Biogenesis of surface-exposed lipoproteins in Gram-negative bacteria

项目摘要 革兰氏阴性菌的外膜是介导细菌相互作用的动态界面 通过在其表面展示蛋白质来吸引宿主。最近发现的表面暴露脂蛋白 SLP在机体铁的获得、粘附、免疫逃避和免疫耐受等病理过程中起着重要作用， 作为有价值的疫苗靶点。尽管它们在生物医学上很重要，但脂蛋白的潜在机制 定位于细胞表面是细菌包膜生物发生的最不为人所知的方面。很长的- 我实验室的长期研究目标是确定脂蛋白靶向和输出的机制， 细菌细胞表面。我们预期具有相似拓扑和/或结构的脂蛋白组 特征共享专用的组装路径。提高我们对分子决定簇的理解 用于出口将能够开发脂蛋白定位的预测计算模型， SLP的基因组鉴定。一个SLP亚家族包括依赖于伴侣β- 桶外膜蛋白（OMP）用于表面暴露。我们发现了RcsF脂蛋白， 大肠杆菌是这种类型的第一个例子。我们进一步发现， 必需的β-桶装配机械（BAM）复合物在RcsF的生物发生中起关键作用， 揭示了BAM复合物在脂蛋白生物合成中的新功能。在这里，我们建议使用 结合遗传学、生物化学和质谱方法来鉴定分子 Bam复合物在细胞表面展示脂蛋白的机制。我们特别 对Bam复合物如何识别脂蛋白并协调脂蛋白表面暴露感兴趣 OMP组装。建议的研究完成后， SLP和革兰氏阴性细胞包膜的生物发生。从建议中获得的知识 研究将使计算模型的制定，以确定新的SLP和急需的 疫苗目标。

PROJECT SUMMARY The outer membrane in Gram-negative bacteria is a dynamic interface that mediates bacterial interaction with the host by displaying proteins on its surface. The recently discovered surface-exposed lipoproteins (SLPs) play critical roles in pathogenesis, including iron acquisition, adhesion, immune evasion and serve as valuable vaccine targets. Despite their biomedical importance, the mechanism underlying lipoprotein localization to the cell surface is the least understood aspect of bacterial envelope biogenesis. The long- term goal of research in my laboratory is to define the mechanism of lipoprotein targeting and export to the bacterial cell surface. We expect that groups of lipoproteins with similar topologies and/or structural features share dedicated assembly pathways. Improving our understanding of molecular determinants for export will enable the development of predictive computational models for lipoprotein localization and genomic identification of SLPs. One SLP subfamily includes lipoproteins that depend on a partner β- barrel outer membrane protein (OMP) for surface exposure. We discovered the RcsF lipoprotein in Escherichia coli as the first example of this type. We further discovered that the highly conserved and essential β-barrel assembly machinery (Bam) complex plays a critical role in the biogenesis of RcsF, uncovering the novel function of the Bam complex in lipoprotein biogenesis. Here, we propose to use a combination of genetics, biochemistry, and mass spectrometry approaches to identify the molecular mechanism by which the Bam complex displays lipoproteins on the cell surface. We are specifically interested in how the Bam complex recognizes lipoproteins and coordinates lipoprotein surface exposure with OMP assembly. The completion of the proposed studies will substantially expand our understating of biogenesis of SLPs and the Gram-negative cell envelope. The knowledge gained from the proposed studies will enable formulation of computational models for identification of novel SLPs and much-needed vaccines targets.