Elucidating how intracellular bacterial pathogens hijack host intercellular communication to promote spread

阐明细胞内细菌病原体如何劫持宿主细胞间通讯以促进传播

• 批准号: 9529899
• 负责人: REBECCA L LAMASON
• 金额: $ 24.9万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9529899
• 关键词: Actins; Advisory Committees; Antigens; Architecture; Bacteria; Bacterial Proteins; Binding; Caveolins; Cell Adhesion; Cell Adhesion Molecules; Cell membrane; Cell physiology; Cell-Cell Adhesion; Cells; Cellular biology; Communication; Complement; Complex; Critical Pathways; Cytoskeleton; Cytosol; Diagnostic; Disease; Endocytosis; Ensure; Future; Genes; Goals; Image; Immune system; Infection; Integration Host Factors; Intercellular Junctions; Invaded; Kinetics; Lead; Learning; Listeria; Listeria monocytogenes; Malignant Neoplasms; Mediating; Membrane; Mentors; Methods; Microbiology; Microscopy; Molecular; Morphology; Mutagenesis; Paper; Pathogenesis; Pathway interactions; Phase; Positioning Attribute; Process; Proteins; Recruitment Activity; Regulation; Research; Research Personnel; Resolution; Rickettsia; Rickettsiales; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Spatial Distribution; System; T-Lymphocyte; Testing; Time; Training; Universities; Variant; Vesicle; Vinculin; Virulence; Work; Writing; base; career; career development; cell motility; experimental study; genome editing; improved; insight; intercellular communication; interest; killings; live cell imaging; live cell microscopy; medical schools; mutant; pathogen; programs; public health relevance; response; student mentoring; success; tool; trafficking; uptake

 DESCRIPTION (provided by applicant): My research is focused on understanding how intracellular bacterial pathogens hijack host pathways of intercellular communication to promote cell-to-cell spread. My interest in the cell biology of host pathogen interactions evolved from my graduate training at Johns Hopkins University School of Medicine. There I studied how T cells use cell signaling pathways to respond to antigen, and how these responses go awry in cancer. I discovered how changes in the subcellular architecture and molecular complexes of positive regulators altered the activation intensity of signals to NF-B. This spurred an interest in understanding additional modes of signaling regulation and led me to investigate how intracellular bacterial pathogens hijack host cell signaling pathways. In particular, I have focuse on the process of cell-to-cell spread, which relies on vesicular-mediated traffic of bacteria between cells and likely involves pathways of intercellular communication. In the long-term, my goal is to discover how bacterial proteins target host cell signaling molecules to enable cell-to-cell spread, and to build a more complete picture of the molecular mechanisms that govern the spreading process. To achieve my long-term goals, I have developed a 2-year training plan that will allow me to learn cutting-edge methods such as genome editing and super resolution microscopy, and to expand my expertise in research fields such as endocytosis and microbiology. These will be essential for the future success of my independent research program. This additional time will allow me to participate in necessary career development activities, including mentoring students, writing papers, and presenting talks. To complete the work during the mentored phase, I have assembled a panel of experts that includes my mentor Dr. Matthew Welch, an expert in cell biology and the actin cytoskeleton, and my co-mentors Dr. Daniel Portnoy, a leader in the field of bacterial pathogenesis, Dr. David Drubin, an expert in membrane trafficking mechanisms, and finally Dr. Xavier Darzacq, a pioneer in super resolution microscopy. The combination of this excellent advisory team as well as the phenomenal facilities provided by UC Berkeley will ensure that I make significant progress towards my research and career goals. This training plan will prepare me for a successful transition into an independent investigator position and lay the groundwork for research to be done during the independent phase. The goal of the research plan outlined in this proposal is to understand how two bacterial pathogens, Rickettsia parkeri and Listeria monocytogenes, hijack host pathways of intercellular communication to promote spread, a process that is crucial for their virulence. These pathogens live in the host cell cytosol and spread from cell to cell by mobilizing the host's actin cytoskeleton for intracellular motility and transport to the plasma membrane. Once at the cell periphery, they induce the formation of membrane protrusions that are engulfed by neighboring cells. Despite recent advances, we lack critical information about how these stages of spread occur. Based on my preliminary work, this proposal will investigate the specific hypothesis that bacterial pathogens promote spread by hijacking proteins normally involved in cell adhesion, membrane remodeling, and endocytosis; processes critically involved in intercellular communication. Furthermore, I hypothesize that distinct host pathways are hijacked by each pathogen. I propose the following experiments to test these hypotheses. In Aim 1, I will identify which endocytic, cell adhesion, and membrane remodeling proteins promote spread, with a focus on the role of caveolin endocytosis in the K99 phase, and a focus on cell adhesion and membrane remodeling factors in the R00 phase. This will define the key host molecules involved in spread. To provide further mechanistic insight, in Aim 2 I will utilize live cell imagig to determine the dynamics of host protein recruitment, and super-resolution imaging to define the subcellular architecture of those host proteins studied in Aim 1. This will reveal the order in which host proteins are recruited, and their spatial organization during spread, providing key insights into their mechanisms of action. Finally, to examine how bacterial effector proteins hijack host factors to promote spread, in Aim 3 I will examine how the R. parkeri secreted factor Sca4 targets the cell adhesion protein vinculin to promote spread. In the K99 phase I will focus on the localization and interaction of these factors. During the R00 phase I will examine the regulation of Sca4 secretion and the role of vinculin in spread. As a whole, this work will delineate how bacterial effectors and host pathways cooperate to enable spread, and reveal similarities and differences in the molecular strategies of spread for diverse pathogens. Discerning the targets of spread should also improve our understanding of basic cellular mechanisms, like vesicular traffic and intercellular communication, and how these go awry in disease.

