Molecular Mechanism of Translocon Assembly into Cell Plasma Membranes

描述（由申请人提供）：蛋白质跨细胞膜转运的复杂过程是细菌发病的一个共同主题。一种特别有效的蛋白质递送分子机器是III型分泌（T3S）系统。T3S系统就像一个注射器，将细菌细胞质中的蛋白质直接注射到靶细胞的细胞质中。一旦进入宿主细胞质，易位的毒素破坏真核细胞过程（例如，阻断吞噬作用），调节宿主的反应，有利于感染。在鼠疫耶尔森菌和铜绿假单胞菌中收集到的证据表明，两种分泌的T3S蛋白插入靶细胞膜并形成一个孔（或易位子），毒素通过该孔易位。尽管最近对这两种蛋白质（易位子）的特征化研究取得了进展，但它们的结构和易位子的组装机制仍不清楚。我已经开发了一套荧光技术，已成功地用于表征各种同聚寡聚细胞溶解毒素的结构和孔隙形成机制。我现在建议将这些技术扩展到多蛋白跨膜复合体，如T3S转座子。荧光方法将与其他生化和生物物理技术（例如，电生理学测量，单分子技术和低温电子显微镜）相结合，以明确地解决T3S易位结构和组装的基本结构方面的问题。通过选择性地将各种探针（例如，对环境敏感的荧光团、交联剂、金纳米粒子、带电基团等）结合到铜绿假单胞菌鼻转运子中，我们将通过实验确定，除其他外：这些蛋白质的哪些片段对确定易位通道的特征至关重要，哪些片段形成针与易位子之间的接触界面，以及易位子如何排列在哺乳动物细胞膜上形成的易位子复合体中。除了阐明易位组装成脂质双层的基本方面外，这些研究可能最终导致新的治疗策略，以阻止蛋白易位并干扰各种威胁人类病原体的细菌定植。

DESCRIPTION (provided by applicant): The complex process of protein translocation across cell membranes is a common theme in bacterial pathogenesis. A particularly efficient molecular machine for pro tein delivery is the type III secretion (T3S) system. The T3S system acts as a syringe that injects proteins from the bacterial cytoplasm directly into the cytoplasm of a target cell. Once into the host cytosol, translocated toxins subvert eukaryotic cellular processes (e.g., blocking phagocytosis), modulating the host response in favor of infection. Collected evidence in Yersinia pestis and Pseudomonas aeruginosa suggests that two secreted T3S proteins insert into the target cell membrane and form a pore (or translocon) through which toxins are translocated. Despite recent advances on the char- acterization of these two proteins (the translocators), their structure and mechanism of assembly of the translocon remain unknown. I have developed a set of fluorescence techniques that have been successfully used to characterize the structure and pore-formation mechanism of various homo- oligomeric cytolytic toxins. I now propose to extent the use of these techniques to multi- protein transmembrane complexes, like the T3S translocon. The fluorescence approach will be combined with other biochemical and biophysical techniques (e.g., electrophysi- ology measurements, single molecule techniques, and cryo-electron microscopy) to un- ambiguously address fundamental structural aspects of the T3S translocon structure and assembly. By selective incorporation of various probes (e.g., environment-sensitive fluorophores, crosslinkers, gold-nanoparticles, charged groups, etc.) in the P. aerugi- nosa translocators, we will experimentally identify, among other things: which segments of these proteins are essential to determine the characteristics of the translocon channel, what segments form the contact interface between the needle and the translocon, and how the translocators are arranged in the translocon complex formed in the mammalian cell membrane. In addition to elucidate fundamental aspects of translocon assembly into lipid bi- layers, these studies may ultimately lead to novel therapeutic strategies that block pro- tein translocation and interfere with bacterial colonization in a broad variety of threaten- ing human pathogens. PUBLIC HEALTH RELEVANCE: The goal of this application is to analyze the structure and mechanism of assembly of the T3S translocon complex employed by pathogenic bacteria to inject virulence factors through the plasma membrane of human cells. Ultimately, the basic research done by us and others will identify how the virulence factors secreted by many different pathogenic bacteria are translocated into the cytosol of the target cell, and hence provide the opportunity to develop new therapeutic agents against pathogens that employ the T3S system while infecting humans. Many of these pathogens are listed as bioterrorism agents by the U.S. Center for Disease Control and Prevention (e.g., Yersinia pestis, Salmonella, Shigella), and pathogens like Pseudomonas aeruginosa, the major cause of death in cystic fibrosis patients, are particularly problematic not only because of their ubiquitous nature but als because of their resistance to most antibiotics.