Role of Borrelia Lpt Homologs in Surface Lipoprotein Secretion

摘要 细菌蛋白质分泌是一个基本的生理过程，它产生细胞膜并维持 它在细菌生命周期中的完整性。在细菌病原体中，各种蛋白质分泌系统具有 已被证明将重要的毒力因子部署到细菌表面，进入环境，甚至直接进入 真核细胞或其他细菌。螺旋体螺旋体，壁虱传播莱姆病的病原体和 复发性发热，有独特的双膜包膜，周围有鞭毛。疏螺旋体表面缺乏 脂多糖，而不是被丰富的、免疫优势的和血清型定义的表面所覆盖 作为传递和致病的关键环节的脂蛋白。最近的一项研究表明，三分之二的人 在莱姆病细菌伯氏疏螺旋体表达的大约130种脂蛋白中，定位于 浮出水面。因此，伯氏杆菌是研究细菌分泌物的理想模式生物。 表面脂蛋白。 多项开创性研究表明：(I)疏螺旋体表面脂蛋白分泌决定因素 通常定位于成熟脂蛋白的N端无序系链区域，(Ii)通过 外膜可以在脂蛋白的C末端开始，并需要至少部分未折叠 构象，和(Iii)疏螺旋体表面脂蛋白最终锚定在外层的表面小叶 膜双层。这些数据支持疏螺旋体表面脂蛋白分泌途径的假设 包括防止表面脂蛋白过早折叠的周质机制和外膜 转位蛋白复合体，允许脂蛋白从周质翻转到表面。 该提议将通过识别和机械地定义组件来检验上述假设 伯氏杆菌表面脂蛋白分泌途径。AIM 1将建立在最近CRISPRi基因沉默的基础上 数据，并利用定点突变和定量蛋白质组学开始确定结构- 伯氏杆菌外膜蛋白促进脂蛋白转位的功能关系 从周质到表面。荧光标记将被用于进一步定义B的封存。 伯氏杆菌脂蛋白在空间和时间上的分布。目标2将使用相同的方法和X射线结晶学 对假设从内层释放表面脂蛋白的周质蛋白进行表征 然后把它们送到外膜上。 总之，这些实验将使用新的方法来进一步阐明新出现的病原体是如何 全球重要性生成并维护它们与主机的接口。这最终将产生更好的工具 诊断和改进预防和治疗战略。

Abstract Bacterial protein secretion is a fundamental physiological process that generates the cell envelope and maintains its integrity throughout the bacterial life cycle. In bacterial pathogens, a variety of protein secretion systems have been shown to deploy important virulence factors to the bacterial surface, into the milieu, or even directly into eukaryotic cells or other bacteria. Borrelia spirochetes, the causative agents of tick-borne Lyme disease and relapsing fever, have a unique double-membrane envelope with periplasmic flagella. The Borrelia surface lacks lipopolysaccharide and is instead covered by abundant, immunodominant and serotype-defining surface lipoproteins that serve as linchpins for transmission and pathogenesis. A recent study has shown that two thirds of the about 130 lipoproteins expressed by the Lyme disease bacterium Borrelia burgdorferi localize to the surface. Therefore, B. burgdorferi is a perfect model organism for investigations into the secretion of bacterial surface lipoproteins. Several seminal studies have demonstrated that (i) Borrelia surface lipoprotein secretion determinants commonly localize to N-terminal disordered tether regions of the mature lipoproteins, (ii) translocation through the outer membrane can initiate at a lipoprotein’s C terminus and requires an at least partially unfolded conformation, and (iii) Borrelia surface lipoproteins are ultimately anchored in the surface leaflet of the outer membrane bilayer. These data support the hypothesis that the Borrelia surface lipoprotein secretion pathway includes a periplasmic mechanism that prevents premature folding of surface lipoprotein and an outer membrane translocon complex that allows for the flipping of lipoproteins from the periplasm to the surface. This proposal will test the above hypothesis by identifying and mechanistically defining the components of the B. burgdorferi surface lipoprotein secretion pathway. Aim 1 will build on recent CRISPRi gene silencing data and employ site-directed mutagenesis and quantitative proteomics to begin determining the structure- function relationships of a B. burgdorferi outer membrane protein shown to facilitate translocation of lipoproteins from the periplasm to the surface. Fluorescent tags will be used to further define the sequestration of the B. burgdorferi lipoproteome in space and time. Aim 2 will use the same approaches as well as X-ray crystallography to characterize periplasmic proteins that are hypothesized to release surface lipoproteins from the inner membrane and deliver them to the outer membrane. Together, these experiments will use novel approaches to further elucidate how emerging pathogens of global importance generate and maintain their interface with the host. This will ultimately yield better tools for diagnostics and improved strategies for prevention and treatment.