 描述（由申请人提供）：我的研究重点是了解细胞内细菌病原体如何劫持宿主细胞间通讯途径，以促进细胞间传播。我在约翰霍普金斯大学医学院的研究生训练中对宿主病原体相互作用的细胞生物学产生了兴趣。在那里，我研究了T细胞如何利用细胞信号通路对抗原做出反应，以及这些反应在癌症中是如何出错的。我发现了亚细胞结构和正调节分子复合物的变化如何改变了NF-κ B B信号的激活强度。这激发了我对了解其他信号调节模式的兴趣，并引导我研究细胞内细菌病原体如何劫持宿主细胞信号通路。特别是，我集中在细胞间传播的过程中，这依赖于囊泡介导的细胞间细菌的交通，并可能涉及细胞间通讯的途径。从长远来看，我的目标是发现细菌蛋白质如何靶向宿主细胞信号分子，以实现细胞间的传播，并建立一个更完整的图片的分子机制，管理传播过程。 为了实现我的长期目标，我制定了一个为期2年的培训计划，这将使我能够学习基因组编辑和超分辨率显微镜等尖端方法，并扩展我在内吞作用和微生物学等研究领域的专业知识。这些将是我的独立研究计划未来成功的关键。这些额外的时间将使我能够参加必要的职业发展活动，包括指导学生，撰写论文和演讲。为了在指导阶段完成这项工作，我召集了一个专家小组，其中包括我的导师Matthew Welch博士，他是细胞生物学和肌动蛋白细胞骨架方面的专家，和我的共同导师丹尼尔波特诺伊博士，他是细菌发病机理领域的领导者，大卫德鲁宾博士，他是膜运输机制方面的专家，最后是泽维尔·达尔扎克博士，他是超分辨率显微镜的先驱。这个优秀的咨询团队以及加州大学伯克利分校提供的惊人的设施相结合，将确保我朝着我的研究和职业目标取得重大进展。该培训计划将为我成功过渡到独立调查员职位做好准备，并为独立阶段的研究奠定基础。 该提案中概述的研究计划的目标是了解两种细菌病原体，立克次氏体和单核细胞增生李斯特菌如何劫持宿主细胞间通讯途径以促进传播，这一过程对其毒力至关重要。这些病原体生活在宿主细胞的胞质溶胶中，并通过动员宿主的肌动蛋白细胞骨架进行细胞内运动和运输到质膜而在细胞间传播。一旦到达细胞周边，它们诱导膜突起的形成，这些突起被邻近细胞吞噬。尽管最近取得了进展，但我们缺乏关于这些传播阶段如何发生的关键信息。基于我的初步工作，本提案将调查细菌病原体通过劫持通常参与细胞粘附，膜重塑和内吞作用的蛋白质来促进传播的具体假设;这些过程关键参与细胞间通讯。此外，我假设不同的宿主途径被每种病原体劫持。我提出以下实验来检验这些假设。在目标1中，我将确定哪些内吞，细胞粘附和膜重塑蛋白促进扩散，重点是小窝蛋白内吞作用在K99阶段，并专注于细胞粘附和膜重塑因子在R 00阶段。这将定义参与传播的关键宿主分子。为了提供进一步的机制见解，在目标2中，我将利用活细胞成像来确定宿主蛋白质募集的动态，并利用超分辨率成像来定义目标1中研究的那些宿主蛋白质的亚细胞结构。这将显示 哪些宿主蛋白被招募，以及它们在传播过程中的空间组织，为它们的作用机制提供了关键的见解。最后，为了研究细菌效应蛋白如何劫持宿主因子以促进传播，在目标3中，我将研究R。parkeri分泌的因子Sca 4靶向细胞粘附蛋白粘着斑蛋白以促进扩散。在K99阶段，我将专注于这些因素的定位和相互作用。在R 00阶段，I将检查Sca 4分泌的调节和黏着斑蛋白在扩散中的作用。作为一个整体，这项工作将描绘细菌效应器和宿主途径如何合作，使传播，并揭示不同病原体传播的分子策略的相似性和差异。识别传播的目标也应该提高我们对基本细胞机制的理解，如囊泡运输和细胞间通讯，以及这些在疾病中是如何出错的。