Role of Borrelia Lpt Homologs in Surface Lipoprotein Secretion

疏螺旋体 Lpt 同源物在表面脂蛋白分泌中的作用

• 批准号: 10742481
• 负责人: WOLFRAM R ZUECKERT
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-23 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10742481
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Amino Acid Sequence; Architecture; Bacteria; Bacterial Proteins; Biological Process; Borrelia; Borrelia burgdorferi; Bypass; C-terminal; CRISPR interference; Cells; Complement; Complex; Data; Disease; Ensure; Eubacterium; Eukaryotic Cell; Flagella; Gene Silencing; Genome; Gram-Negative Bacteria; Homologous Gene; Investigation; Life Cycle Stages; Lipopolysaccharides; Lipoprotein (a); Lipoproteins; Lyme Disease; Mediating; Membrane; Membrane Proteins; Modeling; Modification; Molecular Conformation; Mutation; N-terminal; Order Spirochaetales; Pathogenesis; Pathway interactions; Peptides; Periplasmic Proteins; Physiological Processes; Prevalence; Prevention; Prevention strategy; Process; Property; Protein Secretion; Proteins; Proteomics; Relapsing Fever; Research; Research Proposals; Role; Seminal; Serotyping; Site-Directed Mutagenesis; Sorting; Structural Models; Structure; Structure-Activity Relationship; Surface; System; Testing; Time; United States; Vaccines; Variant; Vector-transmitted infectious disease; Virulence Factors; X-Ray Crystallography; cell envelope; diagnostic tool; dimer; emerging pathogen; experimental study; improved; in silico; innovation; insight; knock-down; model organism; monomer; mutant; novel strategies; pathogen; pathogenic bacteria; periplasm; premature; prevent; routine Bacterial stain; tick-borne; translocase; transmission process; vector tick

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.

抽象的 细菌蛋白分泌是一个基本的生理过程，生成细胞包膜并保持 它在整个细菌生命周期中的完整性。在细菌病原体中，多种蛋白质分泌系统具有 被证明将重要病毒因素部署到细菌表面，环境中，甚至直接进入 真核细胞或其他细菌。伯罗里亚螺旋体，tick传播莱姆病的灾难性药物和 发烧，具有周质鞭毛的独特双膜包膜。疏浆表面缺乏 脂多糖，而是被丰富的，免疫主导和血清型定义的表面覆盖 脂蛋白是用于传播和发病机理的素金蛋白。最近的一项研究表明三分之二 在莱姆病细菌表达的大约130种脂蛋白中 表面。因此，B. burgdorferi是研究细菌分泌的完美模型生物 表面脂蛋白。 几项第二项研究表明（i）疏疏促渗透表面脂蛋白分泌决定 通常位于成熟脂蛋白的N末端无序的绑带区域，（ii）通过 外膜可以在脂蛋白的C末端启动，并且至少需要部分展开 构象，（iii）漏硼表面脂蛋白最终锚定在外面的表面小叶中 膜双层。这些数据支持以下假设 包括一种前质机制，可防止表面脂蛋白和外膜的过早折叠 易转基体络合物可以使脂蛋白从周围汇聚到表面。 该建议将通过识别和机械定义组件来检验上述假设 B. burgdorferi表面脂蛋白分泌途径的。 AIM 1将基于最近的Crispri基因沉默 数据和员工定位的诱变和定量蛋白质组学开始确定结构 伯氏芽孢杆菌外膜蛋白的功能关系可促进脂蛋白的易位 从周期到表面。荧光标签将用于进一步定义B的封存。 布尔格德富里脂蛋白在时空中。 AIM 2将使用相同的方法以及X射线晶体学 为了表征外周蛋白，这些蛋白被认为从内部释放表面脂蛋白 膜并将其输送到外膜。 这些实验将共同采用新颖的方法来进一步阐明新兴病原体 全球重要性生成并维护与主机的接口。这最终将为您提供更好的工具 诊断和改进的预防和治疗策略。