Cellular Intoxication Pathway of Cytolethal Distending Toxin

描述(申请人提供)：细胞内作用的细菌蛋白毒素已经进化出各种方式来与哺乳动物细胞相互作用并进入哺乳动物细胞，利用现有的宿主细胞过程。对细菌毒素内化机制的研究在细胞生物学的几个领域取得了根本性的进展，包括受体生物学、内吞作用的机制和途径、囊泡运输和膜转运。在这里，我们建议对一种名为细胞致死性膨胀毒素(CDTS)的新兴毒素家族中的一种成员进行研究，这种毒素在细胞内定位、调节活性和整体结构方面都是不寻常的。CDT是一种多亚单位毒素，由一组不同的致病性革兰氏阴性细菌产生，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源三聚体复合体，一般由三个相邻基因(cdtA、cdtB、cdtC)在一个操纵子中编码。为了对细胞发挥周期调节作用，CDT必须从细胞表面被提取并在细胞内运输，最终导致酶亚基CDtB定位到细胞核。然而，CDT利用现有的吸收和运输途径获得胞浆并最终进入细胞核的分子细节和机制还知之甚少。该应用程序解决了目前知识上的这些空白，并代表了加州大学洛杉矶分校(布拉德利博士)和伊利诺伊大学(布兰克博士)的研究人员合作提出的一项建议，以调查CDT细胞中毒的分子基础。提出了两个目标，以解决关于细胞中毒所需的CDT的细胞表面结合(目标1)和细胞内运输(目标2)的假设。目的1研究宿主(Aim 1.1)和毒素(Aim 1.2)对初始CDT细胞结合的分子决定因素。目标2利用假说驱动(目标2.1)和正向遗传(目标2.2)方法，直接关注宿主细胞对CDT摄取和贩运的需求。重要的是，CDT的异三聚体性质以及催化的CDtB亚单位定位于核，是CDTS的独特特征。因此，识别对CDT中毒很重要的宿主细胞决定因素和运输途径有望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，并为深入了解毒素与宿主的相互作用提供依据。 公共卫生相关性：这里提出的研究旨在了解细菌蛋白毒素与哺乳动物细胞相互作用、进入和改变其特性的机制。我们将重点放在细胞致死膨胀毒素上，这种毒素是由几种在人类疾病中很重要的病原体产生的。这些研究将在细胞生物学的几个领域产生基础知识，包括受体生物学、蛋白质摄取机制和细胞内囊泡运输途径，这些知识反过来将转化为设计、生产和使用新的治疗药物来阻断这些毒素的作用。

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.