Listeria Monocytogenes Infection of the Brain

描述（由申请人提供）： 单核细胞增生李斯特菌（Listeria monocytogenes，Lm）是一种革兰氏阳性的细胞内致病菌，能够侵入多种宿主细胞类型。结核分枝杆菌感染可导致人类严重疾病，最常影响免疫功能低下的个体、孕妇和老年人。特别值得关注的是Lm侵入中枢神经系统（CNS）的能力，导致危及生命的脑膜炎和脑炎。促进Lm脑感染的必要因素的身份仍不清楚。我们已经发现，Lm表面蛋白，InlF，是需要在小鼠的大脑中成功的殖民化。此外，亲和层析和质谱分析的结果表明，InlF与脑内皮细胞表面蛋白，波形蛋白，剪接因子Sfpq和核蛋白AHNAK相互作用。该建议的重点是阐明InlF如何介导Lm感染的大脑。我们假设InlF与波形蛋白相互作用，伴随着Sfpq和AHNAK，介导Lm进入脑细胞，并且在波形蛋白不存在的情况下，脑的Lm感染将被抑制。 由于InIF-波形蛋白相互作用对于成功的脑定殖的重要性，在体内严重受损。我们进一步假设InIF是穿过BBB以在脑中建立Lm感染所必需的。在目的I中，将使用庆大霉素保护测定来确定波形蛋白对于InIF介导的培养细胞系的侵袭的需求。使用表达绿色荧光蛋白（GFP）的Lm菌株和InIF包被的绿色荧光珠的荧光显微镜将用于更直接地检查InIF与宿主细胞相互作用的能力。此外，将采用生物化学方法来证明直接蛋白结合并鉴定参与InIF-波形蛋白蛋白-蛋白相互作用的区域。 在目的II中，InIF-波形蛋白相互作用对体内Lm感染的贡献将通过波形蛋白或AHNAK敲除小鼠中的感染研究来确定。将用从正常和敲除小鼠培养的原代脑细胞进行体外感染测定和荧光显微镜检查，以确定InIF是否介导不同原代脑细胞类型的感染以及波形蛋白和AHNAK对感染的重要性。在目的III中，我们将确定InIF是否通过直接侵袭或细胞间扩散介导BBB通过。将使用原代小鼠脑微血管内皮细胞和脑内皮细胞系进行庆大霉素保护试验，以确定直接侵入构成BBB的细胞是否需要InIF。将使用Lm感染的单核细胞和脑内皮细胞的异源性扩散测定来评估InIF在细胞间扩散到脑中的重要性。最后，用细胞间扩散缺陷的<$actA或<$actA/<$inlF Lm菌株感染小鼠将确定InlF通过直接侵袭与细胞间扩散对脑定殖的体内贡献。提出的研究将提供对促进Lm侵袭脑的蛋白质-蛋白质相互作用的深入了解，并可能确定用于预防Lm和其他细胞内病原体感染脑的新靶点。

DESCRIPTION (provided by applicant): Listeria monocytogenes (Lm) is a Gram-positive 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 discovered that an Lm surface protein, InlF, is required for successful colonization of the brain in mice. Moreover, the results of affinity chromatography and mass spectrometry analysis indicate that InlF interacts with the brain endothelial cell surface protein, Vimentin, splicing factor Sfpq and nucleoprotein AHNAK. The focus of this proposal is to elucidate how InlF mediates Lm infection of the brain. We hypothesize that InlF interacts with Vimentin, accompanied by Sfpq and AHNAK, to mediate entry of Lm into brain cells and that in the absence of Vimentin, Lm infection of the brain will be severely compromised in vivo due to the importance of InlF-Vimentin interactions for successful brain colonization. We further hypothesize that InlF is required for passage across the BBB to establish an Lm infection in the brain. In Aim I, gentamicin protection assays will be used to determine the requirement of Vimentin for InlF-mediated invasion of cultured cell lines. Fluorescence microscopy with Lm strains expressing green fluorescent protein (GFP) and InlF-coated green-fluorescent beads will be used to more directly examine the ability of InlF to interact with host cells. In addition, a biochemical approach will be taken to demonstrate direct protein binding and identify the regions involved in the InlF-Vimentin protein-protein interaction. In Aim II, the contribution of InlF-Vimentin interactions to Lm infection in vivo will be determine by infection studies in Vimentin or AHNAK 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 infection of distinct primary brain cell types and the importance of Vimentin and AHNAK for infection. In Aim III, we will determine if InlF mediates BBB passage by direct invasion or cell-to-cell spread. Gentamicin protection assays with primary mouse brain microvascular endothelial cells and brain endothelial cell lines will be performed to determine if InlF is required for direct invasion of cells that constitute the BBB. Heterologous spread assays with Lm infected monocytes and brain endothelial cells will be used to assess the importance of InlF in cell-to-cell spread into the brain. Finally, infection of mice with cell-to-cell spread defective ¿actA or ¿actA/¿inlF Lm strains will determine the in vivo contribution of InlF for colonization of the brain by direct invasion versus cell-to-cell spread. Te proposed studies will provide insights into the protein-protein interactions facilitating Lm invasin of the brain and may identify novel targets for preventing infections of the brain by Lm and other intracellular pathogens.