Cellular Intoxication Pathway of Cytolethal Distending Toxin

描述（由申请人提供）：细胞内作用的细菌蛋白毒素已经进化出各种与哺乳动物细胞相互作用并进入哺乳动物细胞的方法，利用现有的宿主细胞过程。对细菌毒素内化机制的研究为细胞生物学的几个领域提供了基础的进展，包括受体生物学、内吞作用的机制和途径、囊泡运输和膜易位。在这里，我们建议研究集中在一个新兴的毒素家族的一个成员，称为细胞致死扩张性毒素（CDTs），它们在细胞内定位，调节活性和整体结构上是不寻常的。CDTs是由多种致病性革兰氏阴性菌产生的多亚基毒素，通过调节宿主细胞周期发挥作用。活性CDT全毒素是由三个蛋白质亚基组成的异三聚体复合物，通常由三个相邻的基因（cdtA, cdtB, cdtC）在单个操纵子中编码。为了对细胞发挥其细胞调节作用，CDTs必须从细胞表面被吸收，并以一种最终导致酶亚基CdtB定位到细胞核的方式在细胞内运输。然而，CDTs利用现有的摄取和运输途径进入细胞质并最终进入细胞核的分子细节和机制尚不清楚。该应用程序解决了这些目前的知识空白，并代表了加州大学洛杉矶分校（Bradley博士）和伊利诺伊大学（Blanke博士）的研究人员之间的合作建议，以研究CDT细胞中毒的分子基础。提出了两个目的，以解决关于细胞中毒所需的CDTs的细胞表面结合（Aim 1）和细胞内运输（Aim 2）的假设。目的1研究宿主（目的1.1）和毒素（目的1.2）对初始cdt细胞结合的分子决定因素。目的2直接关注宿主细胞摄取和运输CDT的需求，利用假设驱动（目的2.1）和正向遗传（目的2.2）方法。重要的是，CDTs的异三聚体性质，以及催化CdtB亚基在细胞核中的定位，是CDTs的独特特征。因此，鉴定宿主细胞决定因素和对CDT中毒很重要的运输途径预计将揭示新的宿主细胞需求和/或将蛋白质从细胞表面运输到细胞核的途径，并为毒素-宿主相互作用提供见解。

DESCRIPTION (provided by applicant): The intracellular-acting bacterial protein toxins have evolved various means to interact with and enter mammalian cells, taking advantage of existing host cellular processes. Studies of the mechanisms by which bacterial toxins are internalized have provided fundamental advances in several areas of cell biology, including receptor biology, mechanisms and pathways of endocytosis, vesicle trafficking, and membrane translocation. Here, we propose studies focused on one member of an emerging family of toxins called the cytolethal distending toxins (CDTs), which are unusual in their intracellular localization, modulatory activities, and overall structures. CDTs are multi-subunit toxins that are generated by a diverse group of pathogenic Gram-negative bacteria and function by modulating the host cell cycle. Active CDT holotoxins are heterotrimeric complexes of three protein subunits, generally encoded by three contiguous genes (cdtA, cdtB, cdtC) in a single operon. To exert their cyclomodulatory effects on cells, CDTs must be taken up from the cell surface and transported intracellularly in a manner that ultimately results in localization of the enzymatic subunit CdtB to the nucleus. However, the molecular details and mechanism by which CDTs exploit existing uptake and transport pathways to gain access to the cytosol, and ultimately the nucleus, are poorly understood. This application addresses these current gaps in knowledge and represents a collaborative proposal between investigators at the University of California at Los Angeles (Dr. Bradley) and the University of Illinois (Dr. Blanke) to investigate the molecular basis of CDT cellular intoxication. Two Aims are proposed that address hypotheses regarding the cell surface binding (Aim 1) and intracellular transport (Aim 2) of CDTs required for cellular intoxication. Aim 1 investigates the molecular determinants of the initial CDT-cell binding contributed by both the host (Aim 1.1) and toxin (Aim 1.2). Aim 2 focuses squarely on the host cellular requirements for uptake and trafficking of CDT, utilizing hypothesis driven (Aim 2.1) and forward genetic (Aim 2.2) approaches. Importantly, the heterotrimeric nature of CDTs, as well as the localization of the catalytic CdtB subunit to the nucleus, are unique features of the CDTs. Therefore, identification of host cell determinants and trafficking pathways that are important for CDT intoxication is predicted to reveal novel host cell requirements and/or routes for transporting proteins from the cell surface to the nucleus and provide insight into toxin-host interactions. PUBLIC HEALTH RELEVANCE: The studies proposed here are aimed at understanding the mechanisms by which bacterial protein toxins interact with, enter, and alter the properties of mammalian cells. We will focus on cytolethal distending toxins, which are produced by several pathogenic bacteria important in human disease. These studies will yield fundamental knowledge in several areas of cell biology, including receptor biology, protein uptake mechanisms, and intracellular vesicle transport pathways, which in turn will be translated into the design, production, and use of new therapeutic drugs to block the action of these toxins.