)外膜途径和(Iii)脂蛋白分选规则与其他双子体细菌中描述的规则显著不同。为了验证这些假说，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，进一步确定脆性脂蛋白分选信号。2.通过免疫共沉淀、亲和纯化和互补实验进一步鉴定伯氏杆菌Lol蛋白同源物的生物学功能。3.利用新的诱变剂和蛋白质组学方法鉴定和鉴定硼质脂蛋白输出机制的其他成分。这些研究将(I)显著增加我们对螺旋体毒力的理解，(Ii)更多地揭示细菌蛋白质输出机制的一般进化，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.