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 细菌蛋白质分泌是产生细胞包膜并维持细胞膜的基本生理过程。 它贯穿于细菌的整个生命周期。在细菌病原体中，有多种蛋白质分泌系统。 已被证明可以将重要的毒力因子部署到细菌表面、环境中，甚至直接部署到细菌表面 真核细胞或其他细菌，蜱传莱姆病的病原体和 回归热，具有独特的双膜缺乏与周质鞭毛表面。 脂多糖，而是被丰富的、免疫显性的、血清型定义的表面覆盖 最近的一项研究表明，三分之二的脂蛋白在传播和发病机制中发挥着作用。 莱姆病细菌伯氏疏螺旋体表达的约 130 种脂蛋白定位于 因此，伯氏疏螺旋体是研究细菌分泌的完美模型生物。 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) 通过易位 外膜可以从脂蛋白的 C 末端开始，并且需要至少部分展开 (iii)疏螺旋体表面脂蛋白最终锚定在外层的表面小叶中。 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和外膜过早折叠的周质机制 易位子复合体，允许脂蛋白从周质翻转到表面。 该提案将通过识别和机械定义组件来测试上述假设 伯氏疏螺旋体表面脂蛋白分泌途径的研究目标 1 将建立在最近的 CRISPRi 基因沉默基础上。 数据并采用定点诱变和定量蛋白质组学来开始确定结构- 伯氏疏螺旋体外膜蛋白的功能关系显示促进脂蛋白易位 从周质到表面的荧光标签将用于进一步确定 B. 目标 2 将使用相同的方法以及 X 射线晶体学。 表征周质蛋白，这些蛋白竞相从内部释放表面脂蛋白 膜并将它们输送到外膜。 这些实验将共同使用新的方法来进一步阐明新出现的病原体是如何 全局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。 诊断和改进的预防和治疗策略。