Spatiotemporal Regulation of Protrusion Dynamics During Intracellular Bacterial Dissemination

细胞内细菌传播过程中突起动力学的时空调节

• 批准号: 10740577
• 负责人: DARREN E HIGGINS
• 金额: $ 20.98万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740577
• 关键词: Actins; Affect; Architecture; Bacteria; Bacterial Proteins; Brain; Cell Line; Cell Proliferation; Cell Separation; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Confocal Microscopy; Cytoskeletal Modeling; Cytoskeleton; Cytosol; Data; Disease; Elderly; Electron Microscopy; Endothelium; Epidermal Growth Factor Receptor; Epithelial Cells; Filopodia; Fluorescence Microscopy; Generations; Genes; Goals; Green Fluorescent Proteins; HepG2; Human; Immunocompromised Host; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Situ; Individual; Infection; Integration Host Factors; Knock-out; Knockout Mice; Knowledge; Length; Life; Listeria monocytogenes; Listeriosis; Mass Spectrum Analysis; Modeling; Morphology; Newborn Infant; Pathogenesis; Pathogenicity; Persons; Phase; Phosphotransferases; Plaque Assay; Plasma Cells; Primary carcinoma of the liver cells; Process; Proteins; Proteomics; Pseudopodia; Public Health; RNA Interference; Receptor Signaling; Regulation; Resolution; Role; Shigella flexneri; Small Interfering RNA; Tail; Time; Transmission Electron Microscopy; Variant; cell motility; conditional knockout; domain mapping; foodborne pathogen; insight; knock-down; live cell imaging; monocyte; mortality; non-tuberculosis mycobacteria; pathogenic bacteria; pregnant; profilin; spatiotemporal; uptake

PROJECT SUMMARY/ABSTRACT Infections by intracellular bacterial pathogens are a leading cause of illness and mortality worldwide. Listeria monocytogenes (Lm) is a gram-positive food-borne pathogen that replicates within the cytosol of host cells and causes listeriosis, a life-threatening disease that affects newborns, immunocompromised individuals, pregnant people, and the elderly. A key aspect of Lm pathogenesis is the ability of bacteria to use actin-based motility to spread into neighboring cells via Lm-induced protrusions. The formation and uptake of Lm protrusions is a multi- stage process that includes protrusion initiation, elongation, stabilization, and engulfment by neighboring cells. The host and bacterial factors required for each step of protrusion dynamics have remained unclear. We have discovered that a host protein, Abi1, localizes to Lm-induced protrusions and is required for efficient Lm cell-to- cell spread in human epithelial cells. Moreover, prior host factor screens have indicated that Abi1 is critical for infection by additional intracellular bacterial pathogens. The focus of this proposal is to determine the requirement of Abi1 and potential host accessory proteins for Lm protrusion dynamics and cell-to-cell spread. In Aim I, the requirement of Abi1 for cell-to-cell spread will be determined by infecting host cells of different lineages in which Abi1 expression has been knocked down or knocked out with Lm strains expressing GFP. Assessment of foci of infections by fluorescence microscopy and plaquing assays will be used to evaluate cell-to-cell spread. Furthermore, immunofluorescence (IF) microscopy will be used to determine the localization of Abi1 during infection and to characterize protrusions within wild-type and Abi1-negative host cells. Transmission electron microscopy will be used to determine differences in the architecture of Lm-induced protrusions in Abi1-negative cells. In addition, spinning disk confocal live-cell imaging with GFP-Abi1 expressing host cells and Lm expressing TagRFP will be used to determine the spatiotemporal localization of Abi1 during Lm infection. In Aim II, we will use host cells expressing full-length and truncated GFP-Abi1 variants with IF microscopy to identify the domain(s) of Abi1 responsible for Abi1 localization to Lm protrusions. Plaquing assays will be performed to determine if Abi1 truncation variants can complement cell-to-cell spread in ABI1 knockout cells. We will determine the requirement of known Abi1 interacting proteins for localization of Abi1 and Lm protrusion formation using RNAi in conjunction with IF and spinning disk confocal microscopy. Plaquing assays in cells depleted for known Abi1 interacting proteins or the Abi1 interacting proteins and Abi1 will be performed to determine the contribution of the accessory proteins in Lm cell-to-cell spread. To identify additional accessory proteins that may play a role in Lm protrusion dynamics and bacterial dissemination, we will determine the complete proteomic profile of isolated Lm-induced protrusions by mass spectrometry. The proposed studies will provide significant insights into the role of Abi1 in regulating Lm protrusion dynamics and cell-to-cell spread and provide fundamental knowledge into a process shared by multiple intracellular bacterial pathogens for dissemination within the host.

项目摘要/摘要 细胞内细菌病原体的感染是世界范围内疾病和死亡的主要原因。李斯特菌 单核细胞增多症（Lm）是一种革兰氏阳性食源性病原体，在宿主细胞的胞质溶胶中复制， 引起脑膜炎，一种危及生命的疾病，影响新生儿，免疫功能低下的个人，孕妇 人和老人。Lm发病机制的一个关键方面是细菌利用基于肌动蛋白的运动能力， 通过Lm诱导的突起扩散到相邻细胞中。Lm突起的形成和摄取是多方面的， 包括突起起始、伸长、稳定和被相邻细胞吞噬的阶段过程。 突出动力学的每个步骤所需的宿主和细菌因素仍然不清楚。我们有 发现宿主蛋白Abi 1定位于Lm诱导的突起，并且是Lm细胞有效地与 细胞在人体上皮细胞中扩散。此外，先前的宿主因子筛选已经表明Abi 1对于 感染额外的细胞内细菌病原体。本提案的重点是确定 Lm突出动力学和细胞间传播对Abi 1和潜在宿主辅助蛋白的需求。在 目的I，通过感染不同谱系的宿主细胞来确定Abi 1在细胞间传播的需求 其中Abi 1表达已被敲低或用表达GFP的Lm菌株敲除。评估 将使用荧光显微镜和空斑试验检测感染病灶，以评价细胞间扩散。 此外，免疫荧光（IF）显微镜将用于确定Abi 1在细胞内的定位。 感染和表征野生型和Abi 1阴性宿主细胞内的突起。透射电子 将使用显微镜来确定Abi 1阴性细胞中Lm诱导的突起结构的差异。 细胞此外，用表达GFP-Abi 1的宿主细胞和表达Lm的宿主细胞进行旋转圆盘共聚焦活细胞成像， TagRFP将用于确定Lm感染期间Abi 1的时空定位。在Aim II中，我们将 使用表达全长和截短的GFP-Abi 1变体的宿主细胞，用IF显微镜鉴定结构域 负责Abi 1定位到Lm突起。将进行空斑试验，以确定是否 Abi 1截短变体可以补充ABI 1敲除细胞中的细胞间传播。康贝特人将以 使用RNAi定位Abi 1和Lm突起形成所需的已知Abi 1相互作用蛋白 与IF和旋转圆盘共聚焦显微镜结合。已知Abi 1耗竭细胞中的空斑试验 相互作用蛋白或Abi 1相互作用蛋白和Abi 1的作用，以确定 Lm细胞间传播中的辅助蛋白。为了鉴定可能在以下方面发挥作用的其他辅助蛋白： LM突出动力学和细菌传播，我们将确定分离的完整蛋白质组谱 用质谱法测定稀土诱导的突起。拟议的研究将提供重要的见解， Abi 1在调节Lm突起动力学和细胞间扩散中的作用，并提供基础知识 转化为多种细胞内细菌病原体共享的过程，用于在宿主内传播。