Mechanisms regulating vimentin-dependent invasion of the brain by Listeria monocytogenes

单核细胞增生李斯特菌依赖波形蛋白入侵大脑的调节机制

• 批准号: 10183151
• 负责人: John Hunter Brumell
• 金额: $ 38.53万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-26 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183151
• 关键词: Affect; Antibodies; Astrocytes; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Blood; Blood - brain barrier anatomy; Brain; Cause of Death; Cell Extracts; Cell surface; Cells; Cellular Membrane; Cultured Cells; Disease; Elderly; Encephalitis; Endothelial Cells; Family member; Fluorescence Microscopy; Gastroenteritis; Gentamicins; Goals; Human; Immunocompromised Host; Immunoprecipitation; In Vitro; Individual; Infection; Infection prevention; Intermediate Filament Proteins; Intestines; Invaded; Knock-out; Knockout Mice; Lead; Length; Life; Ligands; Listeria monocytogenes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Meningitis; Multigene Family; Mus; Neuraxis; Neurons; Pathway interactions; Phagocytes; Play; Pregnant Women; Protein Family; Proteins; Role; Surface; Vimentin; blood-brain barrier crossing; brain cell; brain endothelial cell; cell type; experimental study; foodborne; gel electrophoresis; in vivo; insight; novel; novel strategies; pathogen; pathogenic bacteria; pathogenic microbe; prevent; protein protein interaction; receptor; repaired

Project Summary Listeria monocytogenes (Lm) is an intracellular bacterial pathogen capable of invading numerous host cell types. Lm infections can lead to severe disease in humans and most often affects immunocompromised individuals, pregnant women, and the elderly. Of particular concern is the ability of Lm to invade the central nervous system (CNS), leading to life-threatening meningitis and encephalitis. The identity of factors necessary to facilitate Lm brain infection has remained unclear. We have recently shown that a Lm surface protein, InlF, is required for successful colonization of the brain in mice. Moreover, we have determined that InlF binds vimentin, a cytosolic intermediate filament protein also present on the surface of brain endothelial cells. We hypothesize that InlF-vimentin interaction is required for Lm passage across the blood-brain barrier (BBB) to establish an infection in the brain. The focus of this proposal is to elucidate how the InlF-vimentin interaction mediates invasion of host cells in vitro and Lm infection of the brain in vivo. In Aim I, in vitro infection assays will be used to determine how InlF interacts with vimentin to mediate invasion of host cells. Confocal fluorescence microscopy and gentamicin protection assays will be used to directly examine the ability of InlF- expressing Lm to interact with brain microvascular endothelial cells. Biochemical approaches will be used to further characterize InlF-vimentin protein binding and identify the regions of vimentin involved in the InlF- vimentin protein-protein interaction. The mechanisms that regulate cell surface exposure of vimentin are unknown. We hypothesize that cellular membrane repair pathways facilitate redistribution of cytosolic vimentin to the surface of host cells. Cell biological studies will be performed to determine the role of membrane repair pathways for the localization of vimentin to the host cell surface. Additionally, potential vimentin co-receptor candidates previously identified by mass spectrometry will be examined to determine their role in InlF-mediated invasion of brain endothelial cells. In Aim II, the contribution of the InlF-vimentin interaction to Lm infection in vivo will be determined by infection studies in normal and vimentin knockout mice. In vitro infection assays and fluorescence microscopy will be performed with primary brain cells cultured from normal and knockout mice to determine if InlF mediates invasion of distinct primary brain cell types (astrocytes and neurons) and determine the importance of vimentin and potential co-receptors for infection. Gentamicin protection assays with primary mouse endothelial cells will also be performed to determine if InlF is required for invasion of primary cells that constitute the BBB. Finally, infection of mice with cell-to-cell spread-defective ΔactA or ΔactA/ΔinlF-derived Lm strains will determine the in vivo contribution of InlF for colonization of the brain by direct invasion vs. cell-to- cell spread. The proposed studies will provide insights into the protein-protein interactions and cellular mechanisms facilitating Lm invasion of the brain and may identify novel targets for preventing infections of the brain by Lm and other microbial pathogens.

项目摘要 单核细胞增多性李斯特菌是一种能侵袭多种宿主的胞内致病菌 单元类型。肺炎衣原体感染可导致人类严重疾病，最常见的是影响免疫功能低下 个人、孕妇和老年人。特别令人担忧的是LM入侵中央的能力 神经系统(CNS)，导致危及生命的脑膜炎和脑炎。必要因素的同一性 促进LM脑部感染的机制尚不清楚。我们最近发现了一种名为Lm表面蛋白InlF， 是成功在小鼠体内定植大脑所必需的。此外，我们已经确定InlF与 波形蛋白是一种胞浆中间丝蛋白，也存在于脑内皮细胞表面。我们 假设LM通过血脑屏障(BBB)需要InlF-Vimentin相互作用 在大脑中建立感染。这一提议的重点是阐明InlF-Vimentin如何相互作用 在体外介导宿主细胞的侵袭，在体内介导脑部的LM感染。在AIM I中，体外感染分析 将用于确定InlF如何与Vimentin相互作用以介导宿主细胞的侵袭。共焦 荧光显微镜和庆大霉素保护试验将用于直接检测InlF- 表达LM与脑微血管内皮细胞相互作用。将使用生化方法来 进一步鉴定InlF-Vimentin蛋白结合，并鉴定参与InlF-Vimentin的区域 波形蛋白-蛋白质相互作用。调节细胞表面波形蛋白暴露的机制是 未知。我们假设细胞膜修复途径促进胞浆波形蛋白的重新分布 到宿主细胞的表面。将进行细胞生物学研究，以确定膜修复的作用 波形蛋白定位于宿主细胞表面的途径。此外，潜在的波形蛋白共受体 之前通过质谱学确定的候选人将接受检查，以确定他们在InlF中介中的角色 脑内皮细胞的侵袭。在AIM II中，InlF-Vimentin相互作用在肺炎衣原体感染中的作用 活体将通过对正常和波形蛋白基因敲除小鼠的感染研究来确定。体外感染检测和 荧光显微镜将对培养自正常和基因敲除小鼠的原代脑细胞进行 确定InlF是否介导不同的原代脑细胞类型(星形胶质细胞和神经元)的侵袭，并确定 波形蛋白和潜在的辅助受体对感染的重要性。庆大霉素保护性试剂盒 还将对小鼠内皮细胞进行检查，以确定InlF是否是侵袭原代细胞所必需的 构成了血脑屏障。最后，小鼠感染细胞间传播缺陷的ΔActA或ΔActA/Δ在F来源的LM中 菌株将确定InlF在体内通过直接侵袭与细胞侵袭对大脑定植的贡献。 细胞扩散。拟议的研究将提供对蛋白质-蛋白质相互作用和细胞 促进肺炎衣原体侵袭大脑的机制，并可能确定预防肺炎衣原体感染的新靶点 脑部受金黄色葡萄球菌等微生物病原体感染。