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

项目摘要 单核细胞增生李斯特菌（Listeriamonocytogenes，Lm）是一种能侵入多种宿主的胞内致病菌 细胞类型。Lm感染可导致严重的疾病在人类和最经常影响免疫功能低下 个人，孕妇和老人。特别值得关注的是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。必要因素的确定 促进Lm脑部感染的机制仍不清楚。我们最近发现Lm表面蛋白InlF， 是在小鼠大脑中成功定植所必需的。此外，我们已经确定， 波形蛋白，一种也存在于脑内皮细胞表面的胞质中间丝蛋白。我们 假设InIF-波形蛋白相互作用是Lm穿过血脑屏障（BBB）所必需的， 在大脑中建立感染。该提案的重点是阐明InIF-波形蛋白相互作用如何影响细胞的增殖和分化。 介导体外宿主细胞的侵袭和体内脑的Lm感染。在目标I中，体外感染测定 将用于确定InIF如何与波形蛋白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查InIF的能力。 表达Lm以与脑微血管内皮细胞相互作用。生物化学方法将用于 进一步表征InIF-波形蛋白结合并鉴定参与InIF-波形蛋白结合的波形蛋白区域。 波形蛋白-蛋白相互作用。调节波形蛋白细胞表面暴露的机制是 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 将检查先前通过质谱法鉴定的候选物以确定它们在InIF介导的细胞凋亡中的作用。 脑内皮细胞的侵袭。在目的II中，研究了InIF-波形蛋白相互作用对Lm感染的贡献。 将通过在正常和波形蛋白敲除小鼠中的感染研究来确定体内感染。体外感染测定和 荧光显微镜将用从正常和敲除小鼠培养的原代脑细胞进行， 确定InIF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 波形蛋白和潜在的共受体对感染的重要性。庆大霉素保护试验， 还将进行小鼠内皮细胞的检测以确定InIF是否是侵袭 构成了BBB。最后，用细胞间扩散缺陷型ΔactA或ΔactA/Δ inlF衍生的Lm感染小鼠， 菌株将通过直接侵袭与细胞间侵袭来确定InIF对脑定殖的体内贡献。 细胞扩散。这些研究将有助于深入了解蛋白质-蛋白质相互作用和细胞生物学行为。 促进Lm侵入大脑的机制，并可能确定预防脑组织感染的新靶点。 脑中的Lm和其他微生物病原体。

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